The Local Expression and Abundance of Insulin-Like Growth Factor (IGF) Binding Proteins in Skeletal Muscle Are Regulated by Age and Gender But Not Loc
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《内分泌学杂志》
United States Department of Agriculture, Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston Texas 77030
Abstract
We wished to determine whether sustained IGF-I production in skeletal muscle increases local IGF binding protein (IGFBP) abundance, thereby mitigating the long-term stimulation of muscle growth by IGF-I. Muscle growth of transgenic mice that overexpress IGF-I in muscle (SIS2) and of wild-type (Wt) mice was compared. At 3, 5, 10, and 20 wk of age, hind-limb muscle weights and IGFBP-3, -4, -5, and -6 mRNA and protein abundances were quantified. Additional mice were injected with IGF-I or LR3-IGF-I, and phosphorylation of the type 1 IGF receptor (IGF-1R) was compared. Muscle mass was 20% greater in SIS2 compared with Wt mice by 10 wk of age (P < 0.01), and this difference was maintained to 20 wk. IGFBP mRNA and protein abundances were unaffected by genotype. IGFBP-4 and -5 protein abundances increased with age, whereas for IGFBP-3 and -6, there was a sexual dimorphic response (P < 0.01); after 5 wk of age, IGFBP-3 decreased in males but increased in females, whereas IGFBP-6 decreased in females and remained unchanged in males. These protein expression patterns resulted from differences at both the transcriptional and posttranscriptional levels. LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than IGF-I at both 5 and 10 wk of age (P < 0.01), regardless of gender or genotype (P > 0.21). Thus, variations in local IGF-I levels do not appear to regulate muscle IGFBP expression. The age- and gender-specific differences in muscle IGFBP expression are not sufficient to alter the response of the muscle to the IGFs but may impact the IGF-independent effects of these IGFBPs.
Introduction
SKELETAL MUSCLE ACCRETION is at its most rapid in early postnatal life and is enabled by its capacity to sustain high rates of protein synthesis and myonuclear accretion. The activation of these processes in the immature muscle is facilitated by IGF-I and -II as evident from the widespread disruption of normal growth that results from null mutations of the IGF-I and -II and the type 1 IGF receptor (IGF-1R) genes (1, 2, 3, 4). Recent studies of transgenic mice that do not express liver IGF-I, the primary source of circulating IGF-I, have demonstrated that local IGFs acting in an autocrine/paracrine manner are principally responsible for postnatal somatic growth (5). This finding supports the substantial data that implicate a distinct anabolic role for local rather than circulating IGFs in skeletal muscle growth (6).
The intracellular actions of IGF-I and -II are mediated primarily through the IGF-1R that is present on the cell membrane. The IGF-1R is a tyrosine kinase receptor and undergoes autophosphorylation upon binding of either IGF-I or -II. The ligand-binding domain of the receptor is on the extracellular surface of the muscle fiber. In the extracellular space, however, the IGFs are mostly complexed to IGF binding proteins (IGFBPs). Because the affinity of the IGFs for the IGFBPs is higher than for the IGF-1R, the biological activity and stability of IGFs are influenced both positively and negatively by the abundance and composition of the IGFBPs present (7).
The IGFBP family consists primarily of six structurally related proteins distributed throughout the vascular and interstitial spaces. Although the IGFBPs are expressed by all tissues in the body, their pattern of expression is tissue specific. Because the consequence of each individual IGFBP for IGF-1R binding varies, the net effect on IGF bioactivity will vary among tissues according to the composition of the IGFBPs present and their relative abundances.
Skeletal muscle produces IGFBP-3, -4, -5, and -6, with IGFBP-3 and -5 being the most abundant (8). IGFBP-3 and -5 can both potentiate and inhibit IGF-I actions; soluble IGFBP-3 and -5 seem to inhibit IGF-I-mediated effects (9, 10), but membrane-bound IGFBP-3 and -5 are observed to potentiate the effects of IGF-I (10, 11). The activity of several IGFBP proteases modulates the activity of the IGFBPs (7). This seems to be particularly true of IGFBP-5 (12), but the biological significance of this cleavage is not well understood. It is clear, however, that proteolyzed fragments of the IGFBPs can retain biological activity (13, 14). IGFBP-4 is generally thought to be inhibitory to IGF-I action and has been observed to prevent the binding of IGF-I to the IGF-1R (15, 16). IGFBP-6 preferentially binds IGF-II (17) and, in so doing, appears to inhibit IGF-II action (18). The effect of individual IGFBPs can vary in different circumstances, but their net effect on the autocrine/paracrine action of IGF-I is, to a large extent, to modify the extent of signaling through the IGF-1R.
Because IGF signaling appears to be critical for the anabolic character of immature muscle, regulation of IGFBP expression will be an important determinant of the magnitude and duration of IGF effects. Although the developmental changes in local IGF and IGF-1R expression have been described, the concurrent changes in muscle IGFBP expression have not. Moreover, the extent to which the expression of the IGFBPs are regulated by the IGFs in vivo, as occurs in vitro (19, 20, 21), is unclear.
Thus, we proposed to characterize the pattern of IGFBP expression as skeletal muscle undergoes maturation and to functionally assess the consequence of these changes for IGF-I signaling. Additionally, to determine whether IGFBP expression is regulated by local IGF, the muscle IGFBP expression patterns in transgenic mice that express IGF-I at high levels in muscle (SIS2) were compared with those in muscles from nontransgenic, wild-type (Wt) mice (22, 23). Given that enhanced local IGF-I expression does not result in a continuous acceleration in muscle growth at least to 52 wk of age (23, 24), we hypothesized that this deceleration in muscle growth rate is, in part, a result of the self-limiting effect of an IGF-I-induced increase in the expression of IGFBPs.
Materials and Methods
Experimental design
The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Male and female homozygous SIS2 (FVB) and Wt control (FVB; Charles River Laboratories, Wilmington, MA) mice were studied at 3, 5, 10, and 20 wk of age (n = 8). Litters were standardized to seven pups per dam at birth. Mice studied at 5, 10, and 20 wk of age were weaned at 3 wk of age and housed individually with ad libitum access to food and water. On the study day, 16 mice (eight males and eight females) of each genotype were studied. Mice were euthanized, blood was collected, and the muscles [quadriceps, gastrocnemius, tibialis anterior (TA), and extensor digitorum longus (EDL)] were dissected quantitatively, weighed, and frozen. These muscles were selected because they have similar metabolic, fiber-type, and growth characteristics (23). Additionally, we had determined that human IGF-I is overexpressed to a similar extent in the quadriceps, gastrocnemius, and TA muscles and at a slightly higher level in the EDL muscle (quadriceps, data not shown; gastrocnemius, TA, and EDL, Ref.23). Because of these similarities, the muscles (excluding the EDL) were pooled before they were analyzed for IGFBP-3, -4, -5, and -6 mRNA and protein abundances. To evaluate the contribution of residual blood contamination on muscle IGFBP, serum samples were analyzed for IGFBP-3 and -5 protein abundances for comparison. All samples were stored at –80 C until analyzed.
IGF-1R activation
The functional significance of the muscle IGFBP concentration and composition for IGF-I signaling was assessed by comparing the degree of activation of the muscle IGF-1R by exogenous IGF-I and Long R3 IGF-I (LR3-IGF-I). The affinity of the IGFBPs for LR3-IGF-I is significantly less than for IGF-I; we reasoned, therefore, that the difference in receptor activation by equimolar quantities of the two peptides would reflect the net functional consequence of the IGFBPs present in the interstitial space. Five- and 10-wk-old mice (n = 8) raised under identical methods were injected iv with 33 μmol/kg (body weight) normal human IGF-I (GroPep Ltd., Adelaide, Australia) and LR3-IGF-I (JRH Biosciences, Lenexa, KS). This dose was chosen because it provides submaximal stimulation of the IGF-1R in muscle and minimal stimulation of liver IGF-1R (25). After exactly 5 min, the mice were euthanized by decapitation, blood was collected for hormone assays, and the quadriceps were dissected and rapidly frozen.
Hormone assays
Serum IGF-I and LR3-IGF-I concentrations were analyzed by RIA using a commercially available kit specific for human IGF-I (DS Laboratories, Inc., Webster, TX). For the LR3-IGF-I assay, the standard curve was generated using recombinant LR-3-IGF-I. The intraassay coefficient of variation of the IGF-I and LR3-IGF-I assays was 4.9 and 5.3%, respectively.
IGFBP Western immunoblots
Muscles were powdered at liquid nitrogen temperature, and a crude muscle soluble protein extract was prepared in buffer (25) that contained 50 mM Tris/HCl (pH 7.5), 3 mM EDTA, 100 mM NaCl, protease inhibitors [Complete Mini (1836153), 1 tab/10ml; Roche Applied Science, Indianapolis, IN], phosphatase inhibitors [(phosphatase inhibitor cocktail 1 (P2850) and 2 (P5726), each at 1:100 vol:vol; Sigma Chemical, St. Louis, MO], and 1% IGEPAL A-630 (USB Corp., Cleveland, OH). The same buffer was used for diluting serum for the analysis of serum IGFBPs. The protein concentration of the extracts was determined using the bicinchoninic acid reagent (26), and equal amounts of protein were resolved on 10% SDS-PAGE gels. Samples were transferred to polyvinylidene fluoride membranes (PALL Co., Pensacola, FL) and incubated with Ponceau S (Sigma) to verify equal loading. All membranes were blocked with 2% BSA in Tris-buffered saline with 0.1% Tween (TBS-T) and probed overnight with a 1/2000 dilution of the appropriate antibody (anti-IGFBP-3 from Santa Cruz Biotechnology, Santa Cruz, CA; anti-IGFBP-4, -5, and -6 from Gropep Limited, Adelaide, Australia). Membranes then were washed in TBS-T and incubated with either horseradish peroxidase-conjugated goat (1/4000 for IGFBP-3; Santa Cruz) or rabbit (1/5000 for IGFBP-4, -5, and -6; Bio-Rad Laboratories, Hercules, CA) secondary antibodies. The horseradish peroxidase activity was detected using ECLplus Western blot detection reagents (Amersham Biosciences Ltd., Piscataway, NJ) and the STORM image and analysis system (Amersham). The blots were quantified using ImageQuant software (Amersham). Identification of the individual IGFBPs was ascertained by analyzing the purified IGFBPs (GroPep). Samples from all genotypes, genders, and age groups were always included on each membrane, so that a valid comparison could be made within individual IGFBPs. A quantitative comparison among IGFBPs is likely to be less accurate because of the differing affinities of individual antibodies for their respective antigens (IGFBPs). For IGFBP-3, however, we developed a standard curve by determining the band intensities of a series of known concentrations of recombinant human IGFBP-3 (Gropep). The standards were diluted in the same buffer as the samples, with the addition of 30 μg soluble catfish muscle protein as a carrier and then processed in the same ways as the muscle samples. Pilot studies were carried out to determine the appropriate concentration range for the standards.
IGF-1R immunoprecipitation and Western blot analysis
Crude muscle membrane preparations were prepared from aliquots of the powdered muscles in buffer containing appropriate phosphatase and protease inhibitors as previously described (27). After determining the protein concentration, equivalent amounts of membrane protein were subjected to Western blot analysis to determine the total receptor abundance. To measure the degree of receptor phosphorylation, the membranes were solubilized and immunoprecipitated using 20 μl anti-IGF-1R antibody (SC-713; Santa Cruz) as previously described (28). Twenty microliters of the precipitated proteins were subjected to Western blot analysis and probed with an antiphosphotyrosine antibody (1/500; Upstate Cell Signaling Solutions, Lake Placid, NY) as described above. Blots were rinsed with TBS-T to remove residual ECLplus activity and reprobed with anti-IGF-1R antibody (1/1000; Santa Cruz).
Quantitative real-time PCR
Total tissue RNA (RNAtot) was extracted from skeletal muscle as previously described (29). Briefly, Ultraspec (1.0 ml/50 mg tissue; Biotecx, Inc., Houston, TX) was added to preweighed (50 mg), powdered, frozen tissue and then homogenized. RNAtot was extracted according to the manufacturer’s protocol and resuspended in nuclease-free water and snap frozen in liquid nitrogen and stored at –80 C until further analysis. The concentration of RNAtot was determined using the Ribo Green assay (Molecular Probes, Eugene, OR).
All samples were DNase treated and reverse transcribed as described by Crosier et al. (30). Briefly, aliquots of RNA were thawed on ice, and 4 μl (2 μg RNAtot) were treated with 15 U DNase I (Roche Molecular Biochemicals, Mannheim, Germany) for 20 min at 37 C. The reaction was stopped with 20 mM EDTA. RT was conducted using random hexamers (Life Technologies, Inc., Grand Island, NY) and SuperScript II reverse transcriptase (Life Technologies) using the manufacturer’s recommended conditions. cDNA was then purified with Qiaquick purification columns (QIAGEN, Inc., Valencia, CA).
Primers and probes were developed from mouse GenBank sequences using Primer Express software (Table 1). Each product sequence was analyzed against the mouse genome using the BLAST program (National Center for Biotechnology Information) to ensure no sequence homology. Electrophoresis of PCR products was performed to ensure that a single product was obtained. Standards were isolated from mouse first-strand cDNA and amplified by PCR. The standard was quantified based on absorbance at 260 nm and diluted to obtain linear range curves for each mRNA.
Each real-time PCR contained cDNA (40 ng GAPDH, 100 ng IGFBP-4, and 150 ng IGFBP-3, -5, and -6), 300 nM of the appropriate forward and reverse primers (custom synthesized by Sigma-Genosys, Woodlands, TX), and 100 nM probe (5'-FAM; 3'-TAMRA), 2.5 mM MgCl, 200 μM dNTP mix, and 1.25 U Hotstart Goldstar polymerase using the Eurogentec Core PCR kit (Eurogentec North America, Inc., Philadelphia, PA) in a final volume of 50 μl. PCRs were performed in 96-well plates using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amount of each transcript, as determined by extrapolation of the cycle threshold (number of PCR cycles required for the fluorescent signal to reach a detection threshold) obtained against the appropriate standard curve, was normalized to GAPDH mRNA levels to control for potential variation between RT reactions. A separate PCR assay was performed to determine the amplification of GAPDH. We demonstrated that GAPDH mRNA expression was similar across genotype, age, and gender (data not shown).
Statistical analyses
Data were subjected to ANOVA using the general linear model procedure of MINITAB (version 13.31; MINITAB Inc., State College, PA). Data were evaluated for the effects of genotype, gender, age, and all appropriate interactions. Genotype, gender, and age were contrasted using a protected least significant difference test (31). The experimental unit for all analyses was individual mouse. Values, where appropriate, are expressed as mean ± 1 SEM, and the significance level for all tests was set at P < 0.05.
Results
Muscle weights
Similar to previous results from our laboratory (23), genotype differences in hind-limb muscle weights emerged between 5 and 10 wk of age. Muscles were 20% heavier in SIS2 mice by 10 wk of age (Fig. 1; P < 0.01), and the difference was maintained thereafter. Differences in muscle weights between genders emerged between 3 and 5 wk of age, by which time muscles were significantly heavier in male than in female mice (P < 0.01). The slope of the muscle growth curve differs between genders only between 3 and 5 wk of age when there was no genotype effect (10.0 ± 1.1 mg/d for males vs. 5.4 ± 0.8 for females; P < 0.05). However, there were no differences in muscle growth rates of males and females between 5 and 20 wk of age regardless of genotype (SIS2: male, 1.38 ± 0.12 mg/d; female, 1.20 ± 0.10 mg/d: Wt: male, 0.91 ± 0.09 mg/d; female, 0.94 ± 0.09 mg/d; gender effect, P > 0.35).
IGFBP protein abundance
There were no significant genotype or genotype interactions on any of the skeletal muscle IGFBP protein abundances (P > 0.38). This lack of genotype effect is illustrated in Fig. 2, in which all age and gender IGFBP data for each genotype have been averaged. To evaluate the gender and age effects (which are similar for both genotypes), data for SIS2 and Wt IGFBP protein abundances have been combined. Changes over time among IGFBPs are illustrated in Fig. 3. IGFBP protein abundances are presented relative to the amount present at 3 wk of age, when neither gender nor genotype differences were present (P > 0.21).
Muscle IGFBP-3 protein abundance responded differently in male and female mice (Fig. 3; gender x age, P < 0.001). IGFBP-3 initially decreased (wk 3 to wk 5; P < 0.01) for both genders. There was an additional decrease in males from 5 to 20 wk of age (P < 0.001), whereas in females, levels increased by 40% from 5 to 20 wk of age (P < 0.03). This resulted in 68% higher IGFBP-3 concentrations in females compared with mice of either genotype at 20 wk of age (P < 0.001). Muscle IGFBP-4 and -5 increased with age (Fig. 3; P < 0.001), regardless of gender (P > 0.10). For IGFBP-6, there was a gender x age interaction (Fig. 3; P < 0.05). IGFBP-6 decreased for both males and females from 3 to 5 wk of age; after 5 wk, IGFBP-6 continued to decrease in females (P < 0.04) but remained unchanged in males (P > 0.10). This resulted in a 50% lower skeletal muscle IGFBP-6 protein abundance at 20 wk of age for female compared with male mice (P < 0.01). No gender, genotype, or age differences were observed in serum IGFBP-3 or -5 protein abundances (Fig. 4; P > 0.10).
IGFBP mRNA abundance
Similar to protein abundances, there were no differences in the IGFBP mRNA abundances between genotypes (P > 0.10), and the data have been combined for analysis. Muscle IGFBP-3 mRNA abundance decreased by 56% from 3 to 20 wk of age (Fig. 5; P < 0.001) and was not significantly different between male and female mice. Muscle IGFBP-4 mRNA abundance increased from 3 to 20 wk of age (Fig. 5; P < 0.001) for both males and females. IGFBP-5 mRNA abundance increased (Fig. 5; P < 0.04) in male mice (P < 0.05) but remained constant in females, so that by 20 wk of age the abundance was 49% lower than in male mice (P < 0.01). No gender, genotype, or age differences were observed in skeletal muscle IGFBP-6 mRNA abundance (Fig. 5; P > 0.10).
IGF-1R phosphorylation
No genotype, gender, or age differences were observed in the activation of the IGF-1R after stimulation by either LR3-IGF-I or IGF-I, as measured by tyrosine phosphorylation (Fig. 6; P > 0.35). However, an equimolar dose of LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than native IGF-I at both 5 and 10 wk of age (P < 0.05). Total IGF-1R protein abundance did not differ between genotypes, genders, or ages (P > 0.41). The plasma concentration of the injected hormones did not differ between the IGF-I and LR3-IGF-I groups (hIGF-I, 105 ± 11 nM; LR3-IGF-I, 122 ± 19 nM; P > 0.25).
Discussion
In the current experiment, we reconfirmed that sustained high levels of muscle IGF-I stimulate muscle growth only transiently with the greatest response occurring between 5 and 10 wk of age despite the continued expression of the transgene (23). The difference in mass at 10 wk was sustained to 20 wk of age, and in our previous studies we determined that the difference remains unchanged to 52 wk of age (23, 32). The plasma concentration of human IGF-I measured in the SIS2 mice was only approximately 4% that of the endogenous mouse IGF-I, indicating that the majority of human IGF-I produced remained in the muscle tissue (23, 32). The enhanced rate of muscle mass accretion, therefore, should have been sustained, but clearly, this did not occur. Because large proportions of IGFBPs in the extracellular fluid are unbound, they are available to regulate the autocrine/paracrine effects of IGF-I. We hypothesized, therefore, that if IGF-I stimulated expression of the IGFBPs, these in turn would inhibit the ability of IGF-I to activate its receptor. We postulated, therefore, that muscle IGFBP levels would be similar in Wt and SIS2 mice up to 5 wk of age, during the time when muscle growth rates in SIS2 mice are higher. However, IGFBP levels would be higher in the SIS2 mice from 10 wk when their muscle growth rates had decreased to match those of Wt mice. To test this hypothesis, we compared the expression of both the mRNA and protein of IGFBP-3, -4, -5, and -6 in the skeletal muscles of SIS2 and Wt mice before (3 wk old), during (5 wk old), and after (10 and 20 wk old) the period of accelerated growth. Moreover, to functionally assess the combined effect of the IGFBPs on IGF-I bioactivity, we measured the phosphorylation of the IGF-1R after stimulation by equimolar amounts of IGF-I and LR3-IGF-I.
To date, it is unclear whether IGFBP-3 is expressed by skeletal muscle. IGFBP-3 expression by mouse muscle cells has been documented in some studies, both in vivo (33), and in vitro (34), but this is not a consistent finding (35, 36, 37). Our data demonstrate that IGFBP-3 mRNA is expressed in the skeletal muscle of mice in vivo and that it decreases during development. Thus, mouse skeletal muscle is similar to human (38), rat (39), bovine (40), and porcine skeletal muscle (41) with respect to IGFBP-3 expression. The decrease in IGFBP-3 mRNA with development in vivo has also been reported in the rat masseter muscle (42) and in pig biceps femoris muscle (43). Similar to the mRNA, the local abundance of IGFBP-3 protein initially decreased. The patterns of muscle and serum IGFBP-3 protein expression were distinctly different, which suggests that the muscle values reflect the local production of the protein. Using recombinant IGFBP-3 as standards, we determined the IGFBP-3 concentration at 3 wk of age to be 1.96 ± 0.19 μg/mg soluble muscle protein. The relative band intensity at 3 wk of age of the IGFBP-4 relative to IGFBP-3 was 0.4:1; IGFBP-5 to IGFBP-3 was 0.43:1, and IGFBP-6 to IGFBP-3 was 0.54:1. From this comparison, it appears that at 3 wk of age IGFBP-3 was the most abundant IGFBP in skeletal muscle. This contrasts with the mature muscle in which IGFBP-5 is the predominant IGFBP. With the onset of puberty, IGFBP-3 protein increased in females but continued to decrease in males. The disparate response of mRNA and protein in females after the onset of puberty suggests a role of sex steroids in the posttranscriptional regulation of IGFBP-3 expression or its posttranslational fate. There have been various investigations on the regulation of IGFBP-3 expression by sex steroids, and these demonstrate that the response is highly dependent on the specific tissue and its stage of differentiation (7).
The in vivo significance for muscle growth of the gender-specific differences in muscle IGFBP-3 expression with age is unclear. When IGFBP-3 was ubiquitously overexpressed in mice (44, 45), muscle growth rate decreased (45). This inhibitory effect of IGFBP-3 on muscle growth is supported by the observation that IGFBP-3 suppresses the proliferation of myogenic cells in culture (46, 47). It is tempting, therefore, to propose that the gender-specific differences in IGFBP-3 may contribute to the reduced muscle growth potential of female mice. However, the muscle mass growth curves demonstrate that the largest divergence in growth between males and females occurs between 3 and 5 wk of age and precedes both puberty and the emergence of the differences in IGFBP-3 protein levels.
There was no difference in IGFBP-3 mRNA or protein expression between genotypes, suggesting that in vivo expression of the gene in skeletal muscle is not regulated by local IGF-I. This finding is consistent with the lack of an in vivo response of skeletal muscle IGFBP-3 to IGF-I in the intact rat (39) and pig (48). Again, however, the expression of IGFBP-3 in response to IGF-I varies depending on species and cell type and, for muscle cells, can vary according to their state of differentiation.
The patterns of IGFBP-4 expression were similar for both the mRNA and protein, exhibiting an increase with age that was not influenced by either genotype or gender. The expression in skeletal muscle of rodents has been documented both in various muscle cell lines (31, 37, 49) and in vivo (33, 35, 36). The increase with age is consistent with the observation that in C2C12 cells the expression of IGFBP-4 increases as differentiation proceeds and is highest in differentiated myotubes (35). When added to proliferating muscle cells, Ewton et al. (50) observed that IGFBP-4 completely inhibited the actions of IGF-I and -II, as it does in other tissues. The increase in muscle IGFBP-4 with age that we observed is commensurate with the overall decline in muscle growth rate, but clearly it is not sufficient to explain the differences in the pattern of muscle growth between genders.
For IGFBP-5, there were gender differences at the level of gene transcription, with males increasing continuously, whereas females demonstrated little change with age. In males, the increase in mRNA abundance translated into a parallel increase in IGFBP-5 protein concentration. In contrast to the constant level of mRNA expression, IGFBP-5 protein in muscle from female mice increased with age and was not significantly different from the concentration in males. Thus, relative to muscles in male mice, the rate of translation of the mRNA in female mice increased and/or the rate of clearance of the IGFBP-5 peptide must have decreased with age. The rate of increase in IGFBP-5 protein expression was greatest between 3 and 5 wk of age and then continued more gradually. By 20 wk of age, levels were almost twice those present at 3 wk, and IGFBP-5 was then the most abundant IGFBP expressed in the muscle. Although IGFBP-5 can inhibit or enhance IGF activity, or it can regulate cellular functions independently of IGF (51), the widespread growth retardation demonstrated by transgenic mice that overexpress IGFBP-5 suggests that its predominant effect is inhibitory. Moreover, the skeletal muscles appeared to be the most severely affected tissue (52). On the basis of this observation, it is possible that the progressive increases with age in IGFBP-5 concentration, together with the increase in IGFBP-4 that we observed, may act to reduce skeletal muscle growth as maturity is attained.
There were no significant differences in IGFBP-5 protein expression between SIS2 and Wt mice. Similar results were reported by Musaro et al. (53) for IGFBP-5 mRNA expression in another transgenic mouse model that targeted IGF-I expression to skeletal muscle. These results are in marked contrast with the increased expression of IGFBP-5 that was observed when IGF-I was infused directly into muscle (54) and the responses observed in a variety of cell types when exposed to IGFs in vitro (37, 51).
IGFBP-6 mRNA was not affected by age, gender, or genotype. This is consistent with the findings of Bayol et al. (35) who demonstrated no change in IGFBP-6 expression in either C2C12 cell cultures or in vivo after denervation of the gastrocnemius muscle. However, we identified a gender-specific response in protein abundance. After an initial decrease from 3 to 5 wk in both male and female mice, IGFBP-6 protein abundance continued to decrease in female but not in male mice. Thus, again there appears to be posttranscriptional regulation of IGFBP-6 expression in female mice. The actions of IGFBP-6 on cell growth appear to be inhibitory and likely are a result of its ability to avidly sequester IGF-II, but with little affinity for IGF-I (18).
In the current study, expression of IGFBP-2 mRNA was very low and, compared with the other IGFBPs, required an additional 10 cycles of amplification to amplify the appropriate band. This contrasted with liver mRNA for which the IGFBP-2 mRNA transcript could be measured readily (data not shown). Although IGFBP-2 is expressed in muscles of rats, birds, and farm animal species, our results are consistent with the observation of an absence of IGFBP-2 expression in mouse skeletal muscle when it is measured by Northern analysis (33, 55), even though it is detectable by PCR (36).
Given the range of changes in IGFBP expression and their diversity of functions, it was not possible to predict their combined effect on IGF-I-regulated muscle growth. We attempted to obtain a functional measure of this by comparing the degree of phosphorylation of the IGF-1R after submaximal stimulation with equimolar amounts of either IGF-I or LR3-IGF-I. LR3-IGF-I has negligible affinity for the IGFBPs while retaining its ability to stimulate the IGF-1R (25). Because the extravascular IGFBPs are present in a large molar excess of the IGFs, we hypothesized that the magnitude of the difference in receptor stimulation would provide an index of the degree of sequestration of IGF-I by the IGFBPs. Indeed, the administration of equimolar amounts of LR3-IGF-I induced greater IGF-1R phosphorylation compared with native IGF-I. However, the difference was similar regardless of genotype, gender, or age. Thus, the net combined effect of the IGFBPs at both 5 and 10 wk of age was to decrease signaling through the IGF-1R, and the differences in relative abundances between 5 and 10 wk of age were not sufficient to produce changes in IGF stimulatory capacity over this time.
The absence of differences in IGFBP expression between the SIS2 and Wt mice shows, for the first time, that local IGF-I does not directly regulate the production of the IGFBPs by the skeletal muscle. This does not exclude the possibility that in normal muscles, extrinsic factors that stimulate local IGF expression could promote IGFBP production independently. Our results demonstrate that the IGFBPs normally present in the muscle mitigate activation of the IGF-1R by IGF-I, as measured by the difference in IGF-1R tyrosine phosphorylation after stimulation by LR3-IGF-I vs. IGF-I. We also observed that this difference in receptor phosphorylation was similar for both genotypes despite the differences in IGF present in the muscle. Thus, in the SIS2 mice, the IGFBPs were able still to attenuate IGF-I activity. These data do not support an independent role for the IGFBPs in the temporal pattern of muscle growth in SIS2 mice.
In addition, we demonstrated that the IGFBPs have a sexually dimorphic, age-dependent pattern of expression that can be regulated at the transcriptional, translational, or posttranslational level, regardless of local IGF-I concentrations. Given the increasing interest in the IGF-independent role of the IGFBPs in the etiology of a number of chronic diseases, our data clearly demonstrate the importance of taking into consideration the normal age, gender, and tissue specificity of the IGFBPs in any evaluation of their expression patterns.
Footnotes
This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01-AR46308 and grants from the United States Department of Agriculture, Agricultural Research Service, under Cooperative Agreement number 58-6250-6001. The contents of this publication do not necessarily reflect the views or policies of the United States Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
First Published Online September 15, 2005
Abbreviations: EDL, Extensor digitorum longus; IGF-1R, type 1 IGF receptor; IGFBP, IGF binding protein; LR3-IGF-I, long R3 IGF-I; RNAtot, total tissue RNA; TA, tibialis anterior; TBS-T, Tris-buffered saline with 0.1% Tween; Wt, wild type.
Accepted for publication September 6, 2005.
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Abstract
We wished to determine whether sustained IGF-I production in skeletal muscle increases local IGF binding protein (IGFBP) abundance, thereby mitigating the long-term stimulation of muscle growth by IGF-I. Muscle growth of transgenic mice that overexpress IGF-I in muscle (SIS2) and of wild-type (Wt) mice was compared. At 3, 5, 10, and 20 wk of age, hind-limb muscle weights and IGFBP-3, -4, -5, and -6 mRNA and protein abundances were quantified. Additional mice were injected with IGF-I or LR3-IGF-I, and phosphorylation of the type 1 IGF receptor (IGF-1R) was compared. Muscle mass was 20% greater in SIS2 compared with Wt mice by 10 wk of age (P < 0.01), and this difference was maintained to 20 wk. IGFBP mRNA and protein abundances were unaffected by genotype. IGFBP-4 and -5 protein abundances increased with age, whereas for IGFBP-3 and -6, there was a sexual dimorphic response (P < 0.01); after 5 wk of age, IGFBP-3 decreased in males but increased in females, whereas IGFBP-6 decreased in females and remained unchanged in males. These protein expression patterns resulted from differences at both the transcriptional and posttranscriptional levels. LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than IGF-I at both 5 and 10 wk of age (P < 0.01), regardless of gender or genotype (P > 0.21). Thus, variations in local IGF-I levels do not appear to regulate muscle IGFBP expression. The age- and gender-specific differences in muscle IGFBP expression are not sufficient to alter the response of the muscle to the IGFs but may impact the IGF-independent effects of these IGFBPs.
Introduction
SKELETAL MUSCLE ACCRETION is at its most rapid in early postnatal life and is enabled by its capacity to sustain high rates of protein synthesis and myonuclear accretion. The activation of these processes in the immature muscle is facilitated by IGF-I and -II as evident from the widespread disruption of normal growth that results from null mutations of the IGF-I and -II and the type 1 IGF receptor (IGF-1R) genes (1, 2, 3, 4). Recent studies of transgenic mice that do not express liver IGF-I, the primary source of circulating IGF-I, have demonstrated that local IGFs acting in an autocrine/paracrine manner are principally responsible for postnatal somatic growth (5). This finding supports the substantial data that implicate a distinct anabolic role for local rather than circulating IGFs in skeletal muscle growth (6).
The intracellular actions of IGF-I and -II are mediated primarily through the IGF-1R that is present on the cell membrane. The IGF-1R is a tyrosine kinase receptor and undergoes autophosphorylation upon binding of either IGF-I or -II. The ligand-binding domain of the receptor is on the extracellular surface of the muscle fiber. In the extracellular space, however, the IGFs are mostly complexed to IGF binding proteins (IGFBPs). Because the affinity of the IGFs for the IGFBPs is higher than for the IGF-1R, the biological activity and stability of IGFs are influenced both positively and negatively by the abundance and composition of the IGFBPs present (7).
The IGFBP family consists primarily of six structurally related proteins distributed throughout the vascular and interstitial spaces. Although the IGFBPs are expressed by all tissues in the body, their pattern of expression is tissue specific. Because the consequence of each individual IGFBP for IGF-1R binding varies, the net effect on IGF bioactivity will vary among tissues according to the composition of the IGFBPs present and their relative abundances.
Skeletal muscle produces IGFBP-3, -4, -5, and -6, with IGFBP-3 and -5 being the most abundant (8). IGFBP-3 and -5 can both potentiate and inhibit IGF-I actions; soluble IGFBP-3 and -5 seem to inhibit IGF-I-mediated effects (9, 10), but membrane-bound IGFBP-3 and -5 are observed to potentiate the effects of IGF-I (10, 11). The activity of several IGFBP proteases modulates the activity of the IGFBPs (7). This seems to be particularly true of IGFBP-5 (12), but the biological significance of this cleavage is not well understood. It is clear, however, that proteolyzed fragments of the IGFBPs can retain biological activity (13, 14). IGFBP-4 is generally thought to be inhibitory to IGF-I action and has been observed to prevent the binding of IGF-I to the IGF-1R (15, 16). IGFBP-6 preferentially binds IGF-II (17) and, in so doing, appears to inhibit IGF-II action (18). The effect of individual IGFBPs can vary in different circumstances, but their net effect on the autocrine/paracrine action of IGF-I is, to a large extent, to modify the extent of signaling through the IGF-1R.
Because IGF signaling appears to be critical for the anabolic character of immature muscle, regulation of IGFBP expression will be an important determinant of the magnitude and duration of IGF effects. Although the developmental changes in local IGF and IGF-1R expression have been described, the concurrent changes in muscle IGFBP expression have not. Moreover, the extent to which the expression of the IGFBPs are regulated by the IGFs in vivo, as occurs in vitro (19, 20, 21), is unclear.
Thus, we proposed to characterize the pattern of IGFBP expression as skeletal muscle undergoes maturation and to functionally assess the consequence of these changes for IGF-I signaling. Additionally, to determine whether IGFBP expression is regulated by local IGF, the muscle IGFBP expression patterns in transgenic mice that express IGF-I at high levels in muscle (SIS2) were compared with those in muscles from nontransgenic, wild-type (Wt) mice (22, 23). Given that enhanced local IGF-I expression does not result in a continuous acceleration in muscle growth at least to 52 wk of age (23, 24), we hypothesized that this deceleration in muscle growth rate is, in part, a result of the self-limiting effect of an IGF-I-induced increase in the expression of IGFBPs.
Materials and Methods
Experimental design
The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Male and female homozygous SIS2 (FVB) and Wt control (FVB; Charles River Laboratories, Wilmington, MA) mice were studied at 3, 5, 10, and 20 wk of age (n = 8). Litters were standardized to seven pups per dam at birth. Mice studied at 5, 10, and 20 wk of age were weaned at 3 wk of age and housed individually with ad libitum access to food and water. On the study day, 16 mice (eight males and eight females) of each genotype were studied. Mice were euthanized, blood was collected, and the muscles [quadriceps, gastrocnemius, tibialis anterior (TA), and extensor digitorum longus (EDL)] were dissected quantitatively, weighed, and frozen. These muscles were selected because they have similar metabolic, fiber-type, and growth characteristics (23). Additionally, we had determined that human IGF-I is overexpressed to a similar extent in the quadriceps, gastrocnemius, and TA muscles and at a slightly higher level in the EDL muscle (quadriceps, data not shown; gastrocnemius, TA, and EDL, Ref.23). Because of these similarities, the muscles (excluding the EDL) were pooled before they were analyzed for IGFBP-3, -4, -5, and -6 mRNA and protein abundances. To evaluate the contribution of residual blood contamination on muscle IGFBP, serum samples were analyzed for IGFBP-3 and -5 protein abundances for comparison. All samples were stored at –80 C until analyzed.
IGF-1R activation
The functional significance of the muscle IGFBP concentration and composition for IGF-I signaling was assessed by comparing the degree of activation of the muscle IGF-1R by exogenous IGF-I and Long R3 IGF-I (LR3-IGF-I). The affinity of the IGFBPs for LR3-IGF-I is significantly less than for IGF-I; we reasoned, therefore, that the difference in receptor activation by equimolar quantities of the two peptides would reflect the net functional consequence of the IGFBPs present in the interstitial space. Five- and 10-wk-old mice (n = 8) raised under identical methods were injected iv with 33 μmol/kg (body weight) normal human IGF-I (GroPep Ltd., Adelaide, Australia) and LR3-IGF-I (JRH Biosciences, Lenexa, KS). This dose was chosen because it provides submaximal stimulation of the IGF-1R in muscle and minimal stimulation of liver IGF-1R (25). After exactly 5 min, the mice were euthanized by decapitation, blood was collected for hormone assays, and the quadriceps were dissected and rapidly frozen.
Hormone assays
Serum IGF-I and LR3-IGF-I concentrations were analyzed by RIA using a commercially available kit specific for human IGF-I (DS Laboratories, Inc., Webster, TX). For the LR3-IGF-I assay, the standard curve was generated using recombinant LR-3-IGF-I. The intraassay coefficient of variation of the IGF-I and LR3-IGF-I assays was 4.9 and 5.3%, respectively.
IGFBP Western immunoblots
Muscles were powdered at liquid nitrogen temperature, and a crude muscle soluble protein extract was prepared in buffer (25) that contained 50 mM Tris/HCl (pH 7.5), 3 mM EDTA, 100 mM NaCl, protease inhibitors [Complete Mini (1836153), 1 tab/10ml; Roche Applied Science, Indianapolis, IN], phosphatase inhibitors [(phosphatase inhibitor cocktail 1 (P2850) and 2 (P5726), each at 1:100 vol:vol; Sigma Chemical, St. Louis, MO], and 1% IGEPAL A-630 (USB Corp., Cleveland, OH). The same buffer was used for diluting serum for the analysis of serum IGFBPs. The protein concentration of the extracts was determined using the bicinchoninic acid reagent (26), and equal amounts of protein were resolved on 10% SDS-PAGE gels. Samples were transferred to polyvinylidene fluoride membranes (PALL Co., Pensacola, FL) and incubated with Ponceau S (Sigma) to verify equal loading. All membranes were blocked with 2% BSA in Tris-buffered saline with 0.1% Tween (TBS-T) and probed overnight with a 1/2000 dilution of the appropriate antibody (anti-IGFBP-3 from Santa Cruz Biotechnology, Santa Cruz, CA; anti-IGFBP-4, -5, and -6 from Gropep Limited, Adelaide, Australia). Membranes then were washed in TBS-T and incubated with either horseradish peroxidase-conjugated goat (1/4000 for IGFBP-3; Santa Cruz) or rabbit (1/5000 for IGFBP-4, -5, and -6; Bio-Rad Laboratories, Hercules, CA) secondary antibodies. The horseradish peroxidase activity was detected using ECLplus Western blot detection reagents (Amersham Biosciences Ltd., Piscataway, NJ) and the STORM image and analysis system (Amersham). The blots were quantified using ImageQuant software (Amersham). Identification of the individual IGFBPs was ascertained by analyzing the purified IGFBPs (GroPep). Samples from all genotypes, genders, and age groups were always included on each membrane, so that a valid comparison could be made within individual IGFBPs. A quantitative comparison among IGFBPs is likely to be less accurate because of the differing affinities of individual antibodies for their respective antigens (IGFBPs). For IGFBP-3, however, we developed a standard curve by determining the band intensities of a series of known concentrations of recombinant human IGFBP-3 (Gropep). The standards were diluted in the same buffer as the samples, with the addition of 30 μg soluble catfish muscle protein as a carrier and then processed in the same ways as the muscle samples. Pilot studies were carried out to determine the appropriate concentration range for the standards.
IGF-1R immunoprecipitation and Western blot analysis
Crude muscle membrane preparations were prepared from aliquots of the powdered muscles in buffer containing appropriate phosphatase and protease inhibitors as previously described (27). After determining the protein concentration, equivalent amounts of membrane protein were subjected to Western blot analysis to determine the total receptor abundance. To measure the degree of receptor phosphorylation, the membranes were solubilized and immunoprecipitated using 20 μl anti-IGF-1R antibody (SC-713; Santa Cruz) as previously described (28). Twenty microliters of the precipitated proteins were subjected to Western blot analysis and probed with an antiphosphotyrosine antibody (1/500; Upstate Cell Signaling Solutions, Lake Placid, NY) as described above. Blots were rinsed with TBS-T to remove residual ECLplus activity and reprobed with anti-IGF-1R antibody (1/1000; Santa Cruz).
Quantitative real-time PCR
Total tissue RNA (RNAtot) was extracted from skeletal muscle as previously described (29). Briefly, Ultraspec (1.0 ml/50 mg tissue; Biotecx, Inc., Houston, TX) was added to preweighed (50 mg), powdered, frozen tissue and then homogenized. RNAtot was extracted according to the manufacturer’s protocol and resuspended in nuclease-free water and snap frozen in liquid nitrogen and stored at –80 C until further analysis. The concentration of RNAtot was determined using the Ribo Green assay (Molecular Probes, Eugene, OR).
All samples were DNase treated and reverse transcribed as described by Crosier et al. (30). Briefly, aliquots of RNA were thawed on ice, and 4 μl (2 μg RNAtot) were treated with 15 U DNase I (Roche Molecular Biochemicals, Mannheim, Germany) for 20 min at 37 C. The reaction was stopped with 20 mM EDTA. RT was conducted using random hexamers (Life Technologies, Inc., Grand Island, NY) and SuperScript II reverse transcriptase (Life Technologies) using the manufacturer’s recommended conditions. cDNA was then purified with Qiaquick purification columns (QIAGEN, Inc., Valencia, CA).
Primers and probes were developed from mouse GenBank sequences using Primer Express software (Table 1). Each product sequence was analyzed against the mouse genome using the BLAST program (National Center for Biotechnology Information) to ensure no sequence homology. Electrophoresis of PCR products was performed to ensure that a single product was obtained. Standards were isolated from mouse first-strand cDNA and amplified by PCR. The standard was quantified based on absorbance at 260 nm and diluted to obtain linear range curves for each mRNA.
Each real-time PCR contained cDNA (40 ng GAPDH, 100 ng IGFBP-4, and 150 ng IGFBP-3, -5, and -6), 300 nM of the appropriate forward and reverse primers (custom synthesized by Sigma-Genosys, Woodlands, TX), and 100 nM probe (5'-FAM; 3'-TAMRA), 2.5 mM MgCl, 200 μM dNTP mix, and 1.25 U Hotstart Goldstar polymerase using the Eurogentec Core PCR kit (Eurogentec North America, Inc., Philadelphia, PA) in a final volume of 50 μl. PCRs were performed in 96-well plates using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amount of each transcript, as determined by extrapolation of the cycle threshold (number of PCR cycles required for the fluorescent signal to reach a detection threshold) obtained against the appropriate standard curve, was normalized to GAPDH mRNA levels to control for potential variation between RT reactions. A separate PCR assay was performed to determine the amplification of GAPDH. We demonstrated that GAPDH mRNA expression was similar across genotype, age, and gender (data not shown).
Statistical analyses
Data were subjected to ANOVA using the general linear model procedure of MINITAB (version 13.31; MINITAB Inc., State College, PA). Data were evaluated for the effects of genotype, gender, age, and all appropriate interactions. Genotype, gender, and age were contrasted using a protected least significant difference test (31). The experimental unit for all analyses was individual mouse. Values, where appropriate, are expressed as mean ± 1 SEM, and the significance level for all tests was set at P < 0.05.
Results
Muscle weights
Similar to previous results from our laboratory (23), genotype differences in hind-limb muscle weights emerged between 5 and 10 wk of age. Muscles were 20% heavier in SIS2 mice by 10 wk of age (Fig. 1; P < 0.01), and the difference was maintained thereafter. Differences in muscle weights between genders emerged between 3 and 5 wk of age, by which time muscles were significantly heavier in male than in female mice (P < 0.01). The slope of the muscle growth curve differs between genders only between 3 and 5 wk of age when there was no genotype effect (10.0 ± 1.1 mg/d for males vs. 5.4 ± 0.8 for females; P < 0.05). However, there were no differences in muscle growth rates of males and females between 5 and 20 wk of age regardless of genotype (SIS2: male, 1.38 ± 0.12 mg/d; female, 1.20 ± 0.10 mg/d: Wt: male, 0.91 ± 0.09 mg/d; female, 0.94 ± 0.09 mg/d; gender effect, P > 0.35).
IGFBP protein abundance
There were no significant genotype or genotype interactions on any of the skeletal muscle IGFBP protein abundances (P > 0.38). This lack of genotype effect is illustrated in Fig. 2, in which all age and gender IGFBP data for each genotype have been averaged. To evaluate the gender and age effects (which are similar for both genotypes), data for SIS2 and Wt IGFBP protein abundances have been combined. Changes over time among IGFBPs are illustrated in Fig. 3. IGFBP protein abundances are presented relative to the amount present at 3 wk of age, when neither gender nor genotype differences were present (P > 0.21).
Muscle IGFBP-3 protein abundance responded differently in male and female mice (Fig. 3; gender x age, P < 0.001). IGFBP-3 initially decreased (wk 3 to wk 5; P < 0.01) for both genders. There was an additional decrease in males from 5 to 20 wk of age (P < 0.001), whereas in females, levels increased by 40% from 5 to 20 wk of age (P < 0.03). This resulted in 68% higher IGFBP-3 concentrations in females compared with mice of either genotype at 20 wk of age (P < 0.001). Muscle IGFBP-4 and -5 increased with age (Fig. 3; P < 0.001), regardless of gender (P > 0.10). For IGFBP-6, there was a gender x age interaction (Fig. 3; P < 0.05). IGFBP-6 decreased for both males and females from 3 to 5 wk of age; after 5 wk, IGFBP-6 continued to decrease in females (P < 0.04) but remained unchanged in males (P > 0.10). This resulted in a 50% lower skeletal muscle IGFBP-6 protein abundance at 20 wk of age for female compared with male mice (P < 0.01). No gender, genotype, or age differences were observed in serum IGFBP-3 or -5 protein abundances (Fig. 4; P > 0.10).
IGFBP mRNA abundance
Similar to protein abundances, there were no differences in the IGFBP mRNA abundances between genotypes (P > 0.10), and the data have been combined for analysis. Muscle IGFBP-3 mRNA abundance decreased by 56% from 3 to 20 wk of age (Fig. 5; P < 0.001) and was not significantly different between male and female mice. Muscle IGFBP-4 mRNA abundance increased from 3 to 20 wk of age (Fig. 5; P < 0.001) for both males and females. IGFBP-5 mRNA abundance increased (Fig. 5; P < 0.04) in male mice (P < 0.05) but remained constant in females, so that by 20 wk of age the abundance was 49% lower than in male mice (P < 0.01). No gender, genotype, or age differences were observed in skeletal muscle IGFBP-6 mRNA abundance (Fig. 5; P > 0.10).
IGF-1R phosphorylation
No genotype, gender, or age differences were observed in the activation of the IGF-1R after stimulation by either LR3-IGF-I or IGF-I, as measured by tyrosine phosphorylation (Fig. 6; P > 0.35). However, an equimolar dose of LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than native IGF-I at both 5 and 10 wk of age (P < 0.05). Total IGF-1R protein abundance did not differ between genotypes, genders, or ages (P > 0.41). The plasma concentration of the injected hormones did not differ between the IGF-I and LR3-IGF-I groups (hIGF-I, 105 ± 11 nM; LR3-IGF-I, 122 ± 19 nM; P > 0.25).
Discussion
In the current experiment, we reconfirmed that sustained high levels of muscle IGF-I stimulate muscle growth only transiently with the greatest response occurring between 5 and 10 wk of age despite the continued expression of the transgene (23). The difference in mass at 10 wk was sustained to 20 wk of age, and in our previous studies we determined that the difference remains unchanged to 52 wk of age (23, 32). The plasma concentration of human IGF-I measured in the SIS2 mice was only approximately 4% that of the endogenous mouse IGF-I, indicating that the majority of human IGF-I produced remained in the muscle tissue (23, 32). The enhanced rate of muscle mass accretion, therefore, should have been sustained, but clearly, this did not occur. Because large proportions of IGFBPs in the extracellular fluid are unbound, they are available to regulate the autocrine/paracrine effects of IGF-I. We hypothesized, therefore, that if IGF-I stimulated expression of the IGFBPs, these in turn would inhibit the ability of IGF-I to activate its receptor. We postulated, therefore, that muscle IGFBP levels would be similar in Wt and SIS2 mice up to 5 wk of age, during the time when muscle growth rates in SIS2 mice are higher. However, IGFBP levels would be higher in the SIS2 mice from 10 wk when their muscle growth rates had decreased to match those of Wt mice. To test this hypothesis, we compared the expression of both the mRNA and protein of IGFBP-3, -4, -5, and -6 in the skeletal muscles of SIS2 and Wt mice before (3 wk old), during (5 wk old), and after (10 and 20 wk old) the period of accelerated growth. Moreover, to functionally assess the combined effect of the IGFBPs on IGF-I bioactivity, we measured the phosphorylation of the IGF-1R after stimulation by equimolar amounts of IGF-I and LR3-IGF-I.
To date, it is unclear whether IGFBP-3 is expressed by skeletal muscle. IGFBP-3 expression by mouse muscle cells has been documented in some studies, both in vivo (33), and in vitro (34), but this is not a consistent finding (35, 36, 37). Our data demonstrate that IGFBP-3 mRNA is expressed in the skeletal muscle of mice in vivo and that it decreases during development. Thus, mouse skeletal muscle is similar to human (38), rat (39), bovine (40), and porcine skeletal muscle (41) with respect to IGFBP-3 expression. The decrease in IGFBP-3 mRNA with development in vivo has also been reported in the rat masseter muscle (42) and in pig biceps femoris muscle (43). Similar to the mRNA, the local abundance of IGFBP-3 protein initially decreased. The patterns of muscle and serum IGFBP-3 protein expression were distinctly different, which suggests that the muscle values reflect the local production of the protein. Using recombinant IGFBP-3 as standards, we determined the IGFBP-3 concentration at 3 wk of age to be 1.96 ± 0.19 μg/mg soluble muscle protein. The relative band intensity at 3 wk of age of the IGFBP-4 relative to IGFBP-3 was 0.4:1; IGFBP-5 to IGFBP-3 was 0.43:1, and IGFBP-6 to IGFBP-3 was 0.54:1. From this comparison, it appears that at 3 wk of age IGFBP-3 was the most abundant IGFBP in skeletal muscle. This contrasts with the mature muscle in which IGFBP-5 is the predominant IGFBP. With the onset of puberty, IGFBP-3 protein increased in females but continued to decrease in males. The disparate response of mRNA and protein in females after the onset of puberty suggests a role of sex steroids in the posttranscriptional regulation of IGFBP-3 expression or its posttranslational fate. There have been various investigations on the regulation of IGFBP-3 expression by sex steroids, and these demonstrate that the response is highly dependent on the specific tissue and its stage of differentiation (7).
The in vivo significance for muscle growth of the gender-specific differences in muscle IGFBP-3 expression with age is unclear. When IGFBP-3 was ubiquitously overexpressed in mice (44, 45), muscle growth rate decreased (45). This inhibitory effect of IGFBP-3 on muscle growth is supported by the observation that IGFBP-3 suppresses the proliferation of myogenic cells in culture (46, 47). It is tempting, therefore, to propose that the gender-specific differences in IGFBP-3 may contribute to the reduced muscle growth potential of female mice. However, the muscle mass growth curves demonstrate that the largest divergence in growth between males and females occurs between 3 and 5 wk of age and precedes both puberty and the emergence of the differences in IGFBP-3 protein levels.
There was no difference in IGFBP-3 mRNA or protein expression between genotypes, suggesting that in vivo expression of the gene in skeletal muscle is not regulated by local IGF-I. This finding is consistent with the lack of an in vivo response of skeletal muscle IGFBP-3 to IGF-I in the intact rat (39) and pig (48). Again, however, the expression of IGFBP-3 in response to IGF-I varies depending on species and cell type and, for muscle cells, can vary according to their state of differentiation.
The patterns of IGFBP-4 expression were similar for both the mRNA and protein, exhibiting an increase with age that was not influenced by either genotype or gender. The expression in skeletal muscle of rodents has been documented both in various muscle cell lines (31, 37, 49) and in vivo (33, 35, 36). The increase with age is consistent with the observation that in C2C12 cells the expression of IGFBP-4 increases as differentiation proceeds and is highest in differentiated myotubes (35). When added to proliferating muscle cells, Ewton et al. (50) observed that IGFBP-4 completely inhibited the actions of IGF-I and -II, as it does in other tissues. The increase in muscle IGFBP-4 with age that we observed is commensurate with the overall decline in muscle growth rate, but clearly it is not sufficient to explain the differences in the pattern of muscle growth between genders.
For IGFBP-5, there were gender differences at the level of gene transcription, with males increasing continuously, whereas females demonstrated little change with age. In males, the increase in mRNA abundance translated into a parallel increase in IGFBP-5 protein concentration. In contrast to the constant level of mRNA expression, IGFBP-5 protein in muscle from female mice increased with age and was not significantly different from the concentration in males. Thus, relative to muscles in male mice, the rate of translation of the mRNA in female mice increased and/or the rate of clearance of the IGFBP-5 peptide must have decreased with age. The rate of increase in IGFBP-5 protein expression was greatest between 3 and 5 wk of age and then continued more gradually. By 20 wk of age, levels were almost twice those present at 3 wk, and IGFBP-5 was then the most abundant IGFBP expressed in the muscle. Although IGFBP-5 can inhibit or enhance IGF activity, or it can regulate cellular functions independently of IGF (51), the widespread growth retardation demonstrated by transgenic mice that overexpress IGFBP-5 suggests that its predominant effect is inhibitory. Moreover, the skeletal muscles appeared to be the most severely affected tissue (52). On the basis of this observation, it is possible that the progressive increases with age in IGFBP-5 concentration, together with the increase in IGFBP-4 that we observed, may act to reduce skeletal muscle growth as maturity is attained.
There were no significant differences in IGFBP-5 protein expression between SIS2 and Wt mice. Similar results were reported by Musaro et al. (53) for IGFBP-5 mRNA expression in another transgenic mouse model that targeted IGF-I expression to skeletal muscle. These results are in marked contrast with the increased expression of IGFBP-5 that was observed when IGF-I was infused directly into muscle (54) and the responses observed in a variety of cell types when exposed to IGFs in vitro (37, 51).
IGFBP-6 mRNA was not affected by age, gender, or genotype. This is consistent with the findings of Bayol et al. (35) who demonstrated no change in IGFBP-6 expression in either C2C12 cell cultures or in vivo after denervation of the gastrocnemius muscle. However, we identified a gender-specific response in protein abundance. After an initial decrease from 3 to 5 wk in both male and female mice, IGFBP-6 protein abundance continued to decrease in female but not in male mice. Thus, again there appears to be posttranscriptional regulation of IGFBP-6 expression in female mice. The actions of IGFBP-6 on cell growth appear to be inhibitory and likely are a result of its ability to avidly sequester IGF-II, but with little affinity for IGF-I (18).
In the current study, expression of IGFBP-2 mRNA was very low and, compared with the other IGFBPs, required an additional 10 cycles of amplification to amplify the appropriate band. This contrasted with liver mRNA for which the IGFBP-2 mRNA transcript could be measured readily (data not shown). Although IGFBP-2 is expressed in muscles of rats, birds, and farm animal species, our results are consistent with the observation of an absence of IGFBP-2 expression in mouse skeletal muscle when it is measured by Northern analysis (33, 55), even though it is detectable by PCR (36).
Given the range of changes in IGFBP expression and their diversity of functions, it was not possible to predict their combined effect on IGF-I-regulated muscle growth. We attempted to obtain a functional measure of this by comparing the degree of phosphorylation of the IGF-1R after submaximal stimulation with equimolar amounts of either IGF-I or LR3-IGF-I. LR3-IGF-I has negligible affinity for the IGFBPs while retaining its ability to stimulate the IGF-1R (25). Because the extravascular IGFBPs are present in a large molar excess of the IGFs, we hypothesized that the magnitude of the difference in receptor stimulation would provide an index of the degree of sequestration of IGF-I by the IGFBPs. Indeed, the administration of equimolar amounts of LR3-IGF-I induced greater IGF-1R phosphorylation compared with native IGF-I. However, the difference was similar regardless of genotype, gender, or age. Thus, the net combined effect of the IGFBPs at both 5 and 10 wk of age was to decrease signaling through the IGF-1R, and the differences in relative abundances between 5 and 10 wk of age were not sufficient to produce changes in IGF stimulatory capacity over this time.
The absence of differences in IGFBP expression between the SIS2 and Wt mice shows, for the first time, that local IGF-I does not directly regulate the production of the IGFBPs by the skeletal muscle. This does not exclude the possibility that in normal muscles, extrinsic factors that stimulate local IGF expression could promote IGFBP production independently. Our results demonstrate that the IGFBPs normally present in the muscle mitigate activation of the IGF-1R by IGF-I, as measured by the difference in IGF-1R tyrosine phosphorylation after stimulation by LR3-IGF-I vs. IGF-I. We also observed that this difference in receptor phosphorylation was similar for both genotypes despite the differences in IGF present in the muscle. Thus, in the SIS2 mice, the IGFBPs were able still to attenuate IGF-I activity. These data do not support an independent role for the IGFBPs in the temporal pattern of muscle growth in SIS2 mice.
In addition, we demonstrated that the IGFBPs have a sexually dimorphic, age-dependent pattern of expression that can be regulated at the transcriptional, translational, or posttranslational level, regardless of local IGF-I concentrations. Given the increasing interest in the IGF-independent role of the IGFBPs in the etiology of a number of chronic diseases, our data clearly demonstrate the importance of taking into consideration the normal age, gender, and tissue specificity of the IGFBPs in any evaluation of their expression patterns.
Footnotes
This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Institute Grant R01-AR46308 and grants from the United States Department of Agriculture, Agricultural Research Service, under Cooperative Agreement number 58-6250-6001. The contents of this publication do not necessarily reflect the views or policies of the United States Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
First Published Online September 15, 2005
Abbreviations: EDL, Extensor digitorum longus; IGF-1R, type 1 IGF receptor; IGFBP, IGF binding protein; LR3-IGF-I, long R3 IGF-I; RNAtot, total tissue RNA; TA, tibialis anterior; TBS-T, Tris-buffered saline with 0.1% Tween; Wt, wild type.
Accepted for publication September 6, 2005.
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