Bone Response to Intermittent Parathyroid Hormone Is Altered in Mice Null for ?-Arrestin2
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内分泌学杂志 2005年第4期
S. L. Ferrari, D. D. Pierroz, V. Glatt, D. S. Goddard, E. N. Bianchi, F. T. Lin, D. Manen and M. L. Bouxsein
Service of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis Prevention, Department of Rehabilitation and Geriatrics, Geneva University Hospital (S.L.F., D.D.P., E.N.B., D.M.), 1211 Geneva 14, Switzerland; Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School (V.G., D.S.G., M.L.B.), Boston, Massachusetts 02215; and Department of Cell Biology, University of Alabama (F.T.L.), Birmingham, Alabama 35294
Address all correspondence and requests for reprints to: Dr. S. L. Ferrari, Service of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis Prevention, Department of Rehabilitation and Geriatrics, Geneva University Hospital, 1211 Geneva 14, Switzerland. E-mail: serge.ferrari@medecine.unige.ch.
Abstract
Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. ?-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using ?-arrestin2–/– (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-B ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5–10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 μg/kg·d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that ?-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.
Introduction
INTERMITTENT PTH (i.e. daily administration) increases bone mass, reduces fracture risk (1, 2), and has recently been approved as a treatment for osteoporosis. At the cellular level, PTH directly stimulate osteoblast-mediated bone formation and also, through the coupling of osteoblasts to osteoclasts, bone resorption (3). Thus, PTH increases bone turnover overall, with net anabolic or catabolic effects on the skeleton depending on the dose and mode of exposure (intermittent or continuous) (4, 5). These differences may be explained at least in part by the opposite effects of PTH on the expression by osteoblasts of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) (6, 7), which, respectively, activate and inhibit osteoclastic bone resorption (8). The molecular mechanisms that regulate PTH activity in bone, however, remain incompletely understood (9).
PTH activity is mediated by a G protein-coupled receptor (PTH1Rc) coupled to both Gs and Gq, and thereby to the adenylyl cyclase-cAMP and the phospholipase C-intracellular calcium/diacylglycerol/inositol-3,4,5-triphosphate signaling pathways (10). Furthermore, PTH may activate MAPK extracellular signal-regulated kinase 1/2 (11). Among these intracellular signaling pathways, cAMP plays an essential role to mediate PTH biological activity on osteoblasts (12, 13), including the effects of PTH on the OPG production (14), and cAMP is required for the effects of PTH on the skeleton (15). Arrestins form a family of four highly homologous cytoplasmic molecules, namely two visual and two nonvisual arrestins (?-arrestin-1 and ?-arrestin2), that regulate the activity of G protein-coupled receptors (GPCRs) in various tissues (16, 17). The class II subfamily of GPCRs interacts principally, although not exclusively, with ?-arrestin2, and we previously reported that this molecule is involved in desensitization of cAMP signaling and internalization of PTH1Rc in response to its cognate agonists (18, 19, 20). ?-Arrestins also activate cAMP phosphodiesterase (PDE), thereby prompting degradation of intracellular cAMP (21). Furthermore, ?-arrestins have been implicated in the regulation of Gq-mediated signaling (22) and may serve as adaptor proteins to initiate the MAPK signaling cascade (23, 24). Together, these observations strongly suggest that arrestins may be involved in the regulation of PTH activity in vivo.
?-Arrestin1–/– and ?-arrestin2–/– (KO) mice are fertile and present no gross phenotypic abnormalities (25, 26). In contrast, their response to pharmacological stimulation by ?-adrenergic receptor agonist (26) and μ-opioid receptor agonist (25, 27) is altered. KO mice have also been reported to be resistant to the development of allergic asthma as a result of impaired chemokine receptor function (28). These considerations led us to hypothesize that absence of ?-arrestin2 would alter the effects of intermittent PTH on bone. To test this hypothesis, we investigated the response to PTH in primary osteoblastic (POB) cells isolated from neonatal calvariae of KO and wild-type (WT) mice and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo.
Materials and Methods
Animals and PTH treatment
KO mice were generated by F. Lin and R. Lefkowitz (25) and subsequently backcrossed six generations onto a C57BL/6J background. At the N6 generation, mice were separately bred as homozygous WT or KO. Mice were maintained under standard nonbarrier conditions and had access to mouse chow [5542, Harlan Teklad, Indianapolis, IN; 2.5% calcium (Ca) and 1.2% phosphate (Pi)] and water ad libitum. Synthetic human PTH-(1–34) (Bachem, Torrance, CA) was dissolved in acidified saline (0.1 N) and 2% heat-inactivated mouse serum. Twelve-week-old male mice received sc injections of PTH (80 μg/kg·d) or vehicle (VEH), five times per week for 4 wk. To evaluate acute changes in serum Ca and Pi, 1-yr-old male mice received a single ip injection of PTH (500 μg/kg) or VEH. Blood was collected retroorbitally at 0, 2, and 8 h postinjection. All animal procedures were approved by the ethics committees on animal care and use at Beth Israel Deaconess Medical Center (Boston, MA) and University of Geneva (Geneva, Switzerland).
Bone measurements
Peripheral dual energy x-ray absorptiometry (pDXA; PIXImus, GE-Lunar Corp., Madison, WI) was used to measure in vivo total body bone mineral content (TB BMC; grams), bone mineral density (BMD) (grams per square centimeter), and percent fat mass of mice at different ages (from 4–52 wk). Quantitative microcomputed tomography (μCT40, Scanco Medical AG, Basserdorf, Switzerland) was used ex vivo to assess trabecular bone morphology in the fifth lumbar vertebrae and distal femoral metaphysis, and cortical bone geometry at the midfemoral diaphysis using a 12-μm isotropic voxel size (29). Morphometric variables were computed from the binarized images using direct, three-dimensional techniques that do not rely on any prior assumptions about the underlying structure (29, 30).
Histomorphometry
Femurs were embedded in methylmethacrylate, and 5 μm-thick sagittal sections were cut with a Polycut E microtome (Leica Corp. Microsystems AG, Glattbrugg, Switzerland) and stained with modified Goldner’s Trichrome. To estimate bone mineralization rate, mice received ip injections of calcein (10 mg/kg) 7 and 2 d before euthanasia. In this case, 8-μm-thick femur sections were cut and mounted unstained for evaluation of calcein fluorescence. Quantitative histomorphometry was performed on the secondary spongiosa of the distal femoral metaphysis using a Leica Q image analyzer at x40 magnification. All parameters were calculated and expressed according to standard formulas and nomenclatures (31)
Serum biochemistry and bone turnover markers
Osteocalcin (Biomedical Technologies, Inc., Stoughton, MA), and tartrate-resistant acid phosphatase (TRACP5B; SBA Sciences, Turku, Finland) were measured by ELISA in accordance with the respective manufacturer’s indications. Serum Ca was measured by the ortho-cresylphtalein, point final method on a DADE-Dimension, RXL apparatus (Diamond Diagnostics, Holliston, MA), and Pi was measured using a standard colorimetric method at 820 nm on a Genova Life Science analyzer (Jenway, Felsted, UK) (32).
POB cultures and cAMP signaling
POB were obtained from neonatal calvaria of whole litters. Briefly, cells were harvested by sequential collagenase type II (3 mg/ml; Sigma-Aldrich Corp., Buchs, Switzerland) digestions of calvaria from 2- to 3-d-old mice. Cells from the third to fifth digestions were pooled and cultured in MEM (BioConcept, Allschwil, Switzerland), supplemented with 10% fetal calf serum, 100 U/ml penicillin/100 μg/ml streptomycin, and amphotericin B. At confluence, cells were split and plated at 6000 cells/cm2. For all signaling and gene expression studies, POB cultures from KO and WT mice were first matched for cell numbers and levels of alkaline phosphatase, a marker of osteoblastic differentiation, on d 14 under standard culture conditions. cAMP was determined by ELISA (Sigma-Aldrich Corp., Basel, Switzerland) in confluent KO and WT POB stimulated for 2–30 min with bovine (b) PTH-(1–34) (0.1–100 nM). Of note, the assays were performed in the absence of PDE inhibitors (isobutylmethylxanthine) (21). cAMP production was normalized by protein content, using the Coomassie Plus assay (Pierce Chemical Co., Lausanne, Switzerland).
RNA isolation and gene expression
To mimic intermittent exposure to PTH, POB were stimulated on d 6 in culture with bPTH-(1–34) at 10 nM or VEH for 6 h, then PTH-containing medium was washed out and replaced with fresh culture medium for 42 h (7, 13). This cycle was repeated twice, i.e. until d 10, after which cells were immediately frozen for subsequent RNA extraction. Alternatively, confluent POB on d 14 were treated with bPTH-(1–34) at 100 nM or VEH for 24 h before mRNA extraction.
Total RNA from WT and KO POB was extracted using TriPure (Roche, Basel, Switzerland) combined with deoxyribonculease treatment (RNase-free DNase Set, Qiagen, Basel, Switzerland) and purification on RNeasy Mini-column (Qiagen). The relative levels of expression of ?-arrestin1, PTH1R, OPG, and RANKL mRNA in KO POB were determined by quantitative real-time PCR. Total RNA (2 μg) was reverse transcribed using high capacity cDNA Archive Kit (Applied Biosystems, Rotkreuz, Switzerland) and was 2-fold diluted.
Quantitative RT-PCR (ABI PRISM 7000, Applied Biosystems) was performed as follows: initial denaturation for 10 min at 95 C and 40 cycles of PCR consisting of 15 sec at 95 C and 1 min at 60 C. The ?-arrestin1 and PTH1R reactions were performed in 25 μl containing 12.5 μl 2x SYBR Green PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland), 100 nM primers, 5 μl cDNA, and H2O to 25 μl. The primers for PTH1R were 5'-AGGGATTTTTTGTTGCCATCA-3' and 5'-GCGGCTCCAAGACTTCCTAA-3', those for ?-arrestin1 were 5'-GGACACGAATCTGGCTTCCA-3' and 5'-ACGATGATGCCCAGGATTTC-3', and those for phosphoprotein were 5'-AATCTCCAGAGGCACCATTG-3' and 5'-GTTCAGCATGTTCAGCAGTG-3'.
For OPG and RANKL mRNA, reactions were performed in 25 μl containing 12.5 μl 2x TaqMan Universal PCR Master Mix, 1.25 μl 20x mix of predesigned primers and TaqMan MGB probes (6-carboxy fluorescein dye labeled, Assays-on-Demand products, Applied Biosystems), and H2O to 25 μl. References for Assays-on-Demand are: for OPG, Mm_00435452_m1; for RANKL, Mm_00441908_m1; and for ?2-microglobulin genes (B2M), Mm_00437762_m1.
The mRNA level was normalized using acidic ribosomal phosphoprotein or B2M as internal standard. The relative expression of targeted mRNA was computed from the cycle threshold values of the target and internal standard genes, according to the manufacturer’s notice (User Bulletin 2, Applied Biosystems).
Statistical analysis
Values are given as the mean ± SEM. P values for differences in gene expression levels in the absence of treatment were computed by t test for unpaired comparisons (two tailed). For treatment experiments, comparisons between KO and WT mice and between PTH and VEH treatments as well as interaction analysis between treatment and genotype were performed using two-factor ANOVA for cross-sectional data and repeated measures ANOVA for longitudinal data. Post hoc analysis between specified groups was performed by Fisher’s protected least squares difference (PLSD). Pearson’s correlation coefficients (R) and P values for the relationship between changes in TB BMC (wk 4 to wk 0) and changes in biochemical markers of bone turnover were computed by simple linear regression analysis.
Results
Effects of PTH in POB
In confluent POB cultures, PTH-(1–34) dose-dependent stimulation of cAMP synthesis was significantly higher in cells isolated from KO compared with WT mice (Fig. 1A). Time-course experiments also indicated that cAMP accumulation was increased within 5 min and was sustained after 30 min of exposure to PTH-(1–34) (100 nM) in KO compared with WT cells (Fig. 1A).
FIG. 1. Effects of PTH on cAMP signaling and gene expression in POB. A, PTH-stimulated cAMP production in POB from KO () and WT mice (). Confluent POB cells were exposed to PTH-(1–34) at the indicated concentrations for 15 min, whereas in the time-course experiment, cells were exposed to 100 nM PTH-(1–34). P < 0.005 comparing KO and WT cells by repeated measures ANOVA in both experiments. Dots and bars represent the mean ± SEM of four replicates from one of five experiments with similar results. B, Expression of ?-arrestin1 and ?-arrestin2 mRNA in a mouse osteoblastic cell line, MC3T3-E1, and POB from WT mice by RT-PCR. C, Expression of ?-arrestin1 and PTH1R mRNA in WT () and KO () POB was evaluated by quantitative RT-PCR. The ribosomal phosphoprotein gene (PO) was used as an internal standard. Bars represent the mean and SEM of three independent experiments performed in triplicate. Differences between WT and KO cells were not significant (by t test). D, Quantitative RT-PCR of OPG and RANKL mRNA expression in WT () and KO () POB exposed to VEH or bPTH-(1–34) (100 nM) intermittently for 6 h or for 24 h. B2M was used as an internal standard. Bars represent the mean ± SEM of three to five separate experiments performed in triplicate. For OPG, P = 0.06, KO vs. WT; P = 0.05, genotype treatment interaction. For RANKL, P = 0.06, KO vs. WT; P = 0.07, genotype treatment interaction. For OPG/RANKL ratio, P = 0.13, KO vs. WT; P = 0.0004, genotype treatment interaction (all by two-factor ANOVA). +, P < 0.05, KO vs. WT within treatment group; *, P < 0.05, PTH vs. VEH within genotype (by Fisher’s PLSD for post hoc comparisons).
Both ?-arrestin1 and ?-arrestin2 mRNAs were expressed in WT POB as well as in MC3T3-E1 cells, a mouse osteoblastic cell line (Fig. 1B). In the absence of PTH, ?-arrestin1 mRNA levels were slightly, but not statistically, significantly higher in KO relative to WT POB (range, 1.0- to 2.2-fold in three independent experiments), whereas PTH1R gene expression itself was unaffected by absence of ?-arrestin2 (Fig. 1C). Moreover, 24-h exposure to PTH-(1–34) (100 nM) did not significantly alter ?-arrestin1 or PTH1R mRNA expression in these cells (data not shown).
PTH induced changes in the levels of expression of OPG and RANKL genes that were modulated by ?-arrestin2, as evaluated by a significant interaction between treatment and genotype in POB (Fig. 1D). Thus, compared with VEH, intermittent exposure to PTH for 6 h between d 6 and 10 in culture significantly increased OPG mRNA and the OPG/RANKL ratio in WT cells, whereas the opposite occurred in KO cells (Fig. 1D). In these conditions, OPG and the OPG/RANKL ratio were significantly lower in KO than WT cells. In contrast, exposure to PTH for 24 h on d 14 significantly decreased the OPG/RANKL ratio compared with the effect of VEH in both WT and KO POB (Fig. 1D).
Effects of intermittent PTH on bone mass and microarchitecture
No alterations in skeletal morphology or size were detected by x-ray (Faxitron, Hewlett-Packard, McMinville, OR) analysis of KO compared with WT mice (data not shown). However, body weight, percent fat, and, to a lesser extent, TB BMC were all slightly, but significantly, lower in male KO than WT mice (Fig. 2A). In young animals, the deficit in TB BMC was present before significant differences in body weight appeared. In adult mice, TB BMC was similar in WT and KO mice after correcting for body weight, indicating that the slightly lower bone mass in the latter was proportional to their reduction in weight. Histological sections of the growth plate in growing (6-wk-old) animals showed no obvious abnormalities in cartilage (Fig. 2B). In contrast, von Kossa staining of histological sections of the distal femur illustrates the lower mineralized trabecular bone volume in KO compared with WT adult mice (Fig. 2C).
FIG. 2. Body composition and structure of bone and cartilage. A, Compared with WT males (), sex- and age-matched KO mice () have lower body weight (P < 0.0001), total body (TB) percent fat (P < 0.0001), and TB BMC (P = 0.04, all by ANOVA), as assessed by pDXA. **, P < 0.005; *, P < 0.05; #, P 0.08 (KO vs. WT mice within age groups (by Fisher’s PLSD for post hoc analysis). Bars are the mean ± SEM of (WT/KO) 14/14, 39/32, 16/14, 18/15, 21/24, and 8/10 mice at 4, 8, 12, 16, 24, and 52 wk, respectively. B, Goldner’s Trichrome staining in the growth plate region of distal femur shows normal cartilage development in 6-wk-old KO mice (magnification, x250). C, Von Kossa staining of distal femur shows decreased mineralized trabecular bone volume in adult KO mice (magnification, x100).
Bone mass and architecture were then compared in adult male mice treated with PTH-(1–34) (80 μg/kg) or VEH daily for 1 month. PTH increased TB BMC by 15 ± 2% and 5 ± 2% above baseline in WT and KO mice, respectively (Fig. 3A). Hence, TB BMC remained significantly lower in KO compared with WT mice after 4-wk treatment with PTH or VEH (P = 0.002). At the end of treatment (16-wk-old mice), most trabecular bone microarchitectural parameters were significantly lower in KO compared with WT mice, and these differences were magnified by PTH treatment (Fig. 3, B–D). Indeed, vertebral trabecular bone volume fraction (percentage) and trabecular thickness were significantly increased by PTH compared with VEH in WT mice, whereas in KO mice, these parameters did not significantly improve (Fig. 3, B and C). Trabecular number was also significantly lower in KO than WT, but did not change with PTH treatment. In contrast, connectivity density was significantly lower in KO than WT mice receiving VEH, but was increased significantly with PTH in both mice. Von Kossa staining of the distal femur metaphysis also illustrates that the anabolic response to PTH was less prominent in both primary and secondary spongiosa of KO compared with WT mice (Fig. 4A).
FIG. 3. Effects of intermittent PTH on bone mass and trabecular and cortical bone architecture. A, TB BMC in WT ( and ) and KO mice ( and ) at baseline (wk 0) and after 4 wk of treatment with VEH ( and , dashed line) or human PTH-(1–34) (80 μg/kg·d; and , solid line) was evaluated by pDXA. P = 0.002, KO vs. WT after 4-wk treatment (by ANOVA); *, P = 0.03 vs. VEH; ***, P < 0.0001 vs. wk 0 (by Fisher’s PLSD for post hoc analysis). B–D, Microcomputed tomography (μ-CT) analysis of trabecular bone in the vertebrae (B), two-dimensional μ-CT images of vertebrae (C), and μ-CT analysis of cortical bone at the midshaft femur (D) after 4 wk of treatment with PTH or VEH, showing differences in architectural parameters between KO () and WT () mice. BV/TV, Trabecular bone volume fraction; Tb.Th., trabecular thickness, Tb.N, trabecular number, Conn. Dens., connectivity density; Cort.Th., cortical thickness. Significant P values comparing KO to WT by two-factor-ANOVA were as follows: BV/TV, P = 0.0006; Tb.Th., P = 0.008; Tb.N., P = 0.009; CSA, P = 0.038; and medullary area, P = 0.034. Comparing KO to WT mice within treatment group: , P < 0.01; +, P < 0.05; and #, P 0.08 (by Fisher’s PLSD for post hoc analysis). Comparing PTH to VEH: *, P < 0.05; and **, P < 0.005. Symbols and bars represent the mean ± SEM of (WT/KO) 8/7 mice in VEH groups and 12/11 mice in PTH groups.
FIG. 4. Effects of PTH on bone histomorphometry. A, Von Kossa staining of metaphyseal region of the distal femur shows the PTH-stimulated increase in trabecular bone architecture (primary and secondary spongiosa) in WT compared with KO mice (magnification, x250). B, Goldner’s Trichrome staining in the secondary spongiosa of distal femur after 4-wk treatment with human PTH-(1–34), showing an increased number of osteoclasts (arrows) in KO compared with WT mice treated (magnification, x400).
At the femur midshaft, cross-sectional area (CSA), medullary area, and, to a lesser extent, cortical bone area were all significantly lower in KO than WT mice, particularly after PTH treatment (Fig. 3D). In contrast, cortical thickness was comparable in WT and KO mice and increased significantly with PTH in both groups.
Bone turnover in response to intermittent PTH
To delineate the cellular mechanisms responsible for decreased net anabolic effects of PTH in KO mice, we assessed biochemical and histological indexes of bone turnover. In the VEH group, osteocalcin did not significantly change over time, but tended to decrease in WT mice while it slightly increased in KO (P interaction = 0.034; Table 1). Compared with either baseline or VEH, PTH significantly increased serum osteocalcin in both WT and KO mice. TRACP5B, a marker of bone resorption, did not change over time with VEH, but significantly increased with PTH in both WT and KO mice (Table 1). Consistent with the known effects of PTH on bone turnover and bone mass, TB BMC gain was positively and significantly correlated with changes in both osteocalcin and TRACP5B in WT mice (Fig. 5). In KO mice, however, TB BMC was more weakly correlated with TRACP5B changes and not at all with osteocalcin (Fig. 5), indicating that increasing bone turnover in these mice did not translate into a net gain in bone mass.
TABLE 1. Biochemical markers before and after 4 wk of treatment with VEH or PTH
FIG. 5. Correlation between changes in TB BMC and biochemical markers of bone turnover. In WT mice (, solid line), the TB BMC gain is positively correlated with the percent change in osteocalcin (r = 0.50; P = 0.029) and TRACP5B (r = 0.58; P = 0.047), calculated between the end and the beginning of treatment. In KO (, dashed line), the regressions did not reach statistical significance (r = 0.17; not significant and r = 0.44; P = 0.15 for osteocalcin and TRACP5B, respectively).
Quantitative histomorphometric analyses of the distal femur showed that in the VEH groups, several osteoclastic and osteoblastic indexes tended to be lower (–25% to –35%; not significantly different) in KO compared with WT mice, with differences in the mineral apposition rate reaching borderline significance (Table 2). PTH treatment increased osteoblast number and activity, i.e. bone formation rate and indexes of mineralization, several-fold in both WT and KO mice. In contrast, a significant interaction occurred between treatment and genotype for osteoclastic indexes; PTH significantly increased osteoclast number and surface (up to 80%) in KO, but not in WT, mice (Table 2 and Fig. 4B).
TABLE 2. Histomorphometry of cancellous bone after 4 wk of treatment with intermittent PTH or VEH
PTH effects on serum Ca and Pi
Serum Ca and Pi were measured before and after 4 wk of treatment with intermittent PTH. In these conditions, the levels of these minerals remained stable over time and were similar in WT (Ca, 2.28 ± 0.04 and 2.26 ± 0.08 mmol/liter; Pi, 2.19 ± 0.11 and 2.14 ± 0.07 mmol/liter, before and after PTH, respectively) and KO mice (Ca, 2.25 ± 0.03 and 2.28 ± 0.07 mmol/liter; Pi, 2.32 ± 0.10 and 2.36 ± 0.16 mmol/liter, before and after PTH, respectively).
To investigate the responsiveness of serum minerals to PTH, we evaluated acute changes in serum Ca and Pi in adult male WT and KO mice injected with a higher PTH dose (500 μg/kg, ip) or VEH. As shown in Fig. 6, the marked decline in serum Pi that occurred 1 h after PTH injection was similar in KO and WT mice. After 1 h, PTH-induced changes in serum Ca were also significantly different from those caused by VEH in both WT and KO mice. However, serum Ca actually increased in KO mice only and was significantly higher than that in WT mice. By 8 h after PTH injection, serum Ca levels had returned to baseline, whereas serum Pi levels had rebounded to values higher than baseline in all mice.
FIG. 6. Acute changes in serum Ca and Pi after a single administration of PTH. Serum Ca (upper panel) and Pi (lower panel) levels in WT ( and ) and KO mice ( and ) were measured before and after ip injection of VEH ( and ) or human PTH (500 μg/kg; and ). P = 0.006 for Ca comparing KO with WT mice after 1 h (by two-factor ANOVA). +, P < 0.05 comparing KO with WT within PTH treatment group (by Fisher’s PLSD for post hoc comparisons). *, P < 0.05; **, P < 0.01 for Ca and Pi changes from baseline comparing PTH and VEH within genotype (by Fisher’s PLSD for post hoc comparisons). Symbols and bars represent the mean ± SEM of (WT/KO) three mice in VEH groups and five/six mice in PTH-treated groups.
Discussion
By analyzing the response to PTH in KO mice, we found that PTH activity in POB and, most importantly, the pharmacological effects of intermittent PTH on the skeleton are modulated by ?-arrestin2. We confirmed that both ?-arrestin1 and ?-arrestin2 genes are expressed in osteoblastic cells (33) and showed that the expression of ?-arrestin1 and PTH1R genes was not significantly altered in KO POB cells. Hence, the absence of ?-arrestin2 alone was sufficient to increase and sustain PTH-stimulated cAMP synthesis in osteoblasts. These observations are in keeping with the role of ?-arrestin2 to mediate rapid uncoupling of PTH1Rc from G protein (19, 20) and to activate PDE4 (21).
Intracellular cAMP appears to be the principal mediator of PTH activity on a number of osteoblastic genes (12, 13, 34, 35), including the expression of OPG (14, 36), an essential inhibitor of osteoclast functions (8). Previous studies using cultured mouse marrow cells showed that intermittent PTH modestly increases OPG and RANKL mRNA expression, whereas prolonged exposure to PTH decreases OPG while increasing RANKL, resulting in a marked decrease in the OPG/RANKL ratio (7). Our observations indicate that in absence of ?-arrestin2, the OPG/RANKL ratio decreases whether POB are exposed to PTH intermittently or for 24 h. Therefore, not only the dose and mode of exposure to PTH per se, but also the intracellular mechanisms that regulate PTH1Rc activity, influence the effects of PTH on the expression of these genes.
Our in vivo data show that mice null for ?-arrestin2 develop an apparently normal skeleton with minimal spontaneous deficits in bone mass and architecture during growth, the former being proportionate to their lower body weight and fat mass when they reach adulthood. More prominent alterations may have been prevented by the modest overexpression of ?-arrestin1, whose functions largely overlap those of ?-arrestin2 in most cells, including osteoblasts (Fig. 1). Yet, upon pharmacological challenges, the role of arrestins in regulating GPCR-mediated signaling has been revealed (16). Hence, mice lacking ?-arrestin1 or -2 present distinct alterations in analgesic, cardiovascular, and/or immune responses (25, 26, 27, 28). We now add evidence that after the administration of intermittent PTH, differences between KO and WT mice in total body bone mass, trabecular bone volume and thickness, and, to a lesser extent, cortical architecture are exaggerated. Trabecular connectivity density, however, was an exception, because it was lower in KO compared with WT mice receiving VEH, but was markedly improved by PTH in the former. Because KO had a more rod-like structure whereas WT had a more plate-like structure, as evaluated by the structural model index (data not shown), it is tempting to speculate that even a small, nonsignificant gain in trabecular thickness in KO mice may translate into significant changes in connectivity density.
Taken together with the DXA and microcomputed tomography data, biochemical markers suggest that for a similar increase in bone turnover, the net gains in bone mass and trabecular bone volume upon intermittent PTH treatment are blunted in KO males. Histomorphometry data also indicate that the absence of ?-arrestin2 results in a stronger stimulation of osteoclasts on trabecular surfaces compared with WT mice. Normally, greater osteoclastic activity is observed in rats treated continuously with PTH compared with those who receive PTH intermittently (4, 37, 38). These catabolic effects of PTH are mediated by an increased expression of RANKL concomitant to a decreased expression of OPG in vivo (6). Thus, by preventing sustained PTH-stimulated cAMP signaling and inhibition of OPG expression in osteoblasts, ?-arrestin2 may help to maintain a positive balance between bone formation and resorption in the bone marrow environment. Whether osteoclastic functions might also be directly affected by the absence of ?-arrestin2 remains to be investigated. Interestingly, the renal Pi response to acute PTH was similar in WT and KO mice, whereas the calcemic response was increased in the latter, indicating that it probably originated mostly from bone. Although sustained PTH-stimulated intracellular signaling in absence of ?-arrestin2 is likely to occur in both bone and kidney, the skeletal phenotypes observed in KO mice appear to result mostly from altered PTH activity on bone.
In contrast with the bone resorption indexes, histomorphometric indexes of bone formation were prominently increased by PTH treatment in both WT and KO mice. These findings are consistent with the idea that both continuous and intermittent PTH stimulate osteoblasts’ bone-forming activity (4, 38) and might explain why we observed a similar cortical thickness response in WT and KO mice. Nevertheless, the bone formation rate, femoral CSA, and bone area remained slightly lower in KO compared with WT mice receiving PTH, which probably reflects differences in (sub) periosteal bone formation by lining osteoblasts (39). Differential effects of altered PTH/PTHrP receptor-mediated signaling on the trabecular and cortical compartments have previously been reported in transgenic mice expressing a constitutively active PTH1R mutant (H223R) in osteoblasts (40). Hence, besides PTH1R-mediated signaling and its regulatory mechanisms, the nature and/or cellular environment of osteoblasts at periosteal and endosteal surfaces are other crucial determinants of the response to PTH. Furthermore, the effects of PTH on trabecular and cortical bone surfaces in KO mice may differ between males and females and between intact and ovariectomized females (41), reflecting the additional influence of gonadal steroids on PTH-induced bone remodeling in different compartments.
In comparison, transgenic mice expressing an inhibitor of GPCR kinases (GRK2 and -3) that increases PTH-stimulated cAMP signaling in vitro also present lower OPG expression and higher bone resorption compared with WT mice (42). Therefore, enhanced osteoclast activation appears to be a common feature in the absence of proper regulation of cAMP signaling in osteoblasts. In GRK mutant mice, however, bone formation is increased, resulting in a higher trabecular bone volume. It is tempting to speculate that such differences in osteoblastic functions between GRKs- and ?-arrestin2-deficient mice may result from alterations in other signaling pathways (23). Indeed, arrestins also inhibit Gq-mediated signaling in response to PTH (22) and may serve as a scaffold to initiate the MAPK signaling cascade (23, 24). Additional studies are needed to compare PTH activity on these signaling pathways in POB from GRK-mutant and KO mice.
In conclusion, in the absence of ?-arrestin2, PTH leads to increased and sustained cAMP signaling, decreased OPG/RANKL expression, and increased osteoclastogenesis, thereby limiting the ability of intermittent PTH to improve bone mass and architecture in male mice. Hence, by regulating PTH activity in osteoblasts, ?-arrestin2 plays an important role to specify the effects of intermittent PTH on the skeleton.
Acknowledgments
We thank Dr. R. J. Lefkowitz for the KO mice, Dr. R. Rizzoli for his critical insights and review of our manuscript, Dr. C. Vadas (AMS Laboratory) for calcium measures, and Mrs. M. Lachize and F. Cavat for their excellent technical work.
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Service of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis Prevention, Department of Rehabilitation and Geriatrics, Geneva University Hospital (S.L.F., D.D.P., E.N.B., D.M.), 1211 Geneva 14, Switzerland; Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School (V.G., D.S.G., M.L.B.), Boston, Massachusetts 02215; and Department of Cell Biology, University of Alabama (F.T.L.), Birmingham, Alabama 35294
Address all correspondence and requests for reprints to: Dr. S. L. Ferrari, Service of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis Prevention, Department of Rehabilitation and Geriatrics, Geneva University Hospital, 1211 Geneva 14, Switzerland. E-mail: serge.ferrari@medecine.unige.ch.
Abstract
Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. ?-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using ?-arrestin2–/– (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-B ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5–10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 μg/kg·d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that ?-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.
Introduction
INTERMITTENT PTH (i.e. daily administration) increases bone mass, reduces fracture risk (1, 2), and has recently been approved as a treatment for osteoporosis. At the cellular level, PTH directly stimulate osteoblast-mediated bone formation and also, through the coupling of osteoblasts to osteoclasts, bone resorption (3). Thus, PTH increases bone turnover overall, with net anabolic or catabolic effects on the skeleton depending on the dose and mode of exposure (intermittent or continuous) (4, 5). These differences may be explained at least in part by the opposite effects of PTH on the expression by osteoblasts of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) (6, 7), which, respectively, activate and inhibit osteoclastic bone resorption (8). The molecular mechanisms that regulate PTH activity in bone, however, remain incompletely understood (9).
PTH activity is mediated by a G protein-coupled receptor (PTH1Rc) coupled to both Gs and Gq, and thereby to the adenylyl cyclase-cAMP and the phospholipase C-intracellular calcium/diacylglycerol/inositol-3,4,5-triphosphate signaling pathways (10). Furthermore, PTH may activate MAPK extracellular signal-regulated kinase 1/2 (11). Among these intracellular signaling pathways, cAMP plays an essential role to mediate PTH biological activity on osteoblasts (12, 13), including the effects of PTH on the OPG production (14), and cAMP is required for the effects of PTH on the skeleton (15). Arrestins form a family of four highly homologous cytoplasmic molecules, namely two visual and two nonvisual arrestins (?-arrestin-1 and ?-arrestin2), that regulate the activity of G protein-coupled receptors (GPCRs) in various tissues (16, 17). The class II subfamily of GPCRs interacts principally, although not exclusively, with ?-arrestin2, and we previously reported that this molecule is involved in desensitization of cAMP signaling and internalization of PTH1Rc in response to its cognate agonists (18, 19, 20). ?-Arrestins also activate cAMP phosphodiesterase (PDE), thereby prompting degradation of intracellular cAMP (21). Furthermore, ?-arrestins have been implicated in the regulation of Gq-mediated signaling (22) and may serve as adaptor proteins to initiate the MAPK signaling cascade (23, 24). Together, these observations strongly suggest that arrestins may be involved in the regulation of PTH activity in vivo.
?-Arrestin1–/– and ?-arrestin2–/– (KO) mice are fertile and present no gross phenotypic abnormalities (25, 26). In contrast, their response to pharmacological stimulation by ?-adrenergic receptor agonist (26) and μ-opioid receptor agonist (25, 27) is altered. KO mice have also been reported to be resistant to the development of allergic asthma as a result of impaired chemokine receptor function (28). These considerations led us to hypothesize that absence of ?-arrestin2 would alter the effects of intermittent PTH on bone. To test this hypothesis, we investigated the response to PTH in primary osteoblastic (POB) cells isolated from neonatal calvariae of KO and wild-type (WT) mice and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo.
Materials and Methods
Animals and PTH treatment
KO mice were generated by F. Lin and R. Lefkowitz (25) and subsequently backcrossed six generations onto a C57BL/6J background. At the N6 generation, mice were separately bred as homozygous WT or KO. Mice were maintained under standard nonbarrier conditions and had access to mouse chow [5542, Harlan Teklad, Indianapolis, IN; 2.5% calcium (Ca) and 1.2% phosphate (Pi)] and water ad libitum. Synthetic human PTH-(1–34) (Bachem, Torrance, CA) was dissolved in acidified saline (0.1 N) and 2% heat-inactivated mouse serum. Twelve-week-old male mice received sc injections of PTH (80 μg/kg·d) or vehicle (VEH), five times per week for 4 wk. To evaluate acute changes in serum Ca and Pi, 1-yr-old male mice received a single ip injection of PTH (500 μg/kg) or VEH. Blood was collected retroorbitally at 0, 2, and 8 h postinjection. All animal procedures were approved by the ethics committees on animal care and use at Beth Israel Deaconess Medical Center (Boston, MA) and University of Geneva (Geneva, Switzerland).
Bone measurements
Peripheral dual energy x-ray absorptiometry (pDXA; PIXImus, GE-Lunar Corp., Madison, WI) was used to measure in vivo total body bone mineral content (TB BMC; grams), bone mineral density (BMD) (grams per square centimeter), and percent fat mass of mice at different ages (from 4–52 wk). Quantitative microcomputed tomography (μCT40, Scanco Medical AG, Basserdorf, Switzerland) was used ex vivo to assess trabecular bone morphology in the fifth lumbar vertebrae and distal femoral metaphysis, and cortical bone geometry at the midfemoral diaphysis using a 12-μm isotropic voxel size (29). Morphometric variables were computed from the binarized images using direct, three-dimensional techniques that do not rely on any prior assumptions about the underlying structure (29, 30).
Histomorphometry
Femurs were embedded in methylmethacrylate, and 5 μm-thick sagittal sections were cut with a Polycut E microtome (Leica Corp. Microsystems AG, Glattbrugg, Switzerland) and stained with modified Goldner’s Trichrome. To estimate bone mineralization rate, mice received ip injections of calcein (10 mg/kg) 7 and 2 d before euthanasia. In this case, 8-μm-thick femur sections were cut and mounted unstained for evaluation of calcein fluorescence. Quantitative histomorphometry was performed on the secondary spongiosa of the distal femoral metaphysis using a Leica Q image analyzer at x40 magnification. All parameters were calculated and expressed according to standard formulas and nomenclatures (31)
Serum biochemistry and bone turnover markers
Osteocalcin (Biomedical Technologies, Inc., Stoughton, MA), and tartrate-resistant acid phosphatase (TRACP5B; SBA Sciences, Turku, Finland) were measured by ELISA in accordance with the respective manufacturer’s indications. Serum Ca was measured by the ortho-cresylphtalein, point final method on a DADE-Dimension, RXL apparatus (Diamond Diagnostics, Holliston, MA), and Pi was measured using a standard colorimetric method at 820 nm on a Genova Life Science analyzer (Jenway, Felsted, UK) (32).
POB cultures and cAMP signaling
POB were obtained from neonatal calvaria of whole litters. Briefly, cells were harvested by sequential collagenase type II (3 mg/ml; Sigma-Aldrich Corp., Buchs, Switzerland) digestions of calvaria from 2- to 3-d-old mice. Cells from the third to fifth digestions were pooled and cultured in MEM (BioConcept, Allschwil, Switzerland), supplemented with 10% fetal calf serum, 100 U/ml penicillin/100 μg/ml streptomycin, and amphotericin B. At confluence, cells were split and plated at 6000 cells/cm2. For all signaling and gene expression studies, POB cultures from KO and WT mice were first matched for cell numbers and levels of alkaline phosphatase, a marker of osteoblastic differentiation, on d 14 under standard culture conditions. cAMP was determined by ELISA (Sigma-Aldrich Corp., Basel, Switzerland) in confluent KO and WT POB stimulated for 2–30 min with bovine (b) PTH-(1–34) (0.1–100 nM). Of note, the assays were performed in the absence of PDE inhibitors (isobutylmethylxanthine) (21). cAMP production was normalized by protein content, using the Coomassie Plus assay (Pierce Chemical Co., Lausanne, Switzerland).
RNA isolation and gene expression
To mimic intermittent exposure to PTH, POB were stimulated on d 6 in culture with bPTH-(1–34) at 10 nM or VEH for 6 h, then PTH-containing medium was washed out and replaced with fresh culture medium for 42 h (7, 13). This cycle was repeated twice, i.e. until d 10, after which cells were immediately frozen for subsequent RNA extraction. Alternatively, confluent POB on d 14 were treated with bPTH-(1–34) at 100 nM or VEH for 24 h before mRNA extraction.
Total RNA from WT and KO POB was extracted using TriPure (Roche, Basel, Switzerland) combined with deoxyribonculease treatment (RNase-free DNase Set, Qiagen, Basel, Switzerland) and purification on RNeasy Mini-column (Qiagen). The relative levels of expression of ?-arrestin1, PTH1R, OPG, and RANKL mRNA in KO POB were determined by quantitative real-time PCR. Total RNA (2 μg) was reverse transcribed using high capacity cDNA Archive Kit (Applied Biosystems, Rotkreuz, Switzerland) and was 2-fold diluted.
Quantitative RT-PCR (ABI PRISM 7000, Applied Biosystems) was performed as follows: initial denaturation for 10 min at 95 C and 40 cycles of PCR consisting of 15 sec at 95 C and 1 min at 60 C. The ?-arrestin1 and PTH1R reactions were performed in 25 μl containing 12.5 μl 2x SYBR Green PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland), 100 nM primers, 5 μl cDNA, and H2O to 25 μl. The primers for PTH1R were 5'-AGGGATTTTTTGTTGCCATCA-3' and 5'-GCGGCTCCAAGACTTCCTAA-3', those for ?-arrestin1 were 5'-GGACACGAATCTGGCTTCCA-3' and 5'-ACGATGATGCCCAGGATTTC-3', and those for phosphoprotein were 5'-AATCTCCAGAGGCACCATTG-3' and 5'-GTTCAGCATGTTCAGCAGTG-3'.
For OPG and RANKL mRNA, reactions were performed in 25 μl containing 12.5 μl 2x TaqMan Universal PCR Master Mix, 1.25 μl 20x mix of predesigned primers and TaqMan MGB probes (6-carboxy fluorescein dye labeled, Assays-on-Demand products, Applied Biosystems), and H2O to 25 μl. References for Assays-on-Demand are: for OPG, Mm_00435452_m1; for RANKL, Mm_00441908_m1; and for ?2-microglobulin genes (B2M), Mm_00437762_m1.
The mRNA level was normalized using acidic ribosomal phosphoprotein or B2M as internal standard. The relative expression of targeted mRNA was computed from the cycle threshold values of the target and internal standard genes, according to the manufacturer’s notice (User Bulletin 2, Applied Biosystems).
Statistical analysis
Values are given as the mean ± SEM. P values for differences in gene expression levels in the absence of treatment were computed by t test for unpaired comparisons (two tailed). For treatment experiments, comparisons between KO and WT mice and between PTH and VEH treatments as well as interaction analysis between treatment and genotype were performed using two-factor ANOVA for cross-sectional data and repeated measures ANOVA for longitudinal data. Post hoc analysis between specified groups was performed by Fisher’s protected least squares difference (PLSD). Pearson’s correlation coefficients (R) and P values for the relationship between changes in TB BMC (wk 4 to wk 0) and changes in biochemical markers of bone turnover were computed by simple linear regression analysis.
Results
Effects of PTH in POB
In confluent POB cultures, PTH-(1–34) dose-dependent stimulation of cAMP synthesis was significantly higher in cells isolated from KO compared with WT mice (Fig. 1A). Time-course experiments also indicated that cAMP accumulation was increased within 5 min and was sustained after 30 min of exposure to PTH-(1–34) (100 nM) in KO compared with WT cells (Fig. 1A).
FIG. 1. Effects of PTH on cAMP signaling and gene expression in POB. A, PTH-stimulated cAMP production in POB from KO () and WT mice (). Confluent POB cells were exposed to PTH-(1–34) at the indicated concentrations for 15 min, whereas in the time-course experiment, cells were exposed to 100 nM PTH-(1–34). P < 0.005 comparing KO and WT cells by repeated measures ANOVA in both experiments. Dots and bars represent the mean ± SEM of four replicates from one of five experiments with similar results. B, Expression of ?-arrestin1 and ?-arrestin2 mRNA in a mouse osteoblastic cell line, MC3T3-E1, and POB from WT mice by RT-PCR. C, Expression of ?-arrestin1 and PTH1R mRNA in WT () and KO () POB was evaluated by quantitative RT-PCR. The ribosomal phosphoprotein gene (PO) was used as an internal standard. Bars represent the mean and SEM of three independent experiments performed in triplicate. Differences between WT and KO cells were not significant (by t test). D, Quantitative RT-PCR of OPG and RANKL mRNA expression in WT () and KO () POB exposed to VEH or bPTH-(1–34) (100 nM) intermittently for 6 h or for 24 h. B2M was used as an internal standard. Bars represent the mean ± SEM of three to five separate experiments performed in triplicate. For OPG, P = 0.06, KO vs. WT; P = 0.05, genotype treatment interaction. For RANKL, P = 0.06, KO vs. WT; P = 0.07, genotype treatment interaction. For OPG/RANKL ratio, P = 0.13, KO vs. WT; P = 0.0004, genotype treatment interaction (all by two-factor ANOVA). +, P < 0.05, KO vs. WT within treatment group; *, P < 0.05, PTH vs. VEH within genotype (by Fisher’s PLSD for post hoc comparisons).
Both ?-arrestin1 and ?-arrestin2 mRNAs were expressed in WT POB as well as in MC3T3-E1 cells, a mouse osteoblastic cell line (Fig. 1B). In the absence of PTH, ?-arrestin1 mRNA levels were slightly, but not statistically, significantly higher in KO relative to WT POB (range, 1.0- to 2.2-fold in three independent experiments), whereas PTH1R gene expression itself was unaffected by absence of ?-arrestin2 (Fig. 1C). Moreover, 24-h exposure to PTH-(1–34) (100 nM) did not significantly alter ?-arrestin1 or PTH1R mRNA expression in these cells (data not shown).
PTH induced changes in the levels of expression of OPG and RANKL genes that were modulated by ?-arrestin2, as evaluated by a significant interaction between treatment and genotype in POB (Fig. 1D). Thus, compared with VEH, intermittent exposure to PTH for 6 h between d 6 and 10 in culture significantly increased OPG mRNA and the OPG/RANKL ratio in WT cells, whereas the opposite occurred in KO cells (Fig. 1D). In these conditions, OPG and the OPG/RANKL ratio were significantly lower in KO than WT cells. In contrast, exposure to PTH for 24 h on d 14 significantly decreased the OPG/RANKL ratio compared with the effect of VEH in both WT and KO POB (Fig. 1D).
Effects of intermittent PTH on bone mass and microarchitecture
No alterations in skeletal morphology or size were detected by x-ray (Faxitron, Hewlett-Packard, McMinville, OR) analysis of KO compared with WT mice (data not shown). However, body weight, percent fat, and, to a lesser extent, TB BMC were all slightly, but significantly, lower in male KO than WT mice (Fig. 2A). In young animals, the deficit in TB BMC was present before significant differences in body weight appeared. In adult mice, TB BMC was similar in WT and KO mice after correcting for body weight, indicating that the slightly lower bone mass in the latter was proportional to their reduction in weight. Histological sections of the growth plate in growing (6-wk-old) animals showed no obvious abnormalities in cartilage (Fig. 2B). In contrast, von Kossa staining of histological sections of the distal femur illustrates the lower mineralized trabecular bone volume in KO compared with WT adult mice (Fig. 2C).
FIG. 2. Body composition and structure of bone and cartilage. A, Compared with WT males (), sex- and age-matched KO mice () have lower body weight (P < 0.0001), total body (TB) percent fat (P < 0.0001), and TB BMC (P = 0.04, all by ANOVA), as assessed by pDXA. **, P < 0.005; *, P < 0.05; #, P 0.08 (KO vs. WT mice within age groups (by Fisher’s PLSD for post hoc analysis). Bars are the mean ± SEM of (WT/KO) 14/14, 39/32, 16/14, 18/15, 21/24, and 8/10 mice at 4, 8, 12, 16, 24, and 52 wk, respectively. B, Goldner’s Trichrome staining in the growth plate region of distal femur shows normal cartilage development in 6-wk-old KO mice (magnification, x250). C, Von Kossa staining of distal femur shows decreased mineralized trabecular bone volume in adult KO mice (magnification, x100).
Bone mass and architecture were then compared in adult male mice treated with PTH-(1–34) (80 μg/kg) or VEH daily for 1 month. PTH increased TB BMC by 15 ± 2% and 5 ± 2% above baseline in WT and KO mice, respectively (Fig. 3A). Hence, TB BMC remained significantly lower in KO compared with WT mice after 4-wk treatment with PTH or VEH (P = 0.002). At the end of treatment (16-wk-old mice), most trabecular bone microarchitectural parameters were significantly lower in KO compared with WT mice, and these differences were magnified by PTH treatment (Fig. 3, B–D). Indeed, vertebral trabecular bone volume fraction (percentage) and trabecular thickness were significantly increased by PTH compared with VEH in WT mice, whereas in KO mice, these parameters did not significantly improve (Fig. 3, B and C). Trabecular number was also significantly lower in KO than WT, but did not change with PTH treatment. In contrast, connectivity density was significantly lower in KO than WT mice receiving VEH, but was increased significantly with PTH in both mice. Von Kossa staining of the distal femur metaphysis also illustrates that the anabolic response to PTH was less prominent in both primary and secondary spongiosa of KO compared with WT mice (Fig. 4A).
FIG. 3. Effects of intermittent PTH on bone mass and trabecular and cortical bone architecture. A, TB BMC in WT ( and ) and KO mice ( and ) at baseline (wk 0) and after 4 wk of treatment with VEH ( and , dashed line) or human PTH-(1–34) (80 μg/kg·d; and , solid line) was evaluated by pDXA. P = 0.002, KO vs. WT after 4-wk treatment (by ANOVA); *, P = 0.03 vs. VEH; ***, P < 0.0001 vs. wk 0 (by Fisher’s PLSD for post hoc analysis). B–D, Microcomputed tomography (μ-CT) analysis of trabecular bone in the vertebrae (B), two-dimensional μ-CT images of vertebrae (C), and μ-CT analysis of cortical bone at the midshaft femur (D) after 4 wk of treatment with PTH or VEH, showing differences in architectural parameters between KO () and WT () mice. BV/TV, Trabecular bone volume fraction; Tb.Th., trabecular thickness, Tb.N, trabecular number, Conn. Dens., connectivity density; Cort.Th., cortical thickness. Significant P values comparing KO to WT by two-factor-ANOVA were as follows: BV/TV, P = 0.0006; Tb.Th., P = 0.008; Tb.N., P = 0.009; CSA, P = 0.038; and medullary area, P = 0.034. Comparing KO to WT mice within treatment group: , P < 0.01; +, P < 0.05; and #, P 0.08 (by Fisher’s PLSD for post hoc analysis). Comparing PTH to VEH: *, P < 0.05; and **, P < 0.005. Symbols and bars represent the mean ± SEM of (WT/KO) 8/7 mice in VEH groups and 12/11 mice in PTH groups.
FIG. 4. Effects of PTH on bone histomorphometry. A, Von Kossa staining of metaphyseal region of the distal femur shows the PTH-stimulated increase in trabecular bone architecture (primary and secondary spongiosa) in WT compared with KO mice (magnification, x250). B, Goldner’s Trichrome staining in the secondary spongiosa of distal femur after 4-wk treatment with human PTH-(1–34), showing an increased number of osteoclasts (arrows) in KO compared with WT mice treated (magnification, x400).
At the femur midshaft, cross-sectional area (CSA), medullary area, and, to a lesser extent, cortical bone area were all significantly lower in KO than WT mice, particularly after PTH treatment (Fig. 3D). In contrast, cortical thickness was comparable in WT and KO mice and increased significantly with PTH in both groups.
Bone turnover in response to intermittent PTH
To delineate the cellular mechanisms responsible for decreased net anabolic effects of PTH in KO mice, we assessed biochemical and histological indexes of bone turnover. In the VEH group, osteocalcin did not significantly change over time, but tended to decrease in WT mice while it slightly increased in KO (P interaction = 0.034; Table 1). Compared with either baseline or VEH, PTH significantly increased serum osteocalcin in both WT and KO mice. TRACP5B, a marker of bone resorption, did not change over time with VEH, but significantly increased with PTH in both WT and KO mice (Table 1). Consistent with the known effects of PTH on bone turnover and bone mass, TB BMC gain was positively and significantly correlated with changes in both osteocalcin and TRACP5B in WT mice (Fig. 5). In KO mice, however, TB BMC was more weakly correlated with TRACP5B changes and not at all with osteocalcin (Fig. 5), indicating that increasing bone turnover in these mice did not translate into a net gain in bone mass.
TABLE 1. Biochemical markers before and after 4 wk of treatment with VEH or PTH
FIG. 5. Correlation between changes in TB BMC and biochemical markers of bone turnover. In WT mice (, solid line), the TB BMC gain is positively correlated with the percent change in osteocalcin (r = 0.50; P = 0.029) and TRACP5B (r = 0.58; P = 0.047), calculated between the end and the beginning of treatment. In KO (, dashed line), the regressions did not reach statistical significance (r = 0.17; not significant and r = 0.44; P = 0.15 for osteocalcin and TRACP5B, respectively).
Quantitative histomorphometric analyses of the distal femur showed that in the VEH groups, several osteoclastic and osteoblastic indexes tended to be lower (–25% to –35%; not significantly different) in KO compared with WT mice, with differences in the mineral apposition rate reaching borderline significance (Table 2). PTH treatment increased osteoblast number and activity, i.e. bone formation rate and indexes of mineralization, several-fold in both WT and KO mice. In contrast, a significant interaction occurred between treatment and genotype for osteoclastic indexes; PTH significantly increased osteoclast number and surface (up to 80%) in KO, but not in WT, mice (Table 2 and Fig. 4B).
TABLE 2. Histomorphometry of cancellous bone after 4 wk of treatment with intermittent PTH or VEH
PTH effects on serum Ca and Pi
Serum Ca and Pi were measured before and after 4 wk of treatment with intermittent PTH. In these conditions, the levels of these minerals remained stable over time and were similar in WT (Ca, 2.28 ± 0.04 and 2.26 ± 0.08 mmol/liter; Pi, 2.19 ± 0.11 and 2.14 ± 0.07 mmol/liter, before and after PTH, respectively) and KO mice (Ca, 2.25 ± 0.03 and 2.28 ± 0.07 mmol/liter; Pi, 2.32 ± 0.10 and 2.36 ± 0.16 mmol/liter, before and after PTH, respectively).
To investigate the responsiveness of serum minerals to PTH, we evaluated acute changes in serum Ca and Pi in adult male WT and KO mice injected with a higher PTH dose (500 μg/kg, ip) or VEH. As shown in Fig. 6, the marked decline in serum Pi that occurred 1 h after PTH injection was similar in KO and WT mice. After 1 h, PTH-induced changes in serum Ca were also significantly different from those caused by VEH in both WT and KO mice. However, serum Ca actually increased in KO mice only and was significantly higher than that in WT mice. By 8 h after PTH injection, serum Ca levels had returned to baseline, whereas serum Pi levels had rebounded to values higher than baseline in all mice.
FIG. 6. Acute changes in serum Ca and Pi after a single administration of PTH. Serum Ca (upper panel) and Pi (lower panel) levels in WT ( and ) and KO mice ( and ) were measured before and after ip injection of VEH ( and ) or human PTH (500 μg/kg; and ). P = 0.006 for Ca comparing KO with WT mice after 1 h (by two-factor ANOVA). +, P < 0.05 comparing KO with WT within PTH treatment group (by Fisher’s PLSD for post hoc comparisons). *, P < 0.05; **, P < 0.01 for Ca and Pi changes from baseline comparing PTH and VEH within genotype (by Fisher’s PLSD for post hoc comparisons). Symbols and bars represent the mean ± SEM of (WT/KO) three mice in VEH groups and five/six mice in PTH-treated groups.
Discussion
By analyzing the response to PTH in KO mice, we found that PTH activity in POB and, most importantly, the pharmacological effects of intermittent PTH on the skeleton are modulated by ?-arrestin2. We confirmed that both ?-arrestin1 and ?-arrestin2 genes are expressed in osteoblastic cells (33) and showed that the expression of ?-arrestin1 and PTH1R genes was not significantly altered in KO POB cells. Hence, the absence of ?-arrestin2 alone was sufficient to increase and sustain PTH-stimulated cAMP synthesis in osteoblasts. These observations are in keeping with the role of ?-arrestin2 to mediate rapid uncoupling of PTH1Rc from G protein (19, 20) and to activate PDE4 (21).
Intracellular cAMP appears to be the principal mediator of PTH activity on a number of osteoblastic genes (12, 13, 34, 35), including the expression of OPG (14, 36), an essential inhibitor of osteoclast functions (8). Previous studies using cultured mouse marrow cells showed that intermittent PTH modestly increases OPG and RANKL mRNA expression, whereas prolonged exposure to PTH decreases OPG while increasing RANKL, resulting in a marked decrease in the OPG/RANKL ratio (7). Our observations indicate that in absence of ?-arrestin2, the OPG/RANKL ratio decreases whether POB are exposed to PTH intermittently or for 24 h. Therefore, not only the dose and mode of exposure to PTH per se, but also the intracellular mechanisms that regulate PTH1Rc activity, influence the effects of PTH on the expression of these genes.
Our in vivo data show that mice null for ?-arrestin2 develop an apparently normal skeleton with minimal spontaneous deficits in bone mass and architecture during growth, the former being proportionate to their lower body weight and fat mass when they reach adulthood. More prominent alterations may have been prevented by the modest overexpression of ?-arrestin1, whose functions largely overlap those of ?-arrestin2 in most cells, including osteoblasts (Fig. 1). Yet, upon pharmacological challenges, the role of arrestins in regulating GPCR-mediated signaling has been revealed (16). Hence, mice lacking ?-arrestin1 or -2 present distinct alterations in analgesic, cardiovascular, and/or immune responses (25, 26, 27, 28). We now add evidence that after the administration of intermittent PTH, differences between KO and WT mice in total body bone mass, trabecular bone volume and thickness, and, to a lesser extent, cortical architecture are exaggerated. Trabecular connectivity density, however, was an exception, because it was lower in KO compared with WT mice receiving VEH, but was markedly improved by PTH in the former. Because KO had a more rod-like structure whereas WT had a more plate-like structure, as evaluated by the structural model index (data not shown), it is tempting to speculate that even a small, nonsignificant gain in trabecular thickness in KO mice may translate into significant changes in connectivity density.
Taken together with the DXA and microcomputed tomography data, biochemical markers suggest that for a similar increase in bone turnover, the net gains in bone mass and trabecular bone volume upon intermittent PTH treatment are blunted in KO males. Histomorphometry data also indicate that the absence of ?-arrestin2 results in a stronger stimulation of osteoclasts on trabecular surfaces compared with WT mice. Normally, greater osteoclastic activity is observed in rats treated continuously with PTH compared with those who receive PTH intermittently (4, 37, 38). These catabolic effects of PTH are mediated by an increased expression of RANKL concomitant to a decreased expression of OPG in vivo (6). Thus, by preventing sustained PTH-stimulated cAMP signaling and inhibition of OPG expression in osteoblasts, ?-arrestin2 may help to maintain a positive balance between bone formation and resorption in the bone marrow environment. Whether osteoclastic functions might also be directly affected by the absence of ?-arrestin2 remains to be investigated. Interestingly, the renal Pi response to acute PTH was similar in WT and KO mice, whereas the calcemic response was increased in the latter, indicating that it probably originated mostly from bone. Although sustained PTH-stimulated intracellular signaling in absence of ?-arrestin2 is likely to occur in both bone and kidney, the skeletal phenotypes observed in KO mice appear to result mostly from altered PTH activity on bone.
In contrast with the bone resorption indexes, histomorphometric indexes of bone formation were prominently increased by PTH treatment in both WT and KO mice. These findings are consistent with the idea that both continuous and intermittent PTH stimulate osteoblasts’ bone-forming activity (4, 38) and might explain why we observed a similar cortical thickness response in WT and KO mice. Nevertheless, the bone formation rate, femoral CSA, and bone area remained slightly lower in KO compared with WT mice receiving PTH, which probably reflects differences in (sub) periosteal bone formation by lining osteoblasts (39). Differential effects of altered PTH/PTHrP receptor-mediated signaling on the trabecular and cortical compartments have previously been reported in transgenic mice expressing a constitutively active PTH1R mutant (H223R) in osteoblasts (40). Hence, besides PTH1R-mediated signaling and its regulatory mechanisms, the nature and/or cellular environment of osteoblasts at periosteal and endosteal surfaces are other crucial determinants of the response to PTH. Furthermore, the effects of PTH on trabecular and cortical bone surfaces in KO mice may differ between males and females and between intact and ovariectomized females (41), reflecting the additional influence of gonadal steroids on PTH-induced bone remodeling in different compartments.
In comparison, transgenic mice expressing an inhibitor of GPCR kinases (GRK2 and -3) that increases PTH-stimulated cAMP signaling in vitro also present lower OPG expression and higher bone resorption compared with WT mice (42). Therefore, enhanced osteoclast activation appears to be a common feature in the absence of proper regulation of cAMP signaling in osteoblasts. In GRK mutant mice, however, bone formation is increased, resulting in a higher trabecular bone volume. It is tempting to speculate that such differences in osteoblastic functions between GRKs- and ?-arrestin2-deficient mice may result from alterations in other signaling pathways (23). Indeed, arrestins also inhibit Gq-mediated signaling in response to PTH (22) and may serve as a scaffold to initiate the MAPK signaling cascade (23, 24). Additional studies are needed to compare PTH activity on these signaling pathways in POB from GRK-mutant and KO mice.
In conclusion, in the absence of ?-arrestin2, PTH leads to increased and sustained cAMP signaling, decreased OPG/RANKL expression, and increased osteoclastogenesis, thereby limiting the ability of intermittent PTH to improve bone mass and architecture in male mice. Hence, by regulating PTH activity in osteoblasts, ?-arrestin2 plays an important role to specify the effects of intermittent PTH on the skeleton.
Acknowledgments
We thank Dr. R. J. Lefkowitz for the KO mice, Dr. R. Rizzoli for his critical insights and review of our manuscript, Dr. C. Vadas (AMS Laboratory) for calcium measures, and Mrs. M. Lachize and F. Cavat for their excellent technical work.
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