Adrenocorticotropin Evokes Transient Elevations in Intracellular Free Calcium ([Ca2+]i) and Increases Basal [Ca2+]i in Resting Chondrocytes
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内分泌学杂志 2005年第7期
Department of Medicine, Winthrop University Hospital (J.F.E., S.P., J.F.A., J.K.Y.), Mineola, New York 11501; Department of Pathology, Texas Tech University Health Sciences Center (C.-L.S.), Lubbock, Texas 79430; Health Sciences Center, State University of New York (J.F.A., J.K.Y.), Stony Brook, New York 11794; and Department of Decision Sciences, St. John’s University (S.P.), Jamaica, New York 11439
Address all correspondence and requests for reprints to: Dr. Jodi F. Evans, Department of Medicine, Winthrop University Hospital, 222 Station Plaza North, Suite 501, Mineola, New York 11501. E-mail: jevans@winthrop.org.
Abstract
Both clinical and in vitro evidence points to the involvement of the melanocortin peptide, ACTH, in the terminal differentiation of chondrocytes. Terminal differentiation along the endochondral pathway is responsible for linear growth, but also plays a role in osteoarthritic cartilage degeneration. Chondrocyte terminal differentiation is associated with an incremental increase in chondrocyte basal intracellular free calcium ([Ca2+]i), and ACTH agonism of melanocortin receptors is known to mobilize [Ca2+]i. Using differentiated resting chondrocytes highly expressing type II collagen and aggrecan, we examined the influence of both ACTH and dexamethasone treatment on matrix gene transcription and [Ca2+]i. Resting chondrocytes treated concurrently with dexamethasone and ACTH expressed matrix gene transcripts in a pattern consistent with that of rapid terminal differentiation. Using the fluorescent Ca2+ indicator, fura-2, we determined that ACTH evokes transient increases in [Ca2+]i and elevates basal Ca2+ levels in resting chondrocytes. The transient increases were initiated intracellularly, were abrogated by the phospholipase C-specific inhibitor, U73122, and were partly attenuated by myo-inositol 1,4,5-triphosphate receptor inhibition via 10 mM caffeine. The initial intracellular release also resulted in store-operated calcium entry, presumably through store-operated channels. Dexamethasone priming increased both the initial ACTH-evoked [Ca2+]i release and the subsequent store-operated calcium entry. These data demonstrate roles for ACTH and glucocorticoid in the regulation of chondrocyte terminal differentiation. Because the actions of ACTH are mediated through known G protein-coupled receptors, the melanocortin receptors, these data may provide a new therapeutic target in the treatment of growth deficiencies and cartilage degeneration.
Introduction
ACTH IS A member of the melanocortin family of peptides derived from proopiomelanocortin (POMC). POMC is posttranslationally processed in the anterior pituitary corticotropes to form ACTH, ?-lipotropic hormone, and pro--MSH. Additional processing into -MSH, CLIP, ?-MSH, and -MSH occurs in the melanotropes of the intermediate lobe of the pituitary (1). ACTH exerts its effects through agonism of the melanocortin receptors. The five melanocortin receptors (MC-R) identified to date are a family of G protein-coupled receptors (GPCR) that activate adenylyl cyclase and mobilize intracellular free calcium ([Ca2+]i) (2). These receptors are distinguishable through their agonist profiles, and the melanocortin-2 receptor (MC2-R) is the primary receptor for ACTH. However, ACTH is capable of activating all five MC-R with differing affinities. In fact, its potency at the MC3-R is nearly equivalent to that of the strongest agonist of this receptor, 2-MSH (2).
The human syndrome of familial glucocorticoid deficiency (FGD) provides clinical evidence of a role for ACTH in the regulation of chondrocyte terminal differentiation. FGD is characterized by an overproduction of ACTH, enhanced linear growth, and advanced bone age (3). Linear growth is accomplished through the process of endochondral ossification, which occurs in the cartilaginous growth plate located at both ends of the vertebrae and long bones (4). With the FGD syndrome, systemic levels of ACTH are extremely elevated due to a mutation in the MC2-R (ACTH) receptor. When FGD patients are treated with glucocorticoid, their ACTH levels are reduced, and their growth patterns normalize somewhat (3). These data suggest a role for ACTH in the regulation of endochondral ossification. Recent in vitro studies support this suggestion. Matrix genes, such as type II collagen (COLL II) and aggrecan (AGR), of chondrocytes and their precursors are directly affected by ACTH treatment. Steady-state mRNA levels of AGR are significantly up-regulated in both resting chondrocytes (RC) and the chondrogenic cell line RCJ3.1C5.18, when treated with ACTH. ACTH also increases the differentiation of these cells along the endochondral pathway (5).
Unlike the chondrocytes of the growth plate, articular chondrocytes in a healthy joint do not undergo terminal differentiation. Damage to the articular cartilage extracellular matrix, however, will induce increased transcription and secretion of type II collagen and AGR by articular chondrocytes (6). This response may lead to long-term changes in the phenotype of the cell (7). In fact, foci of articular chondrocytes in osteoarthritic cartilage, typically near sites of cartilage surface lesions, undergo terminal differentiation in a manner consistent with endochondral differentiation (8).
Interestingly, two major risk factors for osteoarthritic cartilage degeneration, age and obesity, are also associated with dysregulation of the hypothalamic-pituitary adrenal axis. Current data point to a relative adrenal insensitivity to ACTH in obesity, resulting in decreased cortisol secretion (9). Attenuated cortisol negative feedback will augment ACTH output during stress and increase the plasma ACTH concentration (10). Thus, elevated ACTH with a paradoxically low cortisol level is observed in obese subjects at baseline and in response to stress (9). Resistance to glucocorticoid feedback in aging, however, is associated with delayed inhibition of ACTH secretion (11, 12, 13), resulting in an increased exposure to both glucocorticoids and ACTH. These data along with the fact that age and obesity are strongly associated with cartilage degeneration suggest a link between dysregulation of hypothalamic-pituitary adrenal axis hormones and hypertrophic differentiation among articular chondrocytes.
Among the cellular events associated with chondrocyte terminal differentiation is an incremental increase in chondrocyte basal [Ca2+]i (14). Calcium signaling is also a required for stimulus-induced increases in AGR synthesis (15). Although the obligatory second messenger for ACTH is known to be cAMP, there is increasing evidence that ACTH agonism of melanocortin receptors also mobilizes [Ca2+]i (16, 17). Using differentiated RC highly expressing COLL II and AGR and therefore phenotypically similar to both chondrocytes of the growth plate and those in a damaged articular matrix, we examined the influence of ACTH and dexamethasone (DEXA) treatment on matrix gene transcription and [Ca2+]i.
Materials and Methods
Animals
Twelve-week-old female Sprague Dawley rats were obtained from Hilltop Animal Laboratories (Scottdale, PA) and housed under local vivarium conditions: 12-h light, 12-h dark cycle (lights on at 0600 h; lights off at 1800 h), 23 C, and 20% humidity. Animals were allowed to acclimate for at least 2 d before being used in the following experiments. Animals were killed under CO2, and their rib cages were removed for subsequent RC isolation. All procedures using animals fall under the Guidelines for the Care and Use of Laboratory Animals, and the institution’s animal care and use committee approved all animal protocols.
RC culture
RC of rat rib were isolated as previously described (5). Briefly, rib cages were removed by sharp dissection, and individual ribs were separated and cleaned of soft tissue. Slices from a 2-mm region just adjacent to the trabecular region were minced, washed in Hanks’ Balanced Salt Solution (Invitrogen Life Technologies, Inc., Gaithersburg, MD), followed by sequential incubations in trypsin and collagenase type II (Invitrogen Life Technologies, Inc.). Filtered cells were centrifuged at 1500 rpm for 5 min and resuspended in low glucose DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin sodium, 100 U/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B. Cells were maintained and passed in this medium to provide adequate cell numbers for experimentation. Cells at no more than passage 4 were used for experiments and were redifferentiated in chondrocyte differentiation medium [high glucose DMEM supplemented with ITS premix (5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenious acid), 40 μg/ml L-proline, 50 μg/ml ascorbic acid 2-phosphate, 100 U/ml penicillin sodium, 100 U/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B].
Northern analysis
Cells were initiated in differentiation medium at 1.4 x 104 cells/cm2 for 24 h before addition of the test agents. Untreated cultures were maintained in this medium throughout the experimental period. Porcine pituitary ACTH-(1–39) (Sigma-Aldrich Corp., St. Louis, MO; 10–7 M) was added to the ACTH cultures, 10–8 M DEXA was added to the DEXA cultures, and both 10–8 M DEXA and 10–7 M ACTH were added to the DEXA plus ACTH cultures after 24 h and at every medium change (twice weekly). Total RNA was extracted from cultures at the times indicated using TRIzol reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions. Ten-microgram aliquots of total RNA were electrophoresed on agarose/formaldehyde gels and transferred to nylon membrane (Sigma-Aldrich Corp.). Membranes were prehybridized in Hybrisol I (Serologicals Corp., Norcross, GA) for 4 h, and hybridization was carried out in the same solution overnight at 45 C. The rat type I collagen (COLL I) and mouse osteopontin (OP) cDNA probes were gifts from Dr. David Rowe (University of Connecticut, Farmington, CT). The human 28S cDNA probe was purchased from American Type Culture Collection (no. 77235; Manassas, VA). The COLL II and AGR probes were created using RT-PCR with rat AGR- and rat COLL II-specific primers and rat growth plate total RNA as a template. The PCR products were cloned into the PCRTrap vector (GenHunter, Corp., Nashville, TN), and the sequence was verified. AGR, COLL I, COLL II, and OP probes were labeled using PCR with promoter-specific primers and [32P]deoxy-CTP. The 28S (American Type Culture Collection) probe was random prime labeled using the RadPrime kit (Roche, Indianapolis, IN) and [32P]dCTP. Probe (5 x 106 cpm) was used in the hybridization. Membranes were washed twice at room temperature in 1x standard saline citrate/0.1% sodium dodecyl sulfate for 15 min each followed by two washes in 0.2x standard saline citrate/0.1% sodium dodecyl sulfate for 30 min each. Membranes were exposed to Kodak X-OMAT LS film (Eastman Kodak Co., Rochester, NY) for 1 h to overnight. Densitometry was performed using SigmaGel 1.0 (Jandel Scientific, San Ramon, CA). Bands were normalized to 28S RNA expression.
[Ca2+]i measurements
The fluorescent calcium indicator, fura-2-acetoxymethyl ester (Molecular Probes, Eugene, OR), was used to measure changes in intracellular calcium. Differentiated cells treated or untreated with DEXA for 16–24 h were trypsinized (0.5% trypsin/EDTA) for 2–3 min, followed by treatment with collagenase (0.02%) for 1–2 min to create a single-cell suspension. Cells were resuspended at a density of 106/ml in Hanks’ Balanced Salt Solution or -MEM containing Ca2+ and supplemented with 25 mM HEPES, 0.1% BSA, and 1.5 μM fura-2-acetoxymethyl ester (Molecular Probes). Cells were incubated at 25 C with gentle stirring and were protected from light for 30 min. This temperature was used to minimize compartmentalization of the fura-2. After loading, cells were washed three times in isotonic buffer with or without 1 mM Ca2+ as described in the text (132 mM NaCl, 5 mM KCl, 5 mM Na2HPO4, 1.2 mM NaH2PO4, and 0.8 mM MgCl2) and resuspended in this buffer at a density of 5 x 105/ml. Changes in [Ca2+]i were determined ratiometrically (340 nm/380 nm excitation, 512 nm emission) in 2-ml aliquots using a spectrofluorometer (model 610 photomultiplier detection system, Photon Technology International, Lawrenceville, NJ). Calcium concentrations were calculated using the equation (18): [Ca2+]i = Kd(F380max/F380min) (R – Rmin)/(Rmax – R). A dissociation constant (Kd) value of 224 nM was assumed for the binding of calcium to fura-2. Rmax and Rmin were determined in each experimental group by the consecutive addition of 30 μM digitonin (Rmax) and 50 mM EGTA (Rmin). Rmax and Rmin are the maximum and minimum F340/F380 ratios, respectively. F380max/F380min = the ratio of fluorescence emission intensity at 380-nm excitation in Ca2+-depleting (F380max) and Ca2+-saturating (F380min) conditions.
All chemicals were obtained from Sigma-Aldrich Corp. unless indicated otherwise. Nifedipine, an antagonist of L-type voltage-sensitive Ca2+ channels, was dissolved in dimethylsulfoxide (DMSO) at 2 mM and used at a final concentration of 10 μM. CdCl2, an inorganic blocker of all high-threshold, voltage-dependent Ca2+ channels, was dissolved in PBS at 0.5 M and used at a final concentration of 1 mM. Calcium channel blockers were added to the cuvette 80 sec before the addition of ACTH. SHU9119 (Bachem, Torrance, CA), the MC3-R/MC4-R antagonist, was dissolved in PBS at 10–3 M and used at the range of concentrations indicated in the text. In experiments involving SHU9119, cells treated with vehicle were incubated at room temperature for the same time period (1 h) as samples containing antagonist. U73122 and U73433 were dissolved in DMSO at 1 mM and used at a final concentration of 10 μM. U73122 is an aminosteroid that inhibits the coupling of G protein-phospholipase C (PLC) activation. U73433, the inactive analog of U73122, was used as a negative control. Preloaded cells were treated for 5 min or 1 h before calcium measurements. Thapsigargin (TG), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-adenosine triphosphatase (Ca2+-ATPase) was dissolved in DMSO at 100 μM and was used at a final concentration of 1–5 μM. Caffeine was dissolved in PBS at a stock concentration of 75 mM and was used at a final concentration of 10 mM. A high volume of caffeine solution was added to the cuvette to achieve the concentration necessary for myo-inositol 1,4,5-triphosphate (IP3) receptor inhibition. To control for volume-induced changes in ACTH response, the same volume of vehicle was used in all experiments involving caffeine.
Statistical analysis
Northern blots were analyzed by three-way ANOVA (ACTH x DEXA x day as the main factors). In the subanalyses, two-way ANOVA was used where appropriate. All other data were analyzed by one-way ANOVA. To control for an inflated type I error, post hoc test P values were adjusted using the Bonferroni correction. All tests were two-tailed, and a nominal significance level of 0.05 was used.
Results
DEXA and ACTH regulate transcription in RC
We have previously reported that ACTH affects transcription patterns in differentiated RC in a manner consistent with increased differentiation along the endochondral pathway (5). Here we show the effect of ACTH (10–7 M) alone, DEXA (10–8 M) alone, and DEXA in addition to ACTH on COLL II, AGR, OP, and COLL I transcription in RC (Fig. 1). A concentration of 10–7 M ACTH was used because in previous experiments it induced a significant transcriptional response in these cells (5). Control cultures highly expressed COLL II and AGR, indicating the differentiated chondrocyte phenotype.
FIG. 1. DEXA and ACTH regulate transcription of matrix genes in RC. Representative Northern blots (A) and corresponding densitometric analysis (B) of COLL II, AGR, OP, and COLL I transcripts in untreated (CON), 10–8 M DEXA-treated (DEXA), and 10–8 M DEXA- plus 10–7 M ACTH-treated (D+A) RC cultures. Total RNA was extracted from the cultures on the days indicated, and 10 μg were used per lane in the Northern blot (see Materials and Methods). In the bar graphs, values are presented as the mean ± SD (n = 3–4 cultures). Data were analyzed by three-way ANOVA and two-way ANOVA where appropriate. Significant main effects and interaction effects are presented in Results.
Three-way ANOVA (ACTH x DEXA x day) of COLL I expression revealed significant effects of ACTH [F(1,30) = 12.26; P = 0.0015] and DEXA [F(1,30) = 245.85; P < 0.0001], but not day [F(2,30) = 2.19; P = 0.129]. No significant interactions were present with COLL I (ACTH x DEXA, ACTH x day, DEXA x day, ACTH x DEXA x day). Analysis of COLL II expression revealed only a significant effect of DEXA [F(1,34) = 256.88; P < 0.0001] and a significant interaction effect of DEXA x day on COLL II expression, i.e. DEXA significantly reduced COLL II expression over time [F(2,34) = 9.67; P = 0.0005]; however, no other significant interaction effects were found.
DEXA alone significantly reduced AGR transcription [F(1,24) = 56.98; P < 0.001], whereas ACTH had a significant opposite effect [F(1, 24) = 23.16; P < 0.0001]. Of particular note is the very significant early increase in AGR transcripts with concurrent treatment. Statistical analysis revealed a significant three-way interaction effect of ACTH x DEXA x day on AGR expression [F(2,24) = 9.92; P = 0.0007]. The data suggest that the source of the interaction was a significant increase in AGR expression with dual treatment on d 3. These data were therefore analyzed at each time point by two-way ANOVA, which showed that the extreme elevation in AGR on d 3 in the dual treatment group was the result of an interaction effect of DEXA x ACTH [F(1,8) = 19.05; P = 0.0024]. No significant two-way interaction was present at the later time points, and DEXA was responsible for the main significant effect [d 7: F(1,8) = 85.33; P < 0.0001; d 10: F(1,8) = 31.87; P = 0.0005]. Analysis of OP expression revealed a significant effect of DEXA alone [F(1,30) = 133.53; P < 0.0001] and ACTH alone [F(1,30) = 33.06; P < 0.0001] and a significant interaction or synergistic effect of DEXA x ACTH [F(1,30) = 24.95; P < 0.0001]. No other significant interaction effects were found with OP expression.
ACTH induces transient elevations in [Ca2+]i and elevates basal [Ca2+]i in resting chondrocytes
DEXA priming of RC increases their sensitivity to ACTH. ACTH affects transcription patterns in differentiated RC in a manner consistent with increased differentiation along the endochondral pathway (5). As discussed, differentiation along this pathway is associated with an incremental increase in basal cytosolic calcium, and ACTH signaling through the melanocortin receptors is known to mobilize [Ca2+]i and increase basal [Ca2+]i (17). Therefore, changes in [Ca2+]i after stimulation with ACTH at a range of 10–8–10–6 M were measured in differentiated RC left untreated or treated with 10–8 M DEXA for 16–24 h. These measurements were made in the absence and presence of 1 mM extracellular Ca2+. Figure 2 shows that ACTH transiently increases [Ca2+]i in RC, which results in an elevation in basal [Ca2+]i. These changes were observed when RC cultures were stimulated in the presence and absence of extracellular calcium; however, the transients were greater in the presence of extracellular calcium (Fig 2, A and B). Additionally, the priming of differentiated RC with DEXA increased ACTH-induced [Ca2+]i elevations in both the presence and absence of extracellular calcium (Fig. 2, B and D). DEXA priming also increased the sensitivity of RC to ACTH, because transients were measurable at lower doses (10–9 M) compared with controls (not shown).
FIG. 2. ACTH induces transient elevations in [Ca2+]i and increases basal [Ca2+]i in RC. DEXA priming increases these elevations. ACTH dose-dependently induced [Ca2+]i transients in the presence (A and B) and absence (C and D) of 1 mM extracellular Ca2+. Pretreatment of the cells with 10–8 M DEXA for 16 h or longer greatly increased these transients (B and D). The time of ACTH addition is shown. The dose of ACTH is indicated either under the corresponding trace or with an arrow. Note the difference in scale for DEXA-primed cells stimulated in the presence of 1 mM extracellular Ca2+ (B). Traces represent the results of three separate experiments.
ACTH-induced transient increases in [Ca2+]i are intracellular in origin and induce store-operated calcium entry (SOCE) in RC
The fact that ACTH induces transient elevations in [Ca2+]i in the absence of extracellular Ca2+ suggests agonist-induced release from an intracellular store(s). However, the transient elevations are greater in the presence of extracellular Ca2+, suggesting a contribution from an extracellular source. To investigate the possible contribution of Ca2+ influx through voltage-dependent calcium channels (VDCC), we used nifedipine, an L-type VDCC-specific antagonist, and CdCl2, an inorganic blocker of all high-threshold VDCCs. Nifedipine or CdCl2 was added to the cuvette of fura-2-loaded RC, either treated with DEXA or left untreated, in the presence of 1 mM Ca2+ 80 sec before the addition of ACTH (Fig. 3). Neither nifedipine nor CdCl2 treatment decreased the ACTH-induced transient elevation in [Ca2+]i compared with the untreated control.
FIG. 3. Blocking of VDCC does not inhibit ACTH-induced increases in [Ca2+]i. Measurements were carried out on RC that had been pretreated with 10–8 M DEXA for 20 h (A) or left untreated (B). Fura-2-loaded RC cells were treated with either 10 μM nifedipine or 1 mM CdCl2 for 80 sec before being stimulated with ACTH in the presence of 1 mM Ca2+. The unlabeled arrow indicates the time of VDCC inhibitor addition. The specific inhibitor is noted to the right of the corresponding trace. The time of ACTH addition (10–8 M for DEXA-treated and 10–7 M for untreated RC) is noted. Traces represent the results of four separate experiments.
Next we investigated the contribution of calcium influx through store-operated channels (SOC). The depletion of intracellular Ca2+ stores, particularly those of the endoplasmic reticulum (ER), triggers a refilling through the opening of SOC at the plasma membrane. This refilling is known as capacitative calcium entry or, more recently, as SOCE (19). Untreated and DEXA-treated RC were stimulated with a range of ACTH concentrations in the absence of extracellular calcium. Ten minutes after stimulation, 2 mM CaCl2 was added to the cuvette, and the subsequent increase in [Ca2+]i was measured (Fig. 4). ACTH dose-dependently increased SOCE, and this was proportionately greater in RC pretreated with DEXA (Fig. 4B).
FIG. 4. The amount of SOCE after ACTH stimulation of RC is dose dependent, and priming with DEXA increases ACTH induced SOCE. RC either untreated (A) or treated with 10–8 M DEXA (B) for 20 h were stimulated in the absence of extracellular Ca2+ with a range of ACTH concentrations. Traces begin 10 min after stimulation. The times of 2 mM CaCl2 addition and the subsequent increase in [Ca2+]i are shown. The dose of ACTH used in the initial stimulation is shown to the right of the corresponding trace, and the time of CaCl2 is indicated with an arrow. Traces represent the results of three separate experiments.
To confirm that ACTH induces SOCE in RC, MnCl2 was used. The Mn2+ ion quenches the fluorescence of the fura-2 calcium indicator. Measurements were made using excitation wavelengths of F340/F360, where F340 shows the change in cytosolic calcium levels, and F360 is the isobestic wavelength for fura-2. It is at the isobestic wavelength that the quenching of the fura-2 fluorescence by Mn2+ can be measured. Mn2+ ions entering through the opened SOCs will cause an increase in fluorescence quenching at the isobestic wavelength. As shown in Fig. 5, ACTH stimulation of RC caused an increase in the intracellular quenching of fura-2 fluorescence by Mn2+, and this increase was dose dependent (not shown). The increase in Mn2+ quenching was also proportionate to the level of the ACTH-induced elevation in [Ca2+]i. Mn2+ quenching in response to ACTH was increased in DEXA-treated cells compared with untreated cells, mirroring the increased ACTH-induced elevation in [Ca2+]i (Fig. 5).
FIG. 5. Increased Mn2+ quenching of fura-2 confirms the opening of SOC after ACTH stimulation. Mn2+ quenching of fura-2 fluorescence at its isobestic wavelength of 360 nm is increased upon stimulation with 10–7 M ACTH in both untreated (A) and 10–8 M DEXA-treated (B) RC. Cell suspensions were prepared in the absence of extracellular Ca2+. Approximately 100 sec after the addition of 100 μM of MnCl2, ACTH (10–7 M) was added to the cuvette. The increase in cytosolic Ca2+ induced by ACTH was recorded at the fura-2 excitatory wavelength of 340 nm. The quenching of the intracellular dye by Mn2+ entry through SOCs was recorded at the fura-2 isobestic wavelength of 360 nm. Traces represent the results of three separate experiments.
SHU9119 dose-dependently attenuates ACTH-induced transient increases in [Ca2+]i
MC3-R is highly expressed by RC (5) and is therefore the suspected mediator of the ACTH signal. To demonstrate the involvement of this receptor, SHU9119, an MC3-R/MC4-R-specific antagonist, was used to block the ACTH-induced transient increases in [Ca2+]i. After fura-2 loading, DEXA-treated RC were incubated for 1 h in the presence of SHU9119 at a range of concentrations. SHU9119 dose-dependently attenuated the ACTH-induced transient increase in [Ca2+]i in both the presence (Fig. 6A) and absence of 1 mM extracellular Ca2+ (Fig. 6B). Consistent with its agouti-mimetic properties, SHU9119 caused a dose-dependent rise in basal [Ca2+]i (20) only in the presence of extracellular Ca2+. Agouti is known to induce a slow rise in basal [Ca2+]i in adipocytes and muscle cells (21), and this rise has been attributed to an influx of Ca2+ through VDCCs (22).
FIG. 6. SHU9119 attenuates ACTH-elicited transient elevations in [Ca2+]i. RC cultures were treated with 10–8 M DEXA for 20 h. Cell suspensions were prepared in the presence (A) or absence (B) of 1 mM extracellular Ca2+ after fura-2 loading and were incubated in the presence of SHU9119 for 1 h. The unlabeled arrow marks the time of ACTH addition (10–8 M in the presence and 10–7 M in the absence of Ca2+). The dose of SHU9119 is indicated under the corresponding trace. Traces represent the results of four separate experiments.
ACTH evokes [Ca2+]i release from TG-insensitive stores
The ER is the major intracellular calcium store in most cell types, and Ca2+ uptake into the ER is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA). Two types of ER Ca2+ stores have been functionally characterized and identified by immunocytochemical localization of receptors (23). One store is sensitive to IP3, and the other is sensitive to ryanodine/caffeine. These intracellular stores are released upon IP3 binding or ryanodine/caffeine binding to their respective receptors. Irreversible inhibition of SERCA with TG depletes the IP3-sensitive store; however, in some cells, TG does not deplete the caffeine- and ryanodine-sensitive store (23). In this study we sought to characterize the intracellular calcium store(s) released by ACTH in RC. RC cultures were treated with 1 μM TG for 5 min, followed by stimulation with ACTH in the absence of extracellular Ca2+. Interestingly, ACTH-induced transients were only partially TG-sensitive (Fig. 7). TG did not abrogate intracellular calcium release at 10–8 M ACTH (10–8 M ACTH, 139.35 ± 38.74 nM; 10–8 M ACTH after TG, 70.87 ± 26.55 nM; n = 4; P 0.01, by Student’s t test). However, ACTH-evoked calcium release after TG-induced Ca2+ leak from the ER does not increase at stimulatory concentrations above 10–7 M (Fig. 7B). Additionally, up to 5 μM TG did not increase the Ca2+ leak from the ER or prevent subsequent calcium release by 10–8 M ACTH (not shown). This suggests the existence of a TG-insensitive calcium store in RC whose release mechanism becomes saturated at 10–7 M ACTH.
FIG. 7. ACTH-evoked release of Ca2+ from TG-insensitive stores in DEXA-treated RC. A, RC cultures were treated with 10–8 M DEXA for 20 h. Five minutes after exposure to the irreversible inhibitor of SERCA, TG, cells were stimulated with ACTH in the range of 10–8–10–6 M. The time of ACTH addition is shown. The dose of ACTH is indicated with arrows pointing to the corresponding trace. The trace showing the response to ACTH after TG was magnified for clarity. The inset shows the full trace, with time of TG (1 μM) and ACTH addition indicated with arrows. Traces represent the results of three separate experiments. B, Bar graph showing peak ACTH-evoked [Ca2+]i release over the range of doses in A after SERCA inhibition with TG. Data are presented as the mean ± SD (n = 4 from two separate experiments). Significant pairwise differences were determined after a significant one-way ANOVA using a Bonferroni correction.
ACTH-evoked calcium release from intracellular stores is PLC dependent
Activation of the canonical signaling pathway of GPCR that elicit ER calcium release results in increased PLC activity and subsequent production of IP3. Activation of the IP3 receptor on the ER membrane by IP3, in turn, stimulates Ca2+ release (24). To test whether ACTH elicits Ca2+ release through this effector pathway, we first inhibited PLC using the specific inhibitor, U73122. When RC were treated with 10 μM U73122 for 3–5 min, ACTH-induced Ca2+ release was inhibited (Fig. 8A). U73122 inhibited Ca2+ release induced by ACTH concentrations as high as 10–6 M (not shown). In this study ATP (10 μM) was used as a positive control. ATP induced transient elevations in [Ca2+]i in a PLC-dependent manner (25, 26). U73122 blocked these transients (Fig. 8C). U73433 is the inactive analog of U73122 and had no effect on either ACTH-induced or ATP-induced calcium release (Fig. 8, B and D). U73122 also did not interfere with TG-mediated intracellular calcium release (not shown). Both U73122 and U73433 caused an early increase in basal cytosolic calcium (Fig. 7), as previously described for these compounds (25). After 1 h of treatment with U73122, however, basal calcium levels were lowered (not shown), consistent with its PLC-inhibitory properties.
FIG. 8. ACTH-evoked [Ca2+]i release in RC is PLC dependent. RC cultures were pretreated with DEXA for 20 h or longer. After loading with fura-2, RC were treated with either U73122 or U73433 for 5 min in the absence of extracellular Ca2+. Traces begin after U73122 or U73433 treatment, and stimulation with 10–8 M ACTH (A) or 10 μM ATP (C) is shown. U73433, the inactive analog of U73122, had no effect on either ACTH- or ATP-stimulated calcium release (B and D). Traces represent the results of three separate experiments.
ACTH-evoked calcium release and subsequent SOCE are partly IP3 receptor dependent
Next, to determine whether the activation of IP3 receptors is responsible for mediating ACTH-induced calcium release, we used caffeine at an inhibitory concentration. Caffeine at high concentrations (10–20 mM) will inhibit IP3 receptor activation (27). RC cultures were treated with 10 mM caffeine or vehicle for 100 sec before the addition of 10–7 M ACTH (Fig. 9A). Caffeine greatly reduced ACTH-induced Ca2+ release, suggesting that at least a portion of the Ca2+ released is via IP3 binding to its receptor. Additionally, caffeine is a known agonist of the ryanodine receptor. Stimulation of this receptor by caffeine and/or ryanodine will also elicit Ca2+ release. Because caffeine itself does not elicit transient elevations in [Ca2+]i, it is unlikely that RC in this differentiation state are expressing ryanodine receptors, and therefore it is unlikely that ACTH evokes Ca2+ release through this mechanism.
FIG. 9. ACTH-evoked [Ca2+]i release and SOCE in RC is at least partly dependent on the IP3 receptor. RC cultures were pretreated with DEXA for 20 h or longer. A, Fura-2-loaded cells were treated with either caffeine (10 mM) or vehicle (Ca2+- and Mg2+-free PBS) at the time indicated by the unlabeled arrow. The specific treatment is indicated to the right of the corresponding trace. After 100 sec, the cells were stimulated with 10–7 M ACTH as indicated. B, Traces begin about 250 sec after fura-2-loaded cells were stimulated with 10–7 M ACTH. Caffeine (10 mM) or vehicle was added 150 sec after ACTH stimulation and 50 sec before traces begin. Specific treatments are indicated to the right of the corresponding trace. CaCl2 (2 mM) was added about 3 min after caffeine or vehicle, as shown. Traces are representative of three separate experiments.
There is substantial evidence that IP3 receptor activation is required for SOCE (19, 27). In RC, the initial intracellular Ca2+ release is at least partly dependent on IP3 activation of its receptor. Therefore, we next determined whether SOCE after ACTH stimulation is IP3 receptor mediated. RC cultures were stimulated with 10–7 M ACTH. After 150 sec, 10 mM caffeine or vehicle was added for 3 min, followed by 2 mM CaCl2. SOCE after ACTH treatment was greatly reduced by the inhibition of IP3 receptor activity (Fig. 9B).
Discussion
In this study we demonstrate that transcriptional patterns in RC are regulated by the pituitary-derived melanocortin, ACTH, and by glucocorticoid. Consistent with the literature, DEXA significantly reduced COLL I and COLL II transcription (28). Although ACTH also significantly reduced COLL I transcription, statistical analysis showed that there was no synergistic effect between DEXA and ACTH on the expression of this gene. Previous data show that ACTH increases the differentiation of RC along the endochondral pathway (5). Thus, the decrease in COLL I transcription by ACTH may be a result of this differentiation effect. Interestingly, when RC cultures are treated with both glucocorticoid and ACTH, the transcriptional pattern resembles that of rapid differentiation along the endochondral pathway. COLL II is rapidly down-regulated concomitant with a very significant increase in AGR transcription. These changes are followed by a significant increase in OP transcription, a marker of hypertrophic terminal differentiation (29).
Increased [Ca2+]i release via oscillatory fluid flow is linked to increases in AGR synthesis in chondrocytes (15, 30) and increased OP transcription in osteoblasts (31). In line with these data, ACTH increases [Ca2+]i release in RC and increases AGR and OP transcription. ACTH and DEXA also interact to increase both AGR and OP transcription, which is consistent with our data showing that DEXA increases the sensitivity of RC to ACTH-induced changes in [Ca2+]i. ACTH-induced transient elevations in [Ca2+]i are greater in cells pretreated with DEXA and are observed at lower doses of ACTH. ACTH signals through the melanocortin receptors, which are a family of GPCR. The canonical GPCR effector pathway that results in intracellular calcium release is initiated by the activation of the G protein -subunit, Gq (Gq). Interestingly, DEXA has been shown to increase Gq expression and PTH-stimulated PLC activity in osteoblast-like cells (32, 33). These data suggest the possibility that DEXA, through increasing the expression of Gq, enhances the downstream effects of the ACTH stimulatory pathway in RC. More data are necessary to confirm this hypothesis.
We have shown for the first time that ACTH dose-dependently evokes transient elevations in [Ca2+]i and increases basal [Ca2+]i through an endogenously expressed melanocortin receptor in RC. Although the MC2-R (or ACTH receptor) is the endogenous receptor for ACTH, it is not likely that this receptor is responsible for mediation of the melanocortin signal in the growth plate or in RC. FGD type I, linked with both elevated ACTH and enhanced growth velocity, is attributed to a mutation in the MC2-R (3, 34), making it unlikely that stimulation of this receptor is responsible for the growth-related changes. It is the MC3-R that is the likely receptor through which the ACTH signal is mediated in these cells. This receptor has been previously detected by immunoblot at significant levels in RC (5). Additionally, the MC3-R knockout mouse model exhibits reduced linear growth (35), suggesting its involvement in endochondral differentiation. Support for this hypothesis comes from the fact that SHU9119, a specific antagonist of MC3-R and MC4-R, dose-dependently attenuates ACTH-evoked transient increases in [Ca2+]i. Although SHU9119 is also an antagonist of the MC4-R, this receptor is not likely responsible for mediation of the ACTH signal. Using immunochemistry, we did not detect MC4-R in growth plate chondrocytes, nor was expression detected in RC protein lysates by immunoblot (not shown). Moreover, in the obese yellow and MC4-R knockout mouse models, which also demonstrate enhanced somatic growth, the MC4-R is compromised (36, 37).
ACTH-evoked transient elevations in [Ca2+]i that ultimately result in elevated basal [Ca2+]i consist of both an intracellular and an extracellular component. Ca2+ is initially released from an intracellular store, followed by SOCE through SOC. Of note is the fact that the initial intracellular release originates from both TG-sensitive and -insensitive stores and is dependent upon PLC activity and IP3 activation of its receptor. IP3-dependent intracellular calcium stores are thought to overlap with that sensitive to TG (23). TG is an irreversible inhibitor of the SERCA family of Ca2+-transporting ATPases (23, 38). SERCAs are responsible for calcium uptake into the ER, which represents the major intracellular Ca2+ store in most cell types. Recently, however, it has become evident that the Golgi apparatus is also an important Ca2+ store (39, 40). Ca2+ uptake in the Golgi is dependent upon both the SERCA type of Ca2+ transport ATPases and the secretory pathway Ca2+-ATPase (SPCA) family of Ca2+ transport ATPases (39, 40, 41, 42). The SPCA are insensitive to TG, and this large Ca2+ store has been described in A7r5 smooth muscle cells and is partially responsive to IP3 (41). These data point to the possibility that the SPCA-type Ca2+-ATPases are expressed in RC, and they are responding to IP3 generated by ACTH antagonism of the endogenous MC3-R. Although a previous report indicated that the SPCA-expressing portion of the Golgi is not sensitive to agonist stimulation (42), the data were not extended to include cells of chondrogenic origin or agonists of the melanocortin family of receptors. Additional support for calcium release via TG-insensitive stores comes from the finding that this response becomes saturated at higher doses of ACTH. In A7r5 smooth muscle cells, IP3 concentrations above 30 μM failed to further increase the percentage of calcium released from the TG-insensitive store (41).
Interestingly, inhibition of the IP3 receptor by caffeine reduces SOCE after ACTH stimulation, which suggests that some SOC opening after ACTH stimulation is dependent upon IP3 receptor activation. Currently, there are three proposed models of SOC opening. The first is the conformational coupling model, which implies direct protein-protein interaction between the ER membrane proteins responsible for Ca2+ release, such as IP3 and ryanodine receptors and plasma membrane SOCs, which results in SOC activation (19, 43, 44). The second model suggests the existence of diffusible messenger, which is formed and released upon ER depletion. This messenger, termed calcium influx factor, would thereby transmit the signal to plasma membrane SOCs (19, 44, 45). The last model is a secretion-like coupling model, which suggests that fusion of secretory vesicles with the plasma membrane may activate SOCE (19, 46). Our data support a role for the IP3 receptor in ACTH-induced SOC activation and point toward the conformational coupling of ER IP3 receptors to plasma membrane SOCs. Whether this coupling requires the IP3 ligand could not be determined using the U73122 inhibitor in a DMSO vehicle. U73122; its inactive analog, U73433; and their vehicle, DMSO, all inhibited SOCE after ACTH stimulation (not shown). This is probably due to DMSO’s effects on membrane fluidity; DMSO is known to decrease the fluidity of phosphatidylethanolamine membranes (47). Phosphatidylethanolamine makes up about 80% of the inner leaflet of the plasma membrane (48), and DMSO is known to stabilize it in the hexagonal II formation (48). This conceivably prevents interactions at the inner surface of the membrane and prevents SOC activation. The three current models of SOC activation all involve inner membrane protein interactions, thereby making DMSO inhibition nonspecific. Thus, more data are necessary to determine the precise mechanism(s) involved in ACTH-induced SOC activation.
In this study we have begun to outline the signaling pathway of the endogenous MC3-R of resting chondrocytes. ACTH agonism of this receptor appears to stimulate the canonical Gq effector pathway, leading to IP3-induced release of Ca2+ from intracellular stores and ultimately SOCE. Although the data presented demonstrate that ACTH-evoked calcium release is dependent upon PLC activity, confirmation of the precise G-subunit involved is still necessary. Interestingly, the priming of RC with DEXA leads to increased activation of this pathway, suggesting glucocorticoid’s involvement in the regulation of proteins along this pathway, i.e. G-subunits. GPCR pathways have recently been identified as potential therapeutic targets (49, 50); thus, we have identified a possible new therapeutic target in the treatment of growth deficiencies and cartilage degeneration.
References
Solomon S 1999 POMC-derived peptides and their biological action. Ann NY Acad Sci 885:22–40
Wikberg JE, Muceniece R, Mandrika I, Prusis P, Lindblom J, Post C, Skottner A 2000 New aspects on the melanocortins and their receptors. Pharmacol Res 42:393–420
Elias LLK, Huebner A, Metherell LA, Canas A, Warnes GL, Bittis MLM, Cianfaranis S, Clayton PE, Savage MO, Clark AJL 2000 Tall stature in familial glucocorticoid deficiency. Clin Endocrinol (Oxf) 53:423–430
Stevens DA, Williams GR 1999 Hormone regulation of chondrocyte differentiation and endochondral bone formation. Mol Cell Endocrinol 151:195–204
Evans JF, Niu Q-T, Canas JA, Shen C-L, Aloia JF, Yeh JK 2004 ACTH enhances chondrogenesis in multipotential progenitor cells and matrix production in chondrocytes. Bone 35:96–107
Squires GR, Okouneff S, Ionescu M, Poole RA 2003 The pathobiology of focal lesion development in aging human articular cartilage and molecular matrix changes characteristic of osteoarthritis. Arthritis Rheum 48:1261–1270
Buckwalter JA, Mankin HJ 1997 Articular cartilage. Part II: Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Joint Surg Am 79:612–632[Free Full Text]
Johnson KA, van Etten D, Nanda N, Graham RM, Terkeltaub RA 2003 Distinct transglutaminase 2-independent and transglutaminase 2-dependent pathways mediate articular chondrocyte hypertrophy. J Biol Chem 272:18824–18832
Jessop DS, Dallman MF, Fleming D, Lightman SL 2001 Resistance to glucocorticoid feedback in obesity. J Clin Endocrinol Metab 86:4109–4114
Veldhuis JD, Iranmanesh A, Naftolowitz D, Tatham N, Cassidy F, Carroll BJ 2001 Corticotropin secretory dynamic in humans under low glucocorticoid feedback. J Clin Endocrinol Metab 86:5554–5563
Boscara M, Paoletta A, Scarpa E, Barzon L, Fusaro P, Fallo F, Sonino N 1998 Age-related changes in glucocorticoid fast-feedback inhibition of adrenocorticotropin in man. J Clin Endocrinol Metab 83:1380–1383
Wilkinson CW, Petrie EC, Murray SR, Colasurdo EA, Raskind MA, Peskind ER 2001 Human glucocorticoid feedback inhibition is reduced in older individuals: evening study. J Clin Endocrinol Metab 86:545–550
Purnell JQ, Brandon DD, Isabelle LM, Loriaux DL, Samuels MH 2004 Association of 24-hour cortisol production rates, cortisol-binding, globulin, and plasma-free cortisol levels with body composition, leptin levels, and aging in adult men and women. J Clin Endocrinol Metab 89:281–287
Zuscik MJ, D’Souza M, Ionescu AM, Gunter KK, Gunter TE, O’Keefe RJ, Schwarz EM, Puzas JE, Rosier RN 2002 Growth plate chondrocyte maturation is regulated by basal intracellular calcium. Exp Cell Res 276:310–319
Parvizi J, Parpura V, Greenleaf JF, Bolander ME 2002 Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J Orthop Res 21:51–57
Gallo-Payet N, Payet MD 2003 Mechanism of action of ACTH: beyond cAMP. Microsc Res Tech 61:275–287
Mountjoy KG, Kong PL, Taylor JA, Willard DH, Wilkison WO 2001 Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells. Physiol Genomics 5:11–19
Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Abeele VF, Lemonnier L, Thebault S, Lepage G, Parys JB, Shuba Y, Skryma R, Prevarskaya N 2004 Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells. J Biol Chem 279:30326–30337
Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168
Zemel, MB, Kim JH, Woychik RP, Michaud EJ, Kadwell SH, Patel IR, Wilkison WO 1995 Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice. Proc Natl Acad Sci USA 92:4733–4737
Kim JH, Mynatt RL, Moore JW, Woychik RP, Moustaid N, Zemel MB 1996 The effects of calcium channel blockade on agouti-induced obesity. FASEB J 10:1646–1652
Neves SR, Ram PT, Iyengar R 2002 G protein pathways. Science 296:1636–1639
Missiaen L, Van Acker K, Parys JB, De Smedt H, Van Baelen K, Weidema AF, Vanoevelen J, Raeymaekers L, Rneders J, Callewaert G, Rizzuto R, Wuytack F 2001 Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store. J Biol Chem 276:39161–39170
Jan C-R, Ho M-C, Wu S-N, Tseng C-J 1998 The phospho inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium. Life Sciences 63:895–908
Missiaen L, Parys JB, De Smedt H, Himpens B, Casteels R 1994 Inhibition of inositol triphosphate-induced calcium release by caffeine is prevented by ATP. Biochem J 15:81–84
Ma H-T, Venkatachalam K, Li H-S, Montell C, Kurosaki T, Patterson RL, Gill DL 2001 Assessment of the role of the inositol-1,4,5-triphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels. J Biol Chem 276:18888–18896
Richardson DW, Dodge GR 2003 Dose-dependent effects of corticosteroids on the expression of matrix related genes in normal and cytokine-treated articular chondrocytes. Inflamm Res 52:39–49
Ferrari D, Kosher RA 2002 Dlz5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol 252:257–270
Alford AI, Yellowley CE, Jacobs CR, Donahue HJ 2003 Increases in cytosolic calcium, but not fluid flow, affect aggrecan mRNA levels in articular chondrocytes. J Cell Biochem 90:938–944
You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR 2001 Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3–E1 osteoblasts. J Biol Chem 276:13365–13371
Mitchell J, Bansal A 1997 Dexamethasone increases Gq-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells. Am J Physiol 273:E528–E535
Cheung R, Mitchell J 2002 Mechanisms of regulation of G11 protein by dexamethasone in osteoblastic UMR 106–01 cells. Am J Physiol 282:E24–E30
Clark AJ, Weber A 1998 Adrenocorticotropin insensitivity syndromes. Endocr Rev 19:828–843
Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan X-M, Yu H, Rosenblum CI, Vongs A, Feng Y, Cao L, Metzger JM, Strack AM, Camacho RE, Mellin TN, Nunes CN, Min W, Fisher J, Gopal-Truter S, MacIntyre DE, Chen HY, Van der Ploeg LHT 2000 Inactivation of the mouse melanocortin-3 recpeotr results in increased fat mass and reduced lean body mass. Nat Genet 26:97–102
Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141
Wolff GL, Roberts DW, Mountjoy KG 1999 Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome. Physiol Genomics 1:151–163
Strehler EE, Treiman M 2004 Calcium pumps of plasma membrane and cell interior. Curr Mol Med 4:323–335
Pinton P, Pozzan T, Rizzuto R 1998 The Golgi apparatus is an inositol 1,4,5-triphosphate-sensitive Ca2+ store with functional properties distinct from those of the endoplasmic reticulum. EMBO J 17:5298–5308
Missiaen L, Van Acker K, Van Baelen K, Raeymaekers L, Wuytack F, Parys JB, De Smedt H, Vanoevelen J, Dode L, Rizzuto R, Callewaert G 2004 Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments. Cell Calcium 36:479–487
Missiaen L, Vanoevelen J, Parys JB, Raeymaekers L, De Smedt H, Callewaert G, Erneux C, Wuytack F 2002 Ca2+ uptake and release properties of a thapsigargin-insensitive nonmitochondrial Ca2+ store in A7r5 and 16HBE140-cells. J Biol Chem 277:6898–6902
Vanoevelen J, Raeymaekers L, Parys JB, De Smedt H, Van Baelen K, Callewaert G, Wuytack F, Missiaen L 2004 Inositol triphosphate producing agonists do not mobilize the thapsigargin insensitive part of the endoplasmic reticulum and Golgi Ca2+ store. Cell Calcium 35:115–121
Vasquez G, Lievremont J-P, Bird GSJ, Putney JW 2001 Human Trp3 forms both inositol triphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci USA 98:11777–11782
Bolotina VM 2004 Store-operated channels: diversity and activation mechanisms (web version of Science STKE). www.stke.org/cgi/content/full/sigtrans;2004/243/pe34
Su Z, Barker DS, Csutora P, Chang T, Shoemaker RL, Marchase RB, Blalock JE 2003 Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor. Am J Cell Physiol 284:C497–C505
Scott CC, Furuya W, Trimble WS, Grinstein S 2003 Activation of store-operated calcium channels. J Biol Chem 278:30534–30539
Kinoshita K, Li SJ, Yamazaki M 2001 The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide. Eur Biophys J 30:207–220
Daleke DL 2003 Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 44:233–242
Kimple RJ, Jones MB, Shutes A, Yerxa BR, Siderovski DP, Willard FS 2003 Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein subunits and regulator of G-protein signaling (RGS) proteins. Combinatorial Chem High Throughput Screening 6:1–9
Liebman C 2004 G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts. Curr Pharmaceut Design 10:1937–1958(Jodi F. Evans, Chwan-L Sh)
Address all correspondence and requests for reprints to: Dr. Jodi F. Evans, Department of Medicine, Winthrop University Hospital, 222 Station Plaza North, Suite 501, Mineola, New York 11501. E-mail: jevans@winthrop.org.
Abstract
Both clinical and in vitro evidence points to the involvement of the melanocortin peptide, ACTH, in the terminal differentiation of chondrocytes. Terminal differentiation along the endochondral pathway is responsible for linear growth, but also plays a role in osteoarthritic cartilage degeneration. Chondrocyte terminal differentiation is associated with an incremental increase in chondrocyte basal intracellular free calcium ([Ca2+]i), and ACTH agonism of melanocortin receptors is known to mobilize [Ca2+]i. Using differentiated resting chondrocytes highly expressing type II collagen and aggrecan, we examined the influence of both ACTH and dexamethasone treatment on matrix gene transcription and [Ca2+]i. Resting chondrocytes treated concurrently with dexamethasone and ACTH expressed matrix gene transcripts in a pattern consistent with that of rapid terminal differentiation. Using the fluorescent Ca2+ indicator, fura-2, we determined that ACTH evokes transient increases in [Ca2+]i and elevates basal Ca2+ levels in resting chondrocytes. The transient increases were initiated intracellularly, were abrogated by the phospholipase C-specific inhibitor, U73122, and were partly attenuated by myo-inositol 1,4,5-triphosphate receptor inhibition via 10 mM caffeine. The initial intracellular release also resulted in store-operated calcium entry, presumably through store-operated channels. Dexamethasone priming increased both the initial ACTH-evoked [Ca2+]i release and the subsequent store-operated calcium entry. These data demonstrate roles for ACTH and glucocorticoid in the regulation of chondrocyte terminal differentiation. Because the actions of ACTH are mediated through known G protein-coupled receptors, the melanocortin receptors, these data may provide a new therapeutic target in the treatment of growth deficiencies and cartilage degeneration.
Introduction
ACTH IS A member of the melanocortin family of peptides derived from proopiomelanocortin (POMC). POMC is posttranslationally processed in the anterior pituitary corticotropes to form ACTH, ?-lipotropic hormone, and pro--MSH. Additional processing into -MSH, CLIP, ?-MSH, and -MSH occurs in the melanotropes of the intermediate lobe of the pituitary (1). ACTH exerts its effects through agonism of the melanocortin receptors. The five melanocortin receptors (MC-R) identified to date are a family of G protein-coupled receptors (GPCR) that activate adenylyl cyclase and mobilize intracellular free calcium ([Ca2+]i) (2). These receptors are distinguishable through their agonist profiles, and the melanocortin-2 receptor (MC2-R) is the primary receptor for ACTH. However, ACTH is capable of activating all five MC-R with differing affinities. In fact, its potency at the MC3-R is nearly equivalent to that of the strongest agonist of this receptor, 2-MSH (2).
The human syndrome of familial glucocorticoid deficiency (FGD) provides clinical evidence of a role for ACTH in the regulation of chondrocyte terminal differentiation. FGD is characterized by an overproduction of ACTH, enhanced linear growth, and advanced bone age (3). Linear growth is accomplished through the process of endochondral ossification, which occurs in the cartilaginous growth plate located at both ends of the vertebrae and long bones (4). With the FGD syndrome, systemic levels of ACTH are extremely elevated due to a mutation in the MC2-R (ACTH) receptor. When FGD patients are treated with glucocorticoid, their ACTH levels are reduced, and their growth patterns normalize somewhat (3). These data suggest a role for ACTH in the regulation of endochondral ossification. Recent in vitro studies support this suggestion. Matrix genes, such as type II collagen (COLL II) and aggrecan (AGR), of chondrocytes and their precursors are directly affected by ACTH treatment. Steady-state mRNA levels of AGR are significantly up-regulated in both resting chondrocytes (RC) and the chondrogenic cell line RCJ3.1C5.18, when treated with ACTH. ACTH also increases the differentiation of these cells along the endochondral pathway (5).
Unlike the chondrocytes of the growth plate, articular chondrocytes in a healthy joint do not undergo terminal differentiation. Damage to the articular cartilage extracellular matrix, however, will induce increased transcription and secretion of type II collagen and AGR by articular chondrocytes (6). This response may lead to long-term changes in the phenotype of the cell (7). In fact, foci of articular chondrocytes in osteoarthritic cartilage, typically near sites of cartilage surface lesions, undergo terminal differentiation in a manner consistent with endochondral differentiation (8).
Interestingly, two major risk factors for osteoarthritic cartilage degeneration, age and obesity, are also associated with dysregulation of the hypothalamic-pituitary adrenal axis. Current data point to a relative adrenal insensitivity to ACTH in obesity, resulting in decreased cortisol secretion (9). Attenuated cortisol negative feedback will augment ACTH output during stress and increase the plasma ACTH concentration (10). Thus, elevated ACTH with a paradoxically low cortisol level is observed in obese subjects at baseline and in response to stress (9). Resistance to glucocorticoid feedback in aging, however, is associated with delayed inhibition of ACTH secretion (11, 12, 13), resulting in an increased exposure to both glucocorticoids and ACTH. These data along with the fact that age and obesity are strongly associated with cartilage degeneration suggest a link between dysregulation of hypothalamic-pituitary adrenal axis hormones and hypertrophic differentiation among articular chondrocytes.
Among the cellular events associated with chondrocyte terminal differentiation is an incremental increase in chondrocyte basal [Ca2+]i (14). Calcium signaling is also a required for stimulus-induced increases in AGR synthesis (15). Although the obligatory second messenger for ACTH is known to be cAMP, there is increasing evidence that ACTH agonism of melanocortin receptors also mobilizes [Ca2+]i (16, 17). Using differentiated RC highly expressing COLL II and AGR and therefore phenotypically similar to both chondrocytes of the growth plate and those in a damaged articular matrix, we examined the influence of ACTH and dexamethasone (DEXA) treatment on matrix gene transcription and [Ca2+]i.
Materials and Methods
Animals
Twelve-week-old female Sprague Dawley rats were obtained from Hilltop Animal Laboratories (Scottdale, PA) and housed under local vivarium conditions: 12-h light, 12-h dark cycle (lights on at 0600 h; lights off at 1800 h), 23 C, and 20% humidity. Animals were allowed to acclimate for at least 2 d before being used in the following experiments. Animals were killed under CO2, and their rib cages were removed for subsequent RC isolation. All procedures using animals fall under the Guidelines for the Care and Use of Laboratory Animals, and the institution’s animal care and use committee approved all animal protocols.
RC culture
RC of rat rib were isolated as previously described (5). Briefly, rib cages were removed by sharp dissection, and individual ribs were separated and cleaned of soft tissue. Slices from a 2-mm region just adjacent to the trabecular region were minced, washed in Hanks’ Balanced Salt Solution (Invitrogen Life Technologies, Inc., Gaithersburg, MD), followed by sequential incubations in trypsin and collagenase type II (Invitrogen Life Technologies, Inc.). Filtered cells were centrifuged at 1500 rpm for 5 min and resuspended in low glucose DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin sodium, 100 U/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B. Cells were maintained and passed in this medium to provide adequate cell numbers for experimentation. Cells at no more than passage 4 were used for experiments and were redifferentiated in chondrocyte differentiation medium [high glucose DMEM supplemented with ITS premix (5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenious acid), 40 μg/ml L-proline, 50 μg/ml ascorbic acid 2-phosphate, 100 U/ml penicillin sodium, 100 U/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B].
Northern analysis
Cells were initiated in differentiation medium at 1.4 x 104 cells/cm2 for 24 h before addition of the test agents. Untreated cultures were maintained in this medium throughout the experimental period. Porcine pituitary ACTH-(1–39) (Sigma-Aldrich Corp., St. Louis, MO; 10–7 M) was added to the ACTH cultures, 10–8 M DEXA was added to the DEXA cultures, and both 10–8 M DEXA and 10–7 M ACTH were added to the DEXA plus ACTH cultures after 24 h and at every medium change (twice weekly). Total RNA was extracted from cultures at the times indicated using TRIzol reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions. Ten-microgram aliquots of total RNA were electrophoresed on agarose/formaldehyde gels and transferred to nylon membrane (Sigma-Aldrich Corp.). Membranes were prehybridized in Hybrisol I (Serologicals Corp., Norcross, GA) for 4 h, and hybridization was carried out in the same solution overnight at 45 C. The rat type I collagen (COLL I) and mouse osteopontin (OP) cDNA probes were gifts from Dr. David Rowe (University of Connecticut, Farmington, CT). The human 28S cDNA probe was purchased from American Type Culture Collection (no. 77235; Manassas, VA). The COLL II and AGR probes were created using RT-PCR with rat AGR- and rat COLL II-specific primers and rat growth plate total RNA as a template. The PCR products were cloned into the PCRTrap vector (GenHunter, Corp., Nashville, TN), and the sequence was verified. AGR, COLL I, COLL II, and OP probes were labeled using PCR with promoter-specific primers and [32P]deoxy-CTP. The 28S (American Type Culture Collection) probe was random prime labeled using the RadPrime kit (Roche, Indianapolis, IN) and [32P]dCTP. Probe (5 x 106 cpm) was used in the hybridization. Membranes were washed twice at room temperature in 1x standard saline citrate/0.1% sodium dodecyl sulfate for 15 min each followed by two washes in 0.2x standard saline citrate/0.1% sodium dodecyl sulfate for 30 min each. Membranes were exposed to Kodak X-OMAT LS film (Eastman Kodak Co., Rochester, NY) for 1 h to overnight. Densitometry was performed using SigmaGel 1.0 (Jandel Scientific, San Ramon, CA). Bands were normalized to 28S RNA expression.
[Ca2+]i measurements
The fluorescent calcium indicator, fura-2-acetoxymethyl ester (Molecular Probes, Eugene, OR), was used to measure changes in intracellular calcium. Differentiated cells treated or untreated with DEXA for 16–24 h were trypsinized (0.5% trypsin/EDTA) for 2–3 min, followed by treatment with collagenase (0.02%) for 1–2 min to create a single-cell suspension. Cells were resuspended at a density of 106/ml in Hanks’ Balanced Salt Solution or -MEM containing Ca2+ and supplemented with 25 mM HEPES, 0.1% BSA, and 1.5 μM fura-2-acetoxymethyl ester (Molecular Probes). Cells were incubated at 25 C with gentle stirring and were protected from light for 30 min. This temperature was used to minimize compartmentalization of the fura-2. After loading, cells were washed three times in isotonic buffer with or without 1 mM Ca2+ as described in the text (132 mM NaCl, 5 mM KCl, 5 mM Na2HPO4, 1.2 mM NaH2PO4, and 0.8 mM MgCl2) and resuspended in this buffer at a density of 5 x 105/ml. Changes in [Ca2+]i were determined ratiometrically (340 nm/380 nm excitation, 512 nm emission) in 2-ml aliquots using a spectrofluorometer (model 610 photomultiplier detection system, Photon Technology International, Lawrenceville, NJ). Calcium concentrations were calculated using the equation (18): [Ca2+]i = Kd(F380max/F380min) (R – Rmin)/(Rmax – R). A dissociation constant (Kd) value of 224 nM was assumed for the binding of calcium to fura-2. Rmax and Rmin were determined in each experimental group by the consecutive addition of 30 μM digitonin (Rmax) and 50 mM EGTA (Rmin). Rmax and Rmin are the maximum and minimum F340/F380 ratios, respectively. F380max/F380min = the ratio of fluorescence emission intensity at 380-nm excitation in Ca2+-depleting (F380max) and Ca2+-saturating (F380min) conditions.
All chemicals were obtained from Sigma-Aldrich Corp. unless indicated otherwise. Nifedipine, an antagonist of L-type voltage-sensitive Ca2+ channels, was dissolved in dimethylsulfoxide (DMSO) at 2 mM and used at a final concentration of 10 μM. CdCl2, an inorganic blocker of all high-threshold, voltage-dependent Ca2+ channels, was dissolved in PBS at 0.5 M and used at a final concentration of 1 mM. Calcium channel blockers were added to the cuvette 80 sec before the addition of ACTH. SHU9119 (Bachem, Torrance, CA), the MC3-R/MC4-R antagonist, was dissolved in PBS at 10–3 M and used at the range of concentrations indicated in the text. In experiments involving SHU9119, cells treated with vehicle were incubated at room temperature for the same time period (1 h) as samples containing antagonist. U73122 and U73433 were dissolved in DMSO at 1 mM and used at a final concentration of 10 μM. U73122 is an aminosteroid that inhibits the coupling of G protein-phospholipase C (PLC) activation. U73433, the inactive analog of U73122, was used as a negative control. Preloaded cells were treated for 5 min or 1 h before calcium measurements. Thapsigargin (TG), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-adenosine triphosphatase (Ca2+-ATPase) was dissolved in DMSO at 100 μM and was used at a final concentration of 1–5 μM. Caffeine was dissolved in PBS at a stock concentration of 75 mM and was used at a final concentration of 10 mM. A high volume of caffeine solution was added to the cuvette to achieve the concentration necessary for myo-inositol 1,4,5-triphosphate (IP3) receptor inhibition. To control for volume-induced changes in ACTH response, the same volume of vehicle was used in all experiments involving caffeine.
Statistical analysis
Northern blots were analyzed by three-way ANOVA (ACTH x DEXA x day as the main factors). In the subanalyses, two-way ANOVA was used where appropriate. All other data were analyzed by one-way ANOVA. To control for an inflated type I error, post hoc test P values were adjusted using the Bonferroni correction. All tests were two-tailed, and a nominal significance level of 0.05 was used.
Results
DEXA and ACTH regulate transcription in RC
We have previously reported that ACTH affects transcription patterns in differentiated RC in a manner consistent with increased differentiation along the endochondral pathway (5). Here we show the effect of ACTH (10–7 M) alone, DEXA (10–8 M) alone, and DEXA in addition to ACTH on COLL II, AGR, OP, and COLL I transcription in RC (Fig. 1). A concentration of 10–7 M ACTH was used because in previous experiments it induced a significant transcriptional response in these cells (5). Control cultures highly expressed COLL II and AGR, indicating the differentiated chondrocyte phenotype.
FIG. 1. DEXA and ACTH regulate transcription of matrix genes in RC. Representative Northern blots (A) and corresponding densitometric analysis (B) of COLL II, AGR, OP, and COLL I transcripts in untreated (CON), 10–8 M DEXA-treated (DEXA), and 10–8 M DEXA- plus 10–7 M ACTH-treated (D+A) RC cultures. Total RNA was extracted from the cultures on the days indicated, and 10 μg were used per lane in the Northern blot (see Materials and Methods). In the bar graphs, values are presented as the mean ± SD (n = 3–4 cultures). Data were analyzed by three-way ANOVA and two-way ANOVA where appropriate. Significant main effects and interaction effects are presented in Results.
Three-way ANOVA (ACTH x DEXA x day) of COLL I expression revealed significant effects of ACTH [F(1,30) = 12.26; P = 0.0015] and DEXA [F(1,30) = 245.85; P < 0.0001], but not day [F(2,30) = 2.19; P = 0.129]. No significant interactions were present with COLL I (ACTH x DEXA, ACTH x day, DEXA x day, ACTH x DEXA x day). Analysis of COLL II expression revealed only a significant effect of DEXA [F(1,34) = 256.88; P < 0.0001] and a significant interaction effect of DEXA x day on COLL II expression, i.e. DEXA significantly reduced COLL II expression over time [F(2,34) = 9.67; P = 0.0005]; however, no other significant interaction effects were found.
DEXA alone significantly reduced AGR transcription [F(1,24) = 56.98; P < 0.001], whereas ACTH had a significant opposite effect [F(1, 24) = 23.16; P < 0.0001]. Of particular note is the very significant early increase in AGR transcripts with concurrent treatment. Statistical analysis revealed a significant three-way interaction effect of ACTH x DEXA x day on AGR expression [F(2,24) = 9.92; P = 0.0007]. The data suggest that the source of the interaction was a significant increase in AGR expression with dual treatment on d 3. These data were therefore analyzed at each time point by two-way ANOVA, which showed that the extreme elevation in AGR on d 3 in the dual treatment group was the result of an interaction effect of DEXA x ACTH [F(1,8) = 19.05; P = 0.0024]. No significant two-way interaction was present at the later time points, and DEXA was responsible for the main significant effect [d 7: F(1,8) = 85.33; P < 0.0001; d 10: F(1,8) = 31.87; P = 0.0005]. Analysis of OP expression revealed a significant effect of DEXA alone [F(1,30) = 133.53; P < 0.0001] and ACTH alone [F(1,30) = 33.06; P < 0.0001] and a significant interaction or synergistic effect of DEXA x ACTH [F(1,30) = 24.95; P < 0.0001]. No other significant interaction effects were found with OP expression.
ACTH induces transient elevations in [Ca2+]i and elevates basal [Ca2+]i in resting chondrocytes
DEXA priming of RC increases their sensitivity to ACTH. ACTH affects transcription patterns in differentiated RC in a manner consistent with increased differentiation along the endochondral pathway (5). As discussed, differentiation along this pathway is associated with an incremental increase in basal cytosolic calcium, and ACTH signaling through the melanocortin receptors is known to mobilize [Ca2+]i and increase basal [Ca2+]i (17). Therefore, changes in [Ca2+]i after stimulation with ACTH at a range of 10–8–10–6 M were measured in differentiated RC left untreated or treated with 10–8 M DEXA for 16–24 h. These measurements were made in the absence and presence of 1 mM extracellular Ca2+. Figure 2 shows that ACTH transiently increases [Ca2+]i in RC, which results in an elevation in basal [Ca2+]i. These changes were observed when RC cultures were stimulated in the presence and absence of extracellular calcium; however, the transients were greater in the presence of extracellular calcium (Fig 2, A and B). Additionally, the priming of differentiated RC with DEXA increased ACTH-induced [Ca2+]i elevations in both the presence and absence of extracellular calcium (Fig. 2, B and D). DEXA priming also increased the sensitivity of RC to ACTH, because transients were measurable at lower doses (10–9 M) compared with controls (not shown).
FIG. 2. ACTH induces transient elevations in [Ca2+]i and increases basal [Ca2+]i in RC. DEXA priming increases these elevations. ACTH dose-dependently induced [Ca2+]i transients in the presence (A and B) and absence (C and D) of 1 mM extracellular Ca2+. Pretreatment of the cells with 10–8 M DEXA for 16 h or longer greatly increased these transients (B and D). The time of ACTH addition is shown. The dose of ACTH is indicated either under the corresponding trace or with an arrow. Note the difference in scale for DEXA-primed cells stimulated in the presence of 1 mM extracellular Ca2+ (B). Traces represent the results of three separate experiments.
ACTH-induced transient increases in [Ca2+]i are intracellular in origin and induce store-operated calcium entry (SOCE) in RC
The fact that ACTH induces transient elevations in [Ca2+]i in the absence of extracellular Ca2+ suggests agonist-induced release from an intracellular store(s). However, the transient elevations are greater in the presence of extracellular Ca2+, suggesting a contribution from an extracellular source. To investigate the possible contribution of Ca2+ influx through voltage-dependent calcium channels (VDCC), we used nifedipine, an L-type VDCC-specific antagonist, and CdCl2, an inorganic blocker of all high-threshold VDCCs. Nifedipine or CdCl2 was added to the cuvette of fura-2-loaded RC, either treated with DEXA or left untreated, in the presence of 1 mM Ca2+ 80 sec before the addition of ACTH (Fig. 3). Neither nifedipine nor CdCl2 treatment decreased the ACTH-induced transient elevation in [Ca2+]i compared with the untreated control.
FIG. 3. Blocking of VDCC does not inhibit ACTH-induced increases in [Ca2+]i. Measurements were carried out on RC that had been pretreated with 10–8 M DEXA for 20 h (A) or left untreated (B). Fura-2-loaded RC cells were treated with either 10 μM nifedipine or 1 mM CdCl2 for 80 sec before being stimulated with ACTH in the presence of 1 mM Ca2+. The unlabeled arrow indicates the time of VDCC inhibitor addition. The specific inhibitor is noted to the right of the corresponding trace. The time of ACTH addition (10–8 M for DEXA-treated and 10–7 M for untreated RC) is noted. Traces represent the results of four separate experiments.
Next we investigated the contribution of calcium influx through store-operated channels (SOC). The depletion of intracellular Ca2+ stores, particularly those of the endoplasmic reticulum (ER), triggers a refilling through the opening of SOC at the plasma membrane. This refilling is known as capacitative calcium entry or, more recently, as SOCE (19). Untreated and DEXA-treated RC were stimulated with a range of ACTH concentrations in the absence of extracellular calcium. Ten minutes after stimulation, 2 mM CaCl2 was added to the cuvette, and the subsequent increase in [Ca2+]i was measured (Fig. 4). ACTH dose-dependently increased SOCE, and this was proportionately greater in RC pretreated with DEXA (Fig. 4B).
FIG. 4. The amount of SOCE after ACTH stimulation of RC is dose dependent, and priming with DEXA increases ACTH induced SOCE. RC either untreated (A) or treated with 10–8 M DEXA (B) for 20 h were stimulated in the absence of extracellular Ca2+ with a range of ACTH concentrations. Traces begin 10 min after stimulation. The times of 2 mM CaCl2 addition and the subsequent increase in [Ca2+]i are shown. The dose of ACTH used in the initial stimulation is shown to the right of the corresponding trace, and the time of CaCl2 is indicated with an arrow. Traces represent the results of three separate experiments.
To confirm that ACTH induces SOCE in RC, MnCl2 was used. The Mn2+ ion quenches the fluorescence of the fura-2 calcium indicator. Measurements were made using excitation wavelengths of F340/F360, where F340 shows the change in cytosolic calcium levels, and F360 is the isobestic wavelength for fura-2. It is at the isobestic wavelength that the quenching of the fura-2 fluorescence by Mn2+ can be measured. Mn2+ ions entering through the opened SOCs will cause an increase in fluorescence quenching at the isobestic wavelength. As shown in Fig. 5, ACTH stimulation of RC caused an increase in the intracellular quenching of fura-2 fluorescence by Mn2+, and this increase was dose dependent (not shown). The increase in Mn2+ quenching was also proportionate to the level of the ACTH-induced elevation in [Ca2+]i. Mn2+ quenching in response to ACTH was increased in DEXA-treated cells compared with untreated cells, mirroring the increased ACTH-induced elevation in [Ca2+]i (Fig. 5).
FIG. 5. Increased Mn2+ quenching of fura-2 confirms the opening of SOC after ACTH stimulation. Mn2+ quenching of fura-2 fluorescence at its isobestic wavelength of 360 nm is increased upon stimulation with 10–7 M ACTH in both untreated (A) and 10–8 M DEXA-treated (B) RC. Cell suspensions were prepared in the absence of extracellular Ca2+. Approximately 100 sec after the addition of 100 μM of MnCl2, ACTH (10–7 M) was added to the cuvette. The increase in cytosolic Ca2+ induced by ACTH was recorded at the fura-2 excitatory wavelength of 340 nm. The quenching of the intracellular dye by Mn2+ entry through SOCs was recorded at the fura-2 isobestic wavelength of 360 nm. Traces represent the results of three separate experiments.
SHU9119 dose-dependently attenuates ACTH-induced transient increases in [Ca2+]i
MC3-R is highly expressed by RC (5) and is therefore the suspected mediator of the ACTH signal. To demonstrate the involvement of this receptor, SHU9119, an MC3-R/MC4-R-specific antagonist, was used to block the ACTH-induced transient increases in [Ca2+]i. After fura-2 loading, DEXA-treated RC were incubated for 1 h in the presence of SHU9119 at a range of concentrations. SHU9119 dose-dependently attenuated the ACTH-induced transient increase in [Ca2+]i in both the presence (Fig. 6A) and absence of 1 mM extracellular Ca2+ (Fig. 6B). Consistent with its agouti-mimetic properties, SHU9119 caused a dose-dependent rise in basal [Ca2+]i (20) only in the presence of extracellular Ca2+. Agouti is known to induce a slow rise in basal [Ca2+]i in adipocytes and muscle cells (21), and this rise has been attributed to an influx of Ca2+ through VDCCs (22).
FIG. 6. SHU9119 attenuates ACTH-elicited transient elevations in [Ca2+]i. RC cultures were treated with 10–8 M DEXA for 20 h. Cell suspensions were prepared in the presence (A) or absence (B) of 1 mM extracellular Ca2+ after fura-2 loading and were incubated in the presence of SHU9119 for 1 h. The unlabeled arrow marks the time of ACTH addition (10–8 M in the presence and 10–7 M in the absence of Ca2+). The dose of SHU9119 is indicated under the corresponding trace. Traces represent the results of four separate experiments.
ACTH evokes [Ca2+]i release from TG-insensitive stores
The ER is the major intracellular calcium store in most cell types, and Ca2+ uptake into the ER is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA). Two types of ER Ca2+ stores have been functionally characterized and identified by immunocytochemical localization of receptors (23). One store is sensitive to IP3, and the other is sensitive to ryanodine/caffeine. These intracellular stores are released upon IP3 binding or ryanodine/caffeine binding to their respective receptors. Irreversible inhibition of SERCA with TG depletes the IP3-sensitive store; however, in some cells, TG does not deplete the caffeine- and ryanodine-sensitive store (23). In this study we sought to characterize the intracellular calcium store(s) released by ACTH in RC. RC cultures were treated with 1 μM TG for 5 min, followed by stimulation with ACTH in the absence of extracellular Ca2+. Interestingly, ACTH-induced transients were only partially TG-sensitive (Fig. 7). TG did not abrogate intracellular calcium release at 10–8 M ACTH (10–8 M ACTH, 139.35 ± 38.74 nM; 10–8 M ACTH after TG, 70.87 ± 26.55 nM; n = 4; P 0.01, by Student’s t test). However, ACTH-evoked calcium release after TG-induced Ca2+ leak from the ER does not increase at stimulatory concentrations above 10–7 M (Fig. 7B). Additionally, up to 5 μM TG did not increase the Ca2+ leak from the ER or prevent subsequent calcium release by 10–8 M ACTH (not shown). This suggests the existence of a TG-insensitive calcium store in RC whose release mechanism becomes saturated at 10–7 M ACTH.
FIG. 7. ACTH-evoked release of Ca2+ from TG-insensitive stores in DEXA-treated RC. A, RC cultures were treated with 10–8 M DEXA for 20 h. Five minutes after exposure to the irreversible inhibitor of SERCA, TG, cells were stimulated with ACTH in the range of 10–8–10–6 M. The time of ACTH addition is shown. The dose of ACTH is indicated with arrows pointing to the corresponding trace. The trace showing the response to ACTH after TG was magnified for clarity. The inset shows the full trace, with time of TG (1 μM) and ACTH addition indicated with arrows. Traces represent the results of three separate experiments. B, Bar graph showing peak ACTH-evoked [Ca2+]i release over the range of doses in A after SERCA inhibition with TG. Data are presented as the mean ± SD (n = 4 from two separate experiments). Significant pairwise differences were determined after a significant one-way ANOVA using a Bonferroni correction.
ACTH-evoked calcium release from intracellular stores is PLC dependent
Activation of the canonical signaling pathway of GPCR that elicit ER calcium release results in increased PLC activity and subsequent production of IP3. Activation of the IP3 receptor on the ER membrane by IP3, in turn, stimulates Ca2+ release (24). To test whether ACTH elicits Ca2+ release through this effector pathway, we first inhibited PLC using the specific inhibitor, U73122. When RC were treated with 10 μM U73122 for 3–5 min, ACTH-induced Ca2+ release was inhibited (Fig. 8A). U73122 inhibited Ca2+ release induced by ACTH concentrations as high as 10–6 M (not shown). In this study ATP (10 μM) was used as a positive control. ATP induced transient elevations in [Ca2+]i in a PLC-dependent manner (25, 26). U73122 blocked these transients (Fig. 8C). U73433 is the inactive analog of U73122 and had no effect on either ACTH-induced or ATP-induced calcium release (Fig. 8, B and D). U73122 also did not interfere with TG-mediated intracellular calcium release (not shown). Both U73122 and U73433 caused an early increase in basal cytosolic calcium (Fig. 7), as previously described for these compounds (25). After 1 h of treatment with U73122, however, basal calcium levels were lowered (not shown), consistent with its PLC-inhibitory properties.
FIG. 8. ACTH-evoked [Ca2+]i release in RC is PLC dependent. RC cultures were pretreated with DEXA for 20 h or longer. After loading with fura-2, RC were treated with either U73122 or U73433 for 5 min in the absence of extracellular Ca2+. Traces begin after U73122 or U73433 treatment, and stimulation with 10–8 M ACTH (A) or 10 μM ATP (C) is shown. U73433, the inactive analog of U73122, had no effect on either ACTH- or ATP-stimulated calcium release (B and D). Traces represent the results of three separate experiments.
ACTH-evoked calcium release and subsequent SOCE are partly IP3 receptor dependent
Next, to determine whether the activation of IP3 receptors is responsible for mediating ACTH-induced calcium release, we used caffeine at an inhibitory concentration. Caffeine at high concentrations (10–20 mM) will inhibit IP3 receptor activation (27). RC cultures were treated with 10 mM caffeine or vehicle for 100 sec before the addition of 10–7 M ACTH (Fig. 9A). Caffeine greatly reduced ACTH-induced Ca2+ release, suggesting that at least a portion of the Ca2+ released is via IP3 binding to its receptor. Additionally, caffeine is a known agonist of the ryanodine receptor. Stimulation of this receptor by caffeine and/or ryanodine will also elicit Ca2+ release. Because caffeine itself does not elicit transient elevations in [Ca2+]i, it is unlikely that RC in this differentiation state are expressing ryanodine receptors, and therefore it is unlikely that ACTH evokes Ca2+ release through this mechanism.
FIG. 9. ACTH-evoked [Ca2+]i release and SOCE in RC is at least partly dependent on the IP3 receptor. RC cultures were pretreated with DEXA for 20 h or longer. A, Fura-2-loaded cells were treated with either caffeine (10 mM) or vehicle (Ca2+- and Mg2+-free PBS) at the time indicated by the unlabeled arrow. The specific treatment is indicated to the right of the corresponding trace. After 100 sec, the cells were stimulated with 10–7 M ACTH as indicated. B, Traces begin about 250 sec after fura-2-loaded cells were stimulated with 10–7 M ACTH. Caffeine (10 mM) or vehicle was added 150 sec after ACTH stimulation and 50 sec before traces begin. Specific treatments are indicated to the right of the corresponding trace. CaCl2 (2 mM) was added about 3 min after caffeine or vehicle, as shown. Traces are representative of three separate experiments.
There is substantial evidence that IP3 receptor activation is required for SOCE (19, 27). In RC, the initial intracellular Ca2+ release is at least partly dependent on IP3 activation of its receptor. Therefore, we next determined whether SOCE after ACTH stimulation is IP3 receptor mediated. RC cultures were stimulated with 10–7 M ACTH. After 150 sec, 10 mM caffeine or vehicle was added for 3 min, followed by 2 mM CaCl2. SOCE after ACTH treatment was greatly reduced by the inhibition of IP3 receptor activity (Fig. 9B).
Discussion
In this study we demonstrate that transcriptional patterns in RC are regulated by the pituitary-derived melanocortin, ACTH, and by glucocorticoid. Consistent with the literature, DEXA significantly reduced COLL I and COLL II transcription (28). Although ACTH also significantly reduced COLL I transcription, statistical analysis showed that there was no synergistic effect between DEXA and ACTH on the expression of this gene. Previous data show that ACTH increases the differentiation of RC along the endochondral pathway (5). Thus, the decrease in COLL I transcription by ACTH may be a result of this differentiation effect. Interestingly, when RC cultures are treated with both glucocorticoid and ACTH, the transcriptional pattern resembles that of rapid differentiation along the endochondral pathway. COLL II is rapidly down-regulated concomitant with a very significant increase in AGR transcription. These changes are followed by a significant increase in OP transcription, a marker of hypertrophic terminal differentiation (29).
Increased [Ca2+]i release via oscillatory fluid flow is linked to increases in AGR synthesis in chondrocytes (15, 30) and increased OP transcription in osteoblasts (31). In line with these data, ACTH increases [Ca2+]i release in RC and increases AGR and OP transcription. ACTH and DEXA also interact to increase both AGR and OP transcription, which is consistent with our data showing that DEXA increases the sensitivity of RC to ACTH-induced changes in [Ca2+]i. ACTH-induced transient elevations in [Ca2+]i are greater in cells pretreated with DEXA and are observed at lower doses of ACTH. ACTH signals through the melanocortin receptors, which are a family of GPCR. The canonical GPCR effector pathway that results in intracellular calcium release is initiated by the activation of the G protein -subunit, Gq (Gq). Interestingly, DEXA has been shown to increase Gq expression and PTH-stimulated PLC activity in osteoblast-like cells (32, 33). These data suggest the possibility that DEXA, through increasing the expression of Gq, enhances the downstream effects of the ACTH stimulatory pathway in RC. More data are necessary to confirm this hypothesis.
We have shown for the first time that ACTH dose-dependently evokes transient elevations in [Ca2+]i and increases basal [Ca2+]i through an endogenously expressed melanocortin receptor in RC. Although the MC2-R (or ACTH receptor) is the endogenous receptor for ACTH, it is not likely that this receptor is responsible for mediation of the melanocortin signal in the growth plate or in RC. FGD type I, linked with both elevated ACTH and enhanced growth velocity, is attributed to a mutation in the MC2-R (3, 34), making it unlikely that stimulation of this receptor is responsible for the growth-related changes. It is the MC3-R that is the likely receptor through which the ACTH signal is mediated in these cells. This receptor has been previously detected by immunoblot at significant levels in RC (5). Additionally, the MC3-R knockout mouse model exhibits reduced linear growth (35), suggesting its involvement in endochondral differentiation. Support for this hypothesis comes from the fact that SHU9119, a specific antagonist of MC3-R and MC4-R, dose-dependently attenuates ACTH-evoked transient increases in [Ca2+]i. Although SHU9119 is also an antagonist of the MC4-R, this receptor is not likely responsible for mediation of the ACTH signal. Using immunochemistry, we did not detect MC4-R in growth plate chondrocytes, nor was expression detected in RC protein lysates by immunoblot (not shown). Moreover, in the obese yellow and MC4-R knockout mouse models, which also demonstrate enhanced somatic growth, the MC4-R is compromised (36, 37).
ACTH-evoked transient elevations in [Ca2+]i that ultimately result in elevated basal [Ca2+]i consist of both an intracellular and an extracellular component. Ca2+ is initially released from an intracellular store, followed by SOCE through SOC. Of note is the fact that the initial intracellular release originates from both TG-sensitive and -insensitive stores and is dependent upon PLC activity and IP3 activation of its receptor. IP3-dependent intracellular calcium stores are thought to overlap with that sensitive to TG (23). TG is an irreversible inhibitor of the SERCA family of Ca2+-transporting ATPases (23, 38). SERCAs are responsible for calcium uptake into the ER, which represents the major intracellular Ca2+ store in most cell types. Recently, however, it has become evident that the Golgi apparatus is also an important Ca2+ store (39, 40). Ca2+ uptake in the Golgi is dependent upon both the SERCA type of Ca2+ transport ATPases and the secretory pathway Ca2+-ATPase (SPCA) family of Ca2+ transport ATPases (39, 40, 41, 42). The SPCA are insensitive to TG, and this large Ca2+ store has been described in A7r5 smooth muscle cells and is partially responsive to IP3 (41). These data point to the possibility that the SPCA-type Ca2+-ATPases are expressed in RC, and they are responding to IP3 generated by ACTH antagonism of the endogenous MC3-R. Although a previous report indicated that the SPCA-expressing portion of the Golgi is not sensitive to agonist stimulation (42), the data were not extended to include cells of chondrogenic origin or agonists of the melanocortin family of receptors. Additional support for calcium release via TG-insensitive stores comes from the finding that this response becomes saturated at higher doses of ACTH. In A7r5 smooth muscle cells, IP3 concentrations above 30 μM failed to further increase the percentage of calcium released from the TG-insensitive store (41).
Interestingly, inhibition of the IP3 receptor by caffeine reduces SOCE after ACTH stimulation, which suggests that some SOC opening after ACTH stimulation is dependent upon IP3 receptor activation. Currently, there are three proposed models of SOC opening. The first is the conformational coupling model, which implies direct protein-protein interaction between the ER membrane proteins responsible for Ca2+ release, such as IP3 and ryanodine receptors and plasma membrane SOCs, which results in SOC activation (19, 43, 44). The second model suggests the existence of diffusible messenger, which is formed and released upon ER depletion. This messenger, termed calcium influx factor, would thereby transmit the signal to plasma membrane SOCs (19, 44, 45). The last model is a secretion-like coupling model, which suggests that fusion of secretory vesicles with the plasma membrane may activate SOCE (19, 46). Our data support a role for the IP3 receptor in ACTH-induced SOC activation and point toward the conformational coupling of ER IP3 receptors to plasma membrane SOCs. Whether this coupling requires the IP3 ligand could not be determined using the U73122 inhibitor in a DMSO vehicle. U73122; its inactive analog, U73433; and their vehicle, DMSO, all inhibited SOCE after ACTH stimulation (not shown). This is probably due to DMSO’s effects on membrane fluidity; DMSO is known to decrease the fluidity of phosphatidylethanolamine membranes (47). Phosphatidylethanolamine makes up about 80% of the inner leaflet of the plasma membrane (48), and DMSO is known to stabilize it in the hexagonal II formation (48). This conceivably prevents interactions at the inner surface of the membrane and prevents SOC activation. The three current models of SOC activation all involve inner membrane protein interactions, thereby making DMSO inhibition nonspecific. Thus, more data are necessary to determine the precise mechanism(s) involved in ACTH-induced SOC activation.
In this study we have begun to outline the signaling pathway of the endogenous MC3-R of resting chondrocytes. ACTH agonism of this receptor appears to stimulate the canonical Gq effector pathway, leading to IP3-induced release of Ca2+ from intracellular stores and ultimately SOCE. Although the data presented demonstrate that ACTH-evoked calcium release is dependent upon PLC activity, confirmation of the precise G-subunit involved is still necessary. Interestingly, the priming of RC with DEXA leads to increased activation of this pathway, suggesting glucocorticoid’s involvement in the regulation of proteins along this pathway, i.e. G-subunits. GPCR pathways have recently been identified as potential therapeutic targets (49, 50); thus, we have identified a possible new therapeutic target in the treatment of growth deficiencies and cartilage degeneration.
References
Solomon S 1999 POMC-derived peptides and their biological action. Ann NY Acad Sci 885:22–40
Wikberg JE, Muceniece R, Mandrika I, Prusis P, Lindblom J, Post C, Skottner A 2000 New aspects on the melanocortins and their receptors. Pharmacol Res 42:393–420
Elias LLK, Huebner A, Metherell LA, Canas A, Warnes GL, Bittis MLM, Cianfaranis S, Clayton PE, Savage MO, Clark AJL 2000 Tall stature in familial glucocorticoid deficiency. Clin Endocrinol (Oxf) 53:423–430
Stevens DA, Williams GR 1999 Hormone regulation of chondrocyte differentiation and endochondral bone formation. Mol Cell Endocrinol 151:195–204
Evans JF, Niu Q-T, Canas JA, Shen C-L, Aloia JF, Yeh JK 2004 ACTH enhances chondrogenesis in multipotential progenitor cells and matrix production in chondrocytes. Bone 35:96–107
Squires GR, Okouneff S, Ionescu M, Poole RA 2003 The pathobiology of focal lesion development in aging human articular cartilage and molecular matrix changes characteristic of osteoarthritis. Arthritis Rheum 48:1261–1270
Buckwalter JA, Mankin HJ 1997 Articular cartilage. Part II: Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Joint Surg Am 79:612–632[Free Full Text]
Johnson KA, van Etten D, Nanda N, Graham RM, Terkeltaub RA 2003 Distinct transglutaminase 2-independent and transglutaminase 2-dependent pathways mediate articular chondrocyte hypertrophy. J Biol Chem 272:18824–18832
Jessop DS, Dallman MF, Fleming D, Lightman SL 2001 Resistance to glucocorticoid feedback in obesity. J Clin Endocrinol Metab 86:4109–4114
Veldhuis JD, Iranmanesh A, Naftolowitz D, Tatham N, Cassidy F, Carroll BJ 2001 Corticotropin secretory dynamic in humans under low glucocorticoid feedback. J Clin Endocrinol Metab 86:5554–5563
Boscara M, Paoletta A, Scarpa E, Barzon L, Fusaro P, Fallo F, Sonino N 1998 Age-related changes in glucocorticoid fast-feedback inhibition of adrenocorticotropin in man. J Clin Endocrinol Metab 83:1380–1383
Wilkinson CW, Petrie EC, Murray SR, Colasurdo EA, Raskind MA, Peskind ER 2001 Human glucocorticoid feedback inhibition is reduced in older individuals: evening study. J Clin Endocrinol Metab 86:545–550
Purnell JQ, Brandon DD, Isabelle LM, Loriaux DL, Samuels MH 2004 Association of 24-hour cortisol production rates, cortisol-binding, globulin, and plasma-free cortisol levels with body composition, leptin levels, and aging in adult men and women. J Clin Endocrinol Metab 89:281–287
Zuscik MJ, D’Souza M, Ionescu AM, Gunter KK, Gunter TE, O’Keefe RJ, Schwarz EM, Puzas JE, Rosier RN 2002 Growth plate chondrocyte maturation is regulated by basal intracellular calcium. Exp Cell Res 276:310–319
Parvizi J, Parpura V, Greenleaf JF, Bolander ME 2002 Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J Orthop Res 21:51–57
Gallo-Payet N, Payet MD 2003 Mechanism of action of ACTH: beyond cAMP. Microsc Res Tech 61:275–287
Mountjoy KG, Kong PL, Taylor JA, Willard DH, Wilkison WO 2001 Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells. Physiol Genomics 5:11–19
Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Abeele VF, Lemonnier L, Thebault S, Lepage G, Parys JB, Shuba Y, Skryma R, Prevarskaya N 2004 Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells. J Biol Chem 279:30326–30337
Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168
Zemel, MB, Kim JH, Woychik RP, Michaud EJ, Kadwell SH, Patel IR, Wilkison WO 1995 Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice. Proc Natl Acad Sci USA 92:4733–4737
Kim JH, Mynatt RL, Moore JW, Woychik RP, Moustaid N, Zemel MB 1996 The effects of calcium channel blockade on agouti-induced obesity. FASEB J 10:1646–1652
Neves SR, Ram PT, Iyengar R 2002 G protein pathways. Science 296:1636–1639
Missiaen L, Van Acker K, Parys JB, De Smedt H, Van Baelen K, Weidema AF, Vanoevelen J, Raeymaekers L, Rneders J, Callewaert G, Rizzuto R, Wuytack F 2001 Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store. J Biol Chem 276:39161–39170
Jan C-R, Ho M-C, Wu S-N, Tseng C-J 1998 The phospho inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium. Life Sciences 63:895–908
Missiaen L, Parys JB, De Smedt H, Himpens B, Casteels R 1994 Inhibition of inositol triphosphate-induced calcium release by caffeine is prevented by ATP. Biochem J 15:81–84
Ma H-T, Venkatachalam K, Li H-S, Montell C, Kurosaki T, Patterson RL, Gill DL 2001 Assessment of the role of the inositol-1,4,5-triphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels. J Biol Chem 276:18888–18896
Richardson DW, Dodge GR 2003 Dose-dependent effects of corticosteroids on the expression of matrix related genes in normal and cytokine-treated articular chondrocytes. Inflamm Res 52:39–49
Ferrari D, Kosher RA 2002 Dlz5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol 252:257–270
Alford AI, Yellowley CE, Jacobs CR, Donahue HJ 2003 Increases in cytosolic calcium, but not fluid flow, affect aggrecan mRNA levels in articular chondrocytes. J Cell Biochem 90:938–944
You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR 2001 Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3–E1 osteoblasts. J Biol Chem 276:13365–13371
Mitchell J, Bansal A 1997 Dexamethasone increases Gq-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells. Am J Physiol 273:E528–E535
Cheung R, Mitchell J 2002 Mechanisms of regulation of G11 protein by dexamethasone in osteoblastic UMR 106–01 cells. Am J Physiol 282:E24–E30
Clark AJ, Weber A 1998 Adrenocorticotropin insensitivity syndromes. Endocr Rev 19:828–843
Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan X-M, Yu H, Rosenblum CI, Vongs A, Feng Y, Cao L, Metzger JM, Strack AM, Camacho RE, Mellin TN, Nunes CN, Min W, Fisher J, Gopal-Truter S, MacIntyre DE, Chen HY, Van der Ploeg LHT 2000 Inactivation of the mouse melanocortin-3 recpeotr results in increased fat mass and reduced lean body mass. Nat Genet 26:97–102
Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141
Wolff GL, Roberts DW, Mountjoy KG 1999 Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome. Physiol Genomics 1:151–163
Strehler EE, Treiman M 2004 Calcium pumps of plasma membrane and cell interior. Curr Mol Med 4:323–335
Pinton P, Pozzan T, Rizzuto R 1998 The Golgi apparatus is an inositol 1,4,5-triphosphate-sensitive Ca2+ store with functional properties distinct from those of the endoplasmic reticulum. EMBO J 17:5298–5308
Missiaen L, Van Acker K, Van Baelen K, Raeymaekers L, Wuytack F, Parys JB, De Smedt H, Vanoevelen J, Dode L, Rizzuto R, Callewaert G 2004 Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments. Cell Calcium 36:479–487
Missiaen L, Vanoevelen J, Parys JB, Raeymaekers L, De Smedt H, Callewaert G, Erneux C, Wuytack F 2002 Ca2+ uptake and release properties of a thapsigargin-insensitive nonmitochondrial Ca2+ store in A7r5 and 16HBE140-cells. J Biol Chem 277:6898–6902
Vanoevelen J, Raeymaekers L, Parys JB, De Smedt H, Van Baelen K, Callewaert G, Wuytack F, Missiaen L 2004 Inositol triphosphate producing agonists do not mobilize the thapsigargin insensitive part of the endoplasmic reticulum and Golgi Ca2+ store. Cell Calcium 35:115–121
Vasquez G, Lievremont J-P, Bird GSJ, Putney JW 2001 Human Trp3 forms both inositol triphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci USA 98:11777–11782
Bolotina VM 2004 Store-operated channels: diversity and activation mechanisms (web version of Science STKE). www.stke.org/cgi/content/full/sigtrans;2004/243/pe34
Su Z, Barker DS, Csutora P, Chang T, Shoemaker RL, Marchase RB, Blalock JE 2003 Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor. Am J Cell Physiol 284:C497–C505
Scott CC, Furuya W, Trimble WS, Grinstein S 2003 Activation of store-operated calcium channels. J Biol Chem 278:30534–30539
Kinoshita K, Li SJ, Yamazaki M 2001 The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide. Eur Biophys J 30:207–220
Daleke DL 2003 Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 44:233–242
Kimple RJ, Jones MB, Shutes A, Yerxa BR, Siderovski DP, Willard FS 2003 Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein subunits and regulator of G-protein signaling (RGS) proteins. Combinatorial Chem High Throughput Screening 6:1–9
Liebman C 2004 G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts. Curr Pharmaceut Design 10:1937–1958(Jodi F. Evans, Chwan-L Sh)