SEARCH

SEARCH BY CITATION

Keywords:

  • skeletal unloading;
  • insulin-like growth factor-I;
  • osteoblast;
  • proliferation;
  • αVβ3 integrin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We showed that unloading markedly diminished the effects of IGF-I to activate its signaling pathways, and the disintegrin echistatin showed a similar block in osteoprogenitor cells. Furthermore, unloading decreased αVβ3 integrin expression. These results show that skeletal unloading induces resistance to IGF-I by inhibiting activation of the IGF-I signaling pathways at least in part through downregulation of integrin signaling.

Introduction: We have previously reported that skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) with respect to bone formation. However, the underlying mechanism remains unclear. The aim of this study was to clarify how skeletal unloading induces resistance to the effects of IGF-I administration in vivo and in vitro with respect to bone formation.

Materials and Methods: We first determined the response of bone to IGF-I administration in vivo during skeletal unloading. We then evaluated the response of osteoprogenitor cells isolated from unloaded bones to IGF-I treatment in vitro with respect to activation of the IGF-I signaling pathways. Finally we examined the potential role of integrins in mediating the responsiveness of osteoprogenitor cells to IGF-I.

Results: IGF-I administration in vivo significantly increased proliferation of osteoblasts. Unloading markedly decreased proliferation and blocked the ability of IGF-I to increase proliferation. On a cellular level, IGF-I treatment in vitro stimulated the activation of its receptor, Ras, ERK1/2 (p44/42 MAPK), and Akt in cultured osteoprogenitor cells from normally loaded bones, but these effects were markedly diminished in cells from unloaded bones. These results were not caused by altered phosphatase activity or changes in receptor binding to IGF-I. Inhibition of the Ras/MAPK pathway was more impacted by unloading than that of Akt. The disintegrin echistatin (an antagonist of the αVβ3 integrin) blocked the ability of IGF-I to stimulate its receptor phosphorylation and osteoblast proliferation, similar to that seen in cells from unloaded bone. Furthermore, unloading significantly decreased the mRNA levels both of αV and β3 integrin subunits in osteoprogenitor cells.

Conclusion: These results indicate that skeletal unloading induces resistance to IGF-I by inhibiting the activation of IGF-I signaling pathways, at least in part, through downregulation of integrin signaling, resulting in decreased proliferation of osteoblasts and their precursors.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

SKELETAL UNLOADING BY space flight and bed rest induces osteopenia in humans.(1) Hindlimb elevation in rats, an established model of skeletal unloading, causes osteopenia by inhibiting bone formation, osteoblast number, and osteoblast maturation in unloaded bones.(2–6) Osteoprogenitor cells, which are considered as the main source of osteoblasts,(7-9) from unloaded bone are impaired with respect to proliferation and differentiation in vitro.(10-12) Although the phenomenon of decreased bone formation during skeletal unloading is well known, the underlying mechanism remains unclear.

Insulin-like growth factor-I (IGF-I) is required for the proliferation of most cell types.(13, 14) In bone, IGF-I is one of the most abundant growth factors and plays an important role in regulating bone formation.(15–18) IGF-I stimulates osteoblast proliferation and promotes bone formation.(15-18) The IGF-I receptor is composed of two α and two β subunits.(19, 20) IGF-I binding to the receptor results in activation of its intrinsic tyrosine kinase, resulting in phosphorylation of multiple sites within the tail of the β subunit.(19, 20) Activation of the IGF-I receptor activates two distinct pathways: the Ras/MAPK pathway and the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway.(19, 20) Activated Ras/MAPK promotes proliferation.(19, 21) Activated PI3K/Akt pathway also mediates cell proliferation,(22) although it has a critical role in regulating apoptosis.(20, 23, 24) In regulating phosphorylation of receptor tyrosine kinases including the IGF-I receptor, protein tyrosine phosphatases (PTPs) have a critical role.(25-27) Src-homology 2 (SH-2)-containing phosphotyrosine phosphatase-2 (SHP-2) is a nontransmembrane protein tyrosine phosphatase (PTP) that contains two Src-homology 2 domains.(28, 29) Recruitment of SHP-2 to the IGF-I receptor results in receptor dephosphorylation in smooth muscle cells(26) and therefore inactivates the receptor. Integrins are cell surface receptors, which are composed of α and β subunits, and each αβ combination has its own binding specificity and signaling properties.(30) Integrins are necessary for optimal activation of growth factor receptors.(30) Blocking ligand occupancy of αVβ3 integrin inhibits the ability of IGF-I to stimulate its receptor phosphorylation in smooth muscle cells.(31, 32) This is associated with an increase in the rate of SHP-2 recruitment to the IGF-I receptor.(33)

We have shown that skeletal unloading induces resistance to IGF-I with respect to bone formation.(34–36) IGF-I administration in vivo stimulates bone growth and bone formation in the normally loaded bone, but not in unloaded bone.(34, 36) Paradoxically, unloading increases the mRNA and protein levels of IGF-I and the mRNA level of the IGF-I receptor, but not the protein levels of IGF-I binding proteins in rat bones.(34) Furthermore, IGF-I administration in vitro increases cell proliferation of the osteoprogenitor cells isolated from loaded bone, but not that of cells from unloaded bone,(35, 36) despite the fact that cultured osteoprogenitor cells from unloaded bone contain normal mRNA levels of the IGF-I receptor.(36) These observations led us to the question: how does skeletal unloading induce resistance to the effects of IGF-I administration in vivo and in vitro with respect to bone formation?

To answer this question, we first determined the response of bone to IGF-I administration in vivo to osteoblast proliferation during skeletal unloading. To minimize the variable of endogenous growth hormone (GH) production and secretion during exogenous IGF-I administration, we chose the GH-deficient dwarf rat.(36) We then evaluated the response of osteoprogenitor cells isolated from unloaded bones to IGF-I treatment in vitro with respect to activation of the IGF-I signal pathways. Finally we examined the potential role of integrins in mediating the responsiveness of osteoprogenitor cells to IGF-I.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animal protocols

Male GH-deficient dwarf rats (dw-4) at 8 weeks of age were purchased from Harlan UK (Bicester, Oxon, UK), fed standard laboratory rat chow (22/5 Rodent Diet (W); Harlan Teklad, Madison, WI, USA) containing 1.13% calcium and 0.94% phosphorus, and maintained on a 12:12-hours light-dark cycle. In vivo experiments were conducted as follows. The rats at 12 weeks of age were randomly divided into four groups of five or six rats each after a period of equilibration in individual housing. Skeletal unloading was achieved using the hindlimb elevation model as previously described.(2) Two of the groups were hindlimb elevated; the other two groups served as the pair-fed controls. Using mini-osmotic pumps (Alza Corp., Palo Alto, CA, USA) implanted in the back of the neck subcutaneously 1 day before beginning hindlimb elevation, the rats were infused with vehicle (10 mM sodium succinate and 140 mM sodium chloride) or 2.5 mg/kg of body weight per day of recombinant human IGF-I, kindly provided by Chiron Corp. (Emeryville, CA, USA), for the duration of the experiment. To determine 5-bromo-2′deoxyuridine (BrdU) incorporation of the osteoblasts and osteocytes, the rats were infused with 6 mg/day of BrdU (Sigma, St Louis, MO, USA) in 50% DMSO and 50% propylene glycol using another mini-osmotic pump implanted, as described above, during the first 7 days of a 14-day unloading period. To evaluate the response of bone marrow osteoprogenitor (BMOp) cells from unloaded bones to IGF-I, the rats were divided into two groups of six rats normally loaded or hindlimb elevated for 7 days. This in vitro experiment was performed twice. The first time was to assess effects of IGF-I and unloading on the activation of the IGF-I receptor, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt. The second time was to assess effects of IGF-I and unloading on the activation of Ras. On day 7 or 14, all animals were killed by exsanguination from the dorsal aorta while under ketamine anesthesia. The tibias were harvested for the study of proliferation in osteoblasts and osteocytes, and the tibias and femurs were harvested for BMOp cell culture. To ensure that these results were not limited to the dw-4 strain, we also examined BMOp cells from tibias and femurs of male Sprague-Dawley or Fischer 344 rats (Simonsen, Gilroy, CA, USA) at 12 weeks of age. These studies were approved by the Animal Use Committee of the San Francisco Veterans Affairs Medical Center, where the studies were performed.

Determination of BrdU-labeled osteoblasts and osteocytes

The tibias were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h at 4°C. Bones were demineralized in 10% EDTA (pH 7.3). Demineralized bones were bisected longitudinally and embedded in paraffin, and 5-μm-thick sections were placed on positively charged glass slides (Fisher Scientific, Pittsburgh, PA, USA). Immunolocalization of BrdU and detection in sections counterstained with hematoxylin were performed using a Zymed BrdU Staining Kit (Zymed Laboratories, South San Francisco, CA, USA) according to the manufacturer's instructions. Sections from tibias of rats without BrdU labeling were used as negative controls. Cells that have dark brown nuclear staining were counted as BrdU-labeled cells. The measurements of BrdU-labeled osteoblasts and osteocytes were performed in the trabecular bone area of the proximal tibial metaphysis in an area beginning 0.5 mm distal to the growth plate/metaphyseal junction to exclude the primary spongiosa. Osteoblasts were identified as cuboidal cells lining the bone surface. Osteocytes were identified as embedded cells in mineralized bone. The ratio of BrdU-labeled osteoblasts per bone surface [BrdU(+)Ob./BS, N/mm] and the ratio of BrdU-labeled osteocytes per bone volume [BrdU(+)osteocytes/BV, N/mm2] were analyzed on a Macintosh computer using a modification of the NIH Image program (developed at the U.S. NIH).

BMOp cell culture

The tibial and femoral BMOp cells, which predominantly express mRNA marking osteoblast differentiation such as alkaline phosphatase and osteocalcin(12) and show enzyme activity of alkaline phosphatase and mineralization,(12, 35, 36) were harvested using techniques previously described.(36) The marrow was collected in primary culture medium (α-MEM containing L-glutamine and nucleosides; Mediatech, Herndon, VA, USA), supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA, USA), 100 U/ml penicillin/streptomycin (Mediatech), and 0.25 μg/ml fungizone (Life Technologies, Rockville, MD, USA). A single-cell suspension was obtained by repeated passage through an 18-gauge needle. A pool of BMOp cells was made from each rat. The cells were plated at 3.0 × 105 cells/cm2 in 10- or 15-cm dishes. Nonadherent cells were removed by aspiration, and the primary medium was replenished on day 5.

Signal induction of IGF-I signal pathways

To evaluate the effects of IGF-I on the activation of the IGF-I signaling pathways, on day 7, the cultures were rinsed twice with PBS and switched to FBS-free medium to serum starve. After 24 h, the cultures were incubated in the medium with or without 10 ng/ml IGF-I for various periods of time at 37°C to induce signaling. To evaluate effects of Na3VO4, a specific inhibitor for PTP,(37, 38) on the activation of the IGF-I receptor during IGF-I stimulation, the cultures were incubated in the medium with or without 1 mM Na3VO4 30 minutes before IGF-I treatment (10 ng/ml for 5 minutes). To evaluate the effects of echistatin, an antagonist of the αVβ3 integrin,(31, 39) on the activation of the IGF-I receptor during IGF-I stimulation, the cultures were incubated in the medium with 10 ng/ml IGF-I for various periods of time at 37°C after pre-incubation with various doses of echistatin (0, 1, 10, or 100 nM) for 12 h.

Immunoprecipitation and Western blotting

The cultured cells were washed once with PBS that contained 0.1% NaF and Na3VO4 and solubilized in lysis buffer consisting of 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 50 mM HEPES, 0.1% NaF, 0.1% Na3VO4, 150 mM NaCl, 100 μg/ml phenylmethane sulfenyl fluoride (PMSF), and protease inhibitor cocktail (Complete Mini; Roche, Mannheim, Germany). The protein concentration in the cell lysates was measured using a BCA protein assay regent kit (Pierce, Rockford, IL, USA). For the phosphorylated IGF-I receptor assay, equivalent protein samples (300–500 μg) of the cell lysates in each assay were immunoprecipitated using rabbit antibody to the IGF-I receptor β chain (C-20; Santa Cruz Biotechnology). Immunoprecipitated material was solubilized in SDS sample buffer (2% SDS, 10 mM DTT, 10% glycerol, 10 mM Tris-HCl, 0.01% bromphenol blue), boiled at 95°C for 5 minutes, and analyzed by SDS-PAGE (with a 7.5% or 4-15% gel). Separated peptides were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat milk, the PVDF membranes were incubated with mouse antibody to p-Tyr (1:1000 dilution; PY99) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:10,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h to quantify phosphorylated IGF-I receptor. For the immunocomplex assay between the IGF-I receptor and SHP-2, immunoprecipitated material using the rabbit antibody to the IGF-I receptor β chain (C-20) was blotted with mouse monoclonal (1:200 dilution, B-1; Santa Cruz Biotechnology or 1:500 dilution; BD Transduction Laboratories, Lexington, KY, USA) or rabbit polyclonal antibody (1:200 dilution, C-18; Santa Cruz Biotechnology) to SHP-2, followed by incubation with secondary antibodies as described above. The blots were developed using a chemiluminescent substrate (SuperSignal West Dura; Pierce) according to the manufacturer's instructions. To measure the other components of the signaling pathways, equivalent protein samples of the cell lysates in SDS sample buffer were electrophoresed by SDS-PAGE (with a 7.5% or 10% gel) and transferred as described above. After blocking, the blots were incubated with rabbit antibodies to IGF-I receptor β chain (1:1000 dilution, C-20), phospho-p44/42 MAPK (1:1000 dilution, phospho-ERK1/2, The202/Tyr204; Cell Signaling Technology, Beverly, MA, USA), p44/42 MAPK (1:1000 dilution, ERK1/2; Cell Signaling Technology), phospho-Akt (1:1000 dilution, Ser473; Cell Signaling Technology), Akt (1:1000 dilution, Cell Signaling Technology), SHP-2 (1:200 dilution, B-1) or actin, which is a housekeeping protein (1:1000 dilution; Sigma), at 4°C overnight, followed by incubation with HRP-conjugated anti-rabbit IgG (1:10,000 dilution; Santa Cruz Biotechnology) or anti-mouse IgG (1:10,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. The blots were developed as described above. In some experiments, the blots were stripped by incubating for 1 h in stripping buffer (2% SDS, 100 mM 2-mercaptorthanol, 62.5 mM Tris-HCl [pH 6.7]) at room temperature, reprobed, and developed. Quantification of all blots was performed on a Macintosh computer using Multi-Analysit software (Bio-Rad Laboratories, Hercules, CA, USA) or Kodak 1D Image Analysis software v3.5 (Eastman Kodak Co., Rochester, NY, USA).

Activated Ras assay

The determination of activated Ras was performed using a Ras Activation Assay kit (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer's instructions. In brief, cultured cells were washed once with PBS containing 0.1% NaF and Na3VO4 and solubilized in lysis buffer consisting of 1% Igepal CA-630, 1 mM EDTA, 25 mM HEPES, 10 mM MgCl2, 25 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 150 mM NaCl. Proteins (200 μg) from the cell lysate were immunoprecipitated using 10 μg of Raf-1 RBD-agarose, which has a high affinity for only activated Ras.(40, 41) Immunoprecipitated material was solubilized in SDS sample buffer, boiled at 95°C for 5 minutes, analyzed by SDS-PAGE (with a 15% gel), and transferred. The PVDF membranes were incubated with mouse antibody to Ras (1:1000 dilution, clone Ras 10) at 4°C overnight, followed by incubation with HRP-conjugated anti-mouse IgG (1:10,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. To determine total amount of Ras, proteins (12 μg) from the cell lysates in SDS sample buffer were electrophoresed by SDS-PAGE (with a 15% gel), transferred, and incubated with mouse antibody to Ras (1:1000 dilution, clone Ras 10), followed by HRP-conjugated anti-mouse IgG. The blots were developed and quantified as described above.

BrdU incorporation in BMOp cells

To determine the effects of echistatin on proliferation during IGF-I stimulation, the cultures of BMOp cells were established as described above and plated at 1 × 106 cells per well in 6-well plates. The cultures were switched to medium containing 1% FBS on day 7. After 24 h, the cells were rinsed once with PBS and switched to FBS-free medium with or without 10 ng/ml IGF-I and with or without various doses of echistatin (1, 10, or 100 nM) for 24 h at 37°C. During the last 4 h of the treatment, the cultures were labeled with 10 μM BrdU. BrdU incorporation during DNA synthesis was measured by means of a Cell Proliferation ELISA BrdU-Kit (Roche). The absorbance (at 370 nm) of the BrdU incorporation in the culture was normalized to cell number by the absorbance of the crystal violet stain of an additional parallel culture that was treated in the same manner.

Quantitative real-time PCR

To determine the mRNA levels of αV and β3 integrin subunits in BMOp cells, the cultures of BMOp cells were established as described above. Total RNA was harvested from day 7 cultured cells by means of an RNA Stat-60 kit (Tel-Test, Friendswood, TX, USA). For each sample, 2 μg of total RNA was reverse-transcribed in 100 μl of a reaction mixture that contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 7.5 mM MgCl2, 1 mM each dNTP, 5 μM random primers (GIBCO BRL, Rockville, MD, USA), 0.4 U/μl RNase inhibitor (Roche), and 2.5 U/μl Moloney murine leukemia virus reverse transcriptase (M-MLV) (GIBCO BRL) at 25°C for 10 minutes, 48°C for 40 minutes, 95°C for 5 minutes, and 4°C (stored). The sequences of the PCR primers for αV and β3 integrin subunits and 18S rRNA of rats are listed in Table 1. These primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA). Primers were synthesized by the Biomolecular Resource Center (University of California, San Francisco, CA, USA). The fluorescence intensity is directly proportional to the accumulation of PCR product and can be detected with an ABI Prism 7900HT (Applied Biosystems). PCR was carried out in triplicate with 20-μl reaction volumes of 10 μl 2× SYBR Green PCR Master Mix (Applied Biosystems), 500 nM of each primer, and 1 μl cDNA template. The cDNA templates for the measurement of 18S rRNA were diluted at 1:1000 because of the abundance of the gene. The PCR reaction was performed in an ABI Prism 7900HT using the following cycle parameters: 1 cycle of 95°C for 12 minutes, 40 cycles of 95°C for 15 s, and 60°C for 1 minute. After the final cycle of PCR amplification, the dissociation curve analysis was performed at 95°C for 30 s, 60°C for 30 s, and slowly heating the samples at 0.25°C/s to 95°C to confirm no primer dimer in the PCR products.(42, 43) Analysis was carried out using the sequence detection software supplied with the ABI Prism 7900HT. The number of PCR cycles (threshold cycles [Ct]) required for the fluorescent intensities to exceed a threshold just above background was calculated for the test reactions.(44) The Ct values were determined for three test reactions in each sample and averaged. The ΔCt values were obtained by subtracting the 18S rRNA (as endogenous control) Ct values from the target gene Ct values of the same samples. The relative quantification of the target genes was given by 2ΔCt.

Table Table 1.. Primers for Real-Time PCR
Thumbnail image of

IGF-I binding assay

To determine the effects of skeletal unloading and echistatin on IGF-I binding to its receptor, the cultures of BMOp cells were established as described above and plated at 1 × 106 cells per well in 12-well plates. The cultures were rinsed twice with PBS and switched to FBS-free medium on day 7. After 12 h, the cultures were incubated in FBS-free medium with or without echistatin (100 nM) for an additional 12 h and exposed to 25 pM125I-IGF-I (specific activity > 74TBq/mmol; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and various doses (0, 0.01, 0.1, 1, 10, or 100 nM) of unlabeled IGF-I for 10 minutes at 37°C. At the end of incubation, cultures were put on ice, rinsed three times with ice-cold PBS, and solubilized in 0.5 M NaOH. The receptor-bound125I-IGF-I activity was counted in a γ-counter.

Statistical analysis

Data are presented as mean ± SD. All data were analyzed using two-factor ANOVA followed by a posthoc Fisher's protected least significant difference (PLSD) test with a StatView 5.0 program (SAS Institute, Cary, NC, USA). Student's t-test was also used for analyzing the effects of IGF-I treatment in vitro on BMOp cells. Statistical significance was stated for p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BrdU incorporation of osteoblasts and osteocytes in vivo

Skeletal unloading induces resistance to IGF-I administration in vivo with respect to bone formation.(34, 36) We first determined the response of bone to IGF-I administration in vivo with respect to BrdU incorporation of osteoblasts and osteocytes during skeletal unloading. BrdU incorporation of the cells was evaluated in the trabecular bone of proximal tibias. We found that IGF-I infusion for 14 days significantly increased BrdU incorporation of osteoblasts and osteocytes (205% and 210% of control, respectively) in the proximal tibias of normally loaded rats (Figs. 1A and 1B). Unloading markedly decreased BrdU incorporation of osteoblasts and osteocytes (17% and 17% of control, respectively) in the proximal tibias in the vehicle-treated rats and blocked the ability of IGF-I to increase the BrdU incorporation (19% and 23% of control, respectively).

thumbnail image

Figure FIG. 1.. Effect of skeletal unloading and IGF-I administration in vivo on BrdU incorporation of osteoblasts and osteocytes. The rats were either unloaded (hindlimb elevated) or normally loaded for 14 days and infused with IGF-I (2.5 mg/kg of body weight) or vehicle during this interval. BrdU was infused during the first 7 days. BrdU incorporation of the osteoblasts and osteocytes was evaluated in the trabecular bone of proximal tibias. (A) The ratios of BrdU-labeled osteoblasts per bone surface [BrdU(+)Ob./BS, N/mm] were evaluated. (B) The ratios of BrdU-labeled osteocytes per bone volume [BrdU(+)osteocytes/BV, N/mm2] were evaluated. Data are means ± SD. Interaction of treatment and loading effects was significant in BrdU(+)Ob./BS or BrdU(+)osteocytes/BV (p < 0.005). *Significantly greater than the other groups (p < 0.0005). #Significantly less than loaded rats (p < 0.005).

Download figure to PowerPoint

These results indicate that skeletal unloading induces resistance to IGF-I administration in vivo with respect to bone formation caused by inhibition of IGF-I-stimulated proliferation of osteoblasts. To determine the mechanism, we examined bone cells in vitro.

Phosphorylation and protein levels of IGF-I receptor in BMOp cells

IGF-I treatment in vitro significantly increased cell proliferation of BMOp cells isolated from normally loaded bones but not that of cells from unloaded bones.(35, 36) To determine whether skeletal unloading alters activation (phosphorylation) and/or protein levels of the IGF-I receptor, we first evaluated the effects of various time points and doses of IGF-I to activate the IGF-I receptor in cultured BMOp cells. We found that 10 ng/ml IGF-I for 5 minutes stimulated phosphorylation of the IGF-I receptor (data not shown). Phosphorylation was only modestly increased by higher (100 ng/ml) concentrations of IGF-I, with little change after 15 minutes. Using these conditions (10 ng/ml IGF-I for 5 minutes), we examined phosphorylation and protein levels of the IGF-I receptor in the BMOp cells isolated from normally loaded or unloaded bones. We found that IGF-I stimulated the phosphorylation of the IGF-I receptor in the BMOp cells from loaded bones, but this effect was markedly diminished in the BMOp cells from unloaded bones (Fig. 2). Unloading did not significantly alter the protein levels of the IGF-I receptor.

thumbnail image

Figure FIG. 2.. Skeletal unloading blocks the ability of IGF-I to stimulate phosphorylation of the IGF-I receptor. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded for 7 days. The BMOp cells were cultured for 7 days, serum deprived for 24 h, and exposed to 10 ng IGF-I/ml or vehicle for 5 minutes. The IGF-I receptor in the lysate was immunoprecipitated with an anti IGF-I receptor antibody, and the phosphorylated form was detected on Western analysis with an anti p-Tyr antibody. Total levels of IGF-I receptor in the lysate (40 μg protein) were determined by Western analysis with an anti-IGF-I receptor antibody. Relative signal intensities of the ratio of phosphorylated to total IGF-I receptor were evaluated. Data are means ± SD of three independent experiments. #Significantly less than cultures of BMOp cells from normally loaded bones (p < 0.05).

Download figure to PowerPoint

Phosphorylation and protein levels of ERK1/2 and Akt in BMOp cells

We next evaluated phosphorylation and protein levels of ERK1/2, a component of the Ras/MAPK pathway, and Akt, a component of the PI3K/Akt pathway, in cultured BMOp cells isolated from loaded or unloaded bones. We found that IGF-I stimulated the phosphorylation of ERK1/2 and Akt in the BMOp cells from loaded bones, but these effects were significantly diminished in BMOp cells from unloaded bones (Fig. 3A). Furthermore, unloading significantly decreased the protein levels of ERK1/2 and Akt (49% and 19% of control, respectively). However, unlike the results for ERK1/2, when normalized to protein levels of Akt, there were no differences in the ratio of phosphorylated to unphosphorylated Akt after IGF-I administration in BMOp cells from loaded or unloaded bones (Fig. 3B). These results indicate that skeletal unloading inhibits IGF-I activation of the Ras/MAPK pathway more than the PI3K/Akt pathway, although both are affected by skeletal unloading. Furthermore, these results are consistent with the in vivo results showing that resistance to IGF-I is seen with respect to osteoblast proliferation (Fig. 1).

thumbnail image

Figure FIG. 3.. Effect of skeletal unloading and IGF-I on phosphorylation and protein levels of ERK1/2 and Akt. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded. The BMOp cells were cultured for 7 days, serum deprived for 24 h, and exposed to 10 ng IGF-I/ml or vehicle for 5 minutes. Phosphorylated and total levels of ERK1/2 (44/42 kDa) in the lysate (10 μg protein) or Akt (60 kDa) in the lysate (15 μg protein) were determined by Western analysis using specific antibodies for the phosphorylated and total forms of ERK1/2 and Akt. (A) Representative Western blot analyses of phosphorylated and total levels of ERK1/2 and Akt, and actin (42 kDa, loading control) levels are shown. (B) Relative signal intensities of the ratio of phosphorylated to total ERK1/2 and Akt were evaluated. Data are means ± SD of three independent experiments. #Significantly less than cultures of BMOp cells from normally loaded bones (p < 0.005).

Download figure to PowerPoint

Activation and protein levels of Ras in BMOp cells

We then examined the activation and protein levels of Ras, which is the first step of the Ras/MAPK pathway,(19, 20) in the BMOp cells isolated from normally loaded or unloaded bones. We found that IGF-I significantly increased activation of Ras in BMOp cells from loaded bones (Fig. 4). Unloading significantly decreased the basal levels of activated Ras in BMOp cells and blocked the ability of IGF-I to increase activated Ras. Unloading did not significantly alter the protein levels of Ras. These results indicate that Ras activation by IGF-I is also impacted by skeletal unloading.

thumbnail image

Figure FIG. 4.. Skeletal unloading decreases Ras activation and blocks the stimulation of Ras activation by IGF-I. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded. The BMOp cells were cultured for 7 days, serum deprived for 24 h, and exposed to 10 ng IGF-I/ml or vehicle for 5 minutes. The activated Ras (21 kDa) was immunoprecipitated with an anti-Raf-1 RBD antibody and detected on Western analysis with an anti-Ras antibody. Total levels of Ras in the lysate were determined by Western analysis with an anti-Ras antibody. Relative signal intensities of the ratio of activated to total Ras were evaluated. Data are means ± SD of three independent experiments. Interaction of treatment and loading effects was significant in the relative signal intensities of activated and total Ras (p < 0.0001). *Significantly greater than other groups (p < 0.0001). #Significantly less than cultures of BMOp cells from normally loaded bones (p < 0.005).

Download figure to PowerPoint

Association of PTPs with phosphorylation of the IGF-I receptor

As shown in the above results, skeletal unloading inhibited the activation of IGF-I signaling pathways beginning with autophosphorylation of the IGF-I receptor. PTPs have a critical role in regulating phosphorylation of receptor tyrosine kinases, including the IGF-I receptor in other tissues.(25–27) To determine whether PTPs significantly affect the activation of the IGF-I receptor in a manner altered by unloading, we first evaluated the association of SHP-2, which is one of the PTPs known to regulate the phosphorylation of the IGF-I receptor in other cell types.(26) In this experiment, we found that phosphorylation of the IGF-I receptor after IGF-I treatment reached a maximum at 5 minutes and slowly decreased (Fig. 5). Unloading decreased the intensity of phosphorylation at each time point without altering the time course of phosphorylation. SHP-2 levels were equivalent in cells from normally loaded and unloaded bone, but recruitment of SHP-2 to the IGF-I receptor was not detected at any time during this time course study (data not shown). Because SHP-2 may not be the only PTP that regulates IGF-I receptor phosphorylation, we next evaluated the effects of Na3VO4, a specific inhibitor for all PTPs, on the activation of the IGF-I receptor in BMOp cells from normally loaded or unloaded bones. We found that Na3VO4 did not significantly alter the phosphorylation levels of the IGF-I receptor in BMOp cells from either loaded or unloaded bones (Fig. 6). These results would seem to exclude the possibility that the failure of IGF-I to activate its receptor in cells from unloaded bone is caused by increased PTP activity.

thumbnail image

Figure FIG. 5.. Time course of the ability of IGF-I to stimulate phosphorylation of the IGF-I receptor. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded. The BMOp cells were cultured for 7 days, serum deprived for 24 h, and exposed to 10 ng IGF-I/ml or vehicle for various periods of time as indicated. The IGF-I receptor in the lysate was immunoprecipitated with an anti IGF-I receptor antibody, and the phosphorylated form was detected on Western analysis with an anti-p-Tyr antibody. After stripping, the total levels of the IGF-I receptor were determined by Western analysis with an anti-IGF-I receptor antibody in the same blot. The ratio of phosphorylated to total IGF-I receptor was determined using the densitometric assessment of the Western blots.

Download figure to PowerPoint

thumbnail image

Figure FIG. 6.. Effect of Na3VO4 on the ability of IGF-I to stimulate phosphorylation of the IGF-I receptor. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded for 7 days. The 7-day cultures were rinsed and incubated in FBS-free medium for 24 h. The cultures were switched to FBS-free medium with or without 1 mM Na3VO4 30 minutes before IGF-I treatment (10 ng/ml for 5 minutes). The IGF-I receptor in the lysate was immunoprecipitated with an anti-IGF-I receptor antibody, and the phosphorylated form was detected on Western analysis with an anti-p-Tyr antibody. After stripping, the total levels of the IGF-I receptor were determined by Western analysis with an anti-IGF-I receptor antibody in the same blot. The ratio of phosphorylated to total IGF-I receptor in the relative signal intensities was evaluated.

Download figure to PowerPoint

Effects of echistatin on phosphorylation of the IGF-I receptor in BMOp cells

The disintegrin echistatin inhibits ligand occupancy of the αVβ3 integrin and reduces IGF-I-stimulated migration, DNA synthesis, and receptor autophosphorylation in smooth muscle cells.(31, 39) The αVβ3 integrin may play an important role in IGF-I receptor phosphorylation; a role that might be altered by skeletal unloading. To test this possibility, we first examined the effects of echistatin, an antagonist of the αVβ3 integrin, on phosphorylation and protein levels of the IGF-I receptor in BMOp cells. We found that echistatin blocked the ability of IGF-I to stimulate phosphorylation of the IGF-I receptor in a dose-dependent fashion (Fig. 7A). Echistatin did not significantly alter the protein levels of IGF-I receptor. SHP-2 recruitment to the IGF-I receptor was not detected in this process (data not shown) in contrast to what has been reported in muscle.(33) Furthermore, in a time course study, phosphorylation inhibited by echistatin (100 nM) was comparable with the inhibition by unloading (Figs. 5 and 7B).

thumbnail image

Figure FIG. 7.. Echistatin blocks the ability of IGF-I to stimulate phosphorylation of the IGF-I receptor. The cultures were switched to FBS-free medium on day 7. After 12 h, the cultures were incubated in FBS-free medium with various doses of echistatin (0, 1, 10, or 100 nM) for an additional 12 h and exposed to 10 ng/ml IGF-I for 5 minutes. The IGF-I receptor in the lysate was immunoprecipitated with an anti-IGF-I receptor antibody, and the phosphorylated form was detected on Western analysis with an anti-p-Tyr antibody. After stripping, total IGF-I receptor was determined by Western analysis using the appropriate antibody. (A) The ratio of phosphorylated to total IGF-I receptor in the relative signal intensities was evaluated. Using the same protocol, the ability of echistatin (100 nM) to block IGF-I stimulation of its receptor phosphorylation as a function of time was evaluated. (B) The ratio of phosphorylated to total IGF-I receptor in the relative signal intensities was evaluated.

Download figure to PowerPoint

Effects of echistatin on BrdU incorporation in BMOp cells

We next examined the effects of echistatin on BrdU incorporation in BMOp cells. We found that echistatin (10 and 100 nM) blocked the ability of IGF-I to stimulate BrdU incorporation in BMOp cells (Fig. 8). Echistatin did not alter the basal levels of BrdU incorporation in BMOp cells. These results indicate that the αVβ3 integrin is involved in the ability of IGF-I to stimulate phosphorylation of its receptor and proliferation in BMOp cells.

thumbnail image

Figure FIG. 8.. Echistatin blocks the ability of IGF-I to stimulate BrdU incorporation in BMOp cells. The cultures were switched to medium containing 1% FBS on day 7. After 24 h, the medium was replaced by FBS-free medium with or without 10 ng/ml IGF-I and with various doses of echistatin (0, 1, 10, or 100 nM) for 24 h. During the last 4 h of the treatment, the cultures were labeled with 10 μM BrdU. Data are means ± SD of triplicate determinations. These values are expressed as a percentage of the control (BMOp cells isolated from normally loaded bones and treated with vehicle). Interaction of IGF-I and echistatin effects was significant in BrdU incorporation (p < 0.01). *Significantly greater than comparable vehicle cultures (p < 0.05).

Download figure to PowerPoint

mRNA levels of αV and β3 integrin subunits in BMOp cells

To determine whether skeletal unloading alters the expression of αVβ3 integrin, we examined the mRNA levels of αV and β3 integrin subunits in BMOp cells isolated from normally loaded or unloaded bones. We found that unloading significantly decreased the mRNA levels of αV and β3 integrin subunits in BMOp cells (17% and 35% of the control level, respectively; Fig. 9).

thumbnail image

Figure FIG. 9.. Skeletal unloading decreases the mRNA levels of αV and β3 integrin subunits in BMOp cells. The BMOp cells were isolated from the tibias and femora of rats that were normally loaded or unloaded. Total RNA was harvested from day 7 cultured cells. Data are means ± SD of four independent BMOp cell pools. These values are expressed as a percentage of the control group (normally loaded rats). #Significantly less than the control group (p < 0.005).

Download figure to PowerPoint

Effects of skeletal unloading and echistatin on IGF-I binding to its receptor

Conceivably, skeletal unloading or echistatin could affect the access of IGF-I to its receptor or alter the affinity of binding of IGF-I to its receptor. Therefore, we evaluated competition between125I-IGF-I and unlabeled IGF-I for binding to its receptor with or without echistatin (100 nM) in BMOp cells isolated from normally loaded or unloaded bones. We found that neither unloading nor echistatin reduced IGF-I binding or the affinity of binding to the IGF-I receptor (Fig. 10)

thumbnail image

Figure FIG. 10.. Effect of skeletal unloading and echistatin on competition between125I-IGF-I and unlabeled IGF-I for binding to its receptor. The BMOp cells were isolated from the tibias and femora of rats that were unloaded or normally loaded for 7 days. The cultures were switched to FBS-free medium on day 7. After 12 h, the cultures were incubated in FBS-free medium with or without echistatin (100 nM) for an additional 12 h and exposed to 25 pM125I-IGF-I and various doses of unlabeled IGF-I for 10 minutes. Data are means in triplicate. Error bars are not shown to simplify presentation, but in all cases, enclosed an SD of less than 10% of the mean. These values are expressed as a percentage of maximal specific125I-IGF-I binding.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Our results in rats indicate that skeletal unloading induces resistance to IGF-I by inhibiting the activation of the IGF-I signaling pathways, leading to the decreased proliferation of osteoblasts and their progenitors.

IGF-I stimulates osteoblast proliferation in cultures of fetal rat calvariae.(15–17) Skeletal unloading decreases preosteoblast proliferation in proximal tibias of rats.(45) In this study, IGF-I administration in vivo increased BrdU incorporation of osteoblasts and osteocytes in the proximal tibias of the normally loaded rats, but unloading markedly decreased BrdU incorporation of cells in the vehicle-treated rats and blocked the ability of IGF-I to increase BrdU incorporation. In the control animals, IGF-I administration for 14 days increased not only the BrdU incorporation into osteoblasts but also into osteocytes, indicating that 14 days is sufficient for proliferating precursors to differentiate first into osteoblasts and then into osteocytes. Of more relevance to this study, the failure of IGF-I to stimulate proliferation in the unloaded bones supports our previous studies in which IGF-I administration in vivo stimulates bone formation in normally loaded bone but not in unloaded bone.(34, 36)

On the cellular level, our data demonstrated that skeletal unloading inhibited the activation of IGF-I signaling pathways. The failure of IGF-I to activate these pathways in cells derived from unloaded bone started with autophosphorylation of the IGF-I receptor, with no significant effect on the protein levels of the receptor. Subsequently, the activation of Ras and the phosphorylation of ERK1/2 and Akt, stimulated by IGF-I, were inhibited in unloaded BMOp cells. However, when normalized to the protein levels, unloading had more profound effects on Ras and ERK1/2 activation than on Akt activation. Ras proto-oncogene proteins are essential components of the Ras/MAPK pathway that controls cell proliferation.(46–49) Ras functions as a critical linker between phosphorylated receptor tyrosine kinases including the IGF-I receptor and the Ras/MAPK cascade downstream consisting of Ras, Raf, MAPK/ERK kinase (MEK), and ERK1/2.(50) These cellular results are consistent with our observations in vivo that IGF-I stimulates cell proliferation of the osteoblastic cells in normally loaded bones but not in unloaded bones.

PTPs have a critical role in regulating phosphorylation of receptor tyrosine kinases, including the IGF-I receptor.(25–27) The primary role of the majority of PTPs is to inhibit signaling, as one might expect based on the stimulatory role of most receptor tyrosine kinases.(25) In this study, skeletal unloading inhibits the activation of IGF-I signaling pathways by a mechanism interfering with IGF-I receptor autophosphorylation. One mechanism found in other systems involves changes in PTP activity, in particular, SHP-2.(26, 33) Our results indicate that this mechanism does not explain the resistance to IGF-I in unloaded bone. Na3VO4, a specific inhibitor of PTPs, did not significantly alter the IGF-I receptor phosphorylation by IGF-I treatment in BMOp cells from either loaded or unloaded bones. Nor could we show a difference in time course of phosphorylation, or more specifically, a recruitment of SHP-2 to the IGF-I receptor in cells from unloaded bone.

Another potential mechanism explaining our results involves integrin signaling. Integrins are cell surface receptors to extracellular matrix components. Several integrin heterodimers including αVβ3 are expressed on the cell surface in osteoblasts.(51, 52) Integrins activate various protein tyrosine kinases such as focal adhesion kinase (FAK) and Src-family kinases that activate Ras/MAPK cascades.(30) Integrins not only signal on their own but are also necessary for optimal activation of growth factor receptors.(30) Occupancy of the αVβ3 integrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor in smooth muscle cells.(31, 32) Our present data demonstrated that echistatin, which blocks occupancy of the αVβ3 integrin,(39) mimicked the results induced by unloading in inhibiting the ability of IGF-I to stimulate phosphorylation of its receptor and proliferation in BMOp cells. Furthermore, unloading significantly reduced the mRNA expression of αV and β3 integrin subunits in BMOp cells. Neither unloading nor echistatin altered IGF-I binding to its receptor. This result indicates that the IGF-I receptor phosphorylation inhibited by unloading or echistatin is because of a postreceptor mechanism, but not to failure of IGF-I binding. These results support the possibility that αVβ3 integrin plays an important role in enhancing the activation of IGF-I signaling pathways, and the resistance to IGF-I induced by unloading can be attributed to a reduction in this integrin/IGF-I receptor interaction. How this interaction takes places is the subject for ongoing investigation.

In conclusion, our data show that skeletal unloading induces resistance to IGF-I by inhibiting the activation of IGF-I signal pathways through downregulation of αVβ3 integrin, resulting in decreased proliferation of osteoblasts and their precursors. Further investigation of these mechanisms should lead to future treatments for osteopenia induced by skeletal unloading.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Scott J Munson for technical assistance. These studies were supported by National Institutes of Health Grant RO1 DK54793 and National Aeronautics and Space Administration Grant NAG 2–1371.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Bikle DD, Halloran BP 1999 The response of bone to unloading. J Bone Miner Metab 17:233244.
  • 2
    Globus RK, Bikle DD, Morey-Holton E 1986 The temporal response of bone to unloading. Endocrinology 118:733742.
  • 3
    Halloran BP, Bikle DD, Harris J, Foskett HC, Morey-Holton E 1993 Skeletal unloading decreases production of 1,25-dihydroxyvitamin D. Am J Physiol 264:E712E716.
  • 4
    Halloran BP, Bikle DD, Harris J, Tanner S, Curren T, Morey-Holton E 1997 Regional responsiveness of the tibia to intermittent administration of parathyroid hormone as affected by skeletal unloading. J Bone Miner Res 12:10681074.
  • 5
    Matsumoto T, Nakayama K, Kodama Y, Fuse H, Nakamura T, Fukumoto S 1998 Effect of mechanical unloading and reloading on periosteal bone formation and gene expression in tail-suspended rapidly growing rats. Bone 22:89S93S.
  • 6
    Dehority W, Halloran BP, Bikle DD, Curren T, Kostenuik PJ, Wronski TJ, Shen Y, Rabkin B, Bouraoui A, Morey-Holton E 1999 Bone and hormonal changes induced by skeletal unloading in the mature male rat. Am J Physiol 276:E62E69.
  • 7
    Friedenstein AJ, Chailakhyan RK, Gerasimov UV 1987 Bone marrow osteogenic stem cells: In vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20:263272.
  • 8
    Owen ME, Cave J, Joyner CJ 1987 Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci 87:731738.
  • 9
    Prockop DJ 1997 Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:7174.
  • 10
    Machwate M, Zerath E, Holy X, Hott M, Modrowski D, Malouvier A, Marie PJ 1993 Skeletal unloading in rat decreases proliferation of rat bone and marrow-derived osteoblastic cells. Am J Physiol 264:E790E799.
  • 11
    Zhang R, Supowit SC, Klein GL, Lu Z, Christensen MD, Lozano R, Simmons DJ 1995 Rat tail suspension reduces messenger RNA level for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells. J Bone Miner Res 10:415423.
  • 12
    Kostenuik PJ, Halloran BP, Morey-Holton ER, Bikle DD 1997 Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells. Am J Physiol 273:E1133E1139.
  • 13
    Van Wyk JJ, Smith EP 1999 Insulin-like growth factors and skeletal growth: Possibilities for therapeutic interventions. J Clin Endocrinol Metab 84:43494354.
  • 14
    LeRoith D 2000 Insulin-like growth factor I receptor signaling-overlapping or redundant pathways? Endocrinology 141:12871288.
  • 15
    Canalis E 1980 Effect of insulinlike growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 66:709719.
  • 16
    Hock JM, Centrella M, Canalis E 1988 Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology 122:254260.
  • 17
    Canalis E, McCarthy T, Centrella M 1988 Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae. Endocrinology 122:2227.
  • 18
    Wergedal JE, Mohan S, Lundy M, Baylink DJ 1990 Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells. J Bone Miner Res 5:179186.
  • 19
    LeRoith D, Werner H, Beitner-Johnson D, Roberts CT Jr 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143163.
  • 20
    LeRoith D, Bondy C, Yakar S, Liu JL, Butler A 2001 The somatomedin hypothesis: 2001. Endocr Rev 22:5374.
  • 21
    Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH 2001 Mitogen-activated protein (MAP) kinase pathways: Regulation and physiological functions. Endocr Rev 22:153183.
  • 22
    Borgatti P, Martelli AM, Bellacosa A, Casto R, Massari L, Capitani S, Neri LM 2000 Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors. FEBS Lett 477:2732.
  • 23
    Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME 1997 Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275:661665.
  • 24
    Franke TF, Kaplan DR, Cantley LC 1997 PI3K: Downstream AKTion blocks apoptosis. Cell 88:435437.
  • 25
    Chernoff J 1999 Protein tyrosine phosphatases as negative regulators of mitogenic signaling. J Cell Physiol 180:173181.
  • 26
    Maile LA, Clemmons DR 2002 Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2. J Biol Chem 277:89558960.
  • 27
    Buckley DA, Cheng A, Kiely PA, Tremblay ML, O'Connor R 2002 Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts. Mol Cell Biol 22:19982010.
  • 28
    Freeman RM Jr, Plutzky J, Neel BG 1992 Identification of a human src homology 2-containing protein-tyrosine-phosphatase: A putative homolog of Drosophila corkscrew. Proc Natl Acad Sci USA 89:1123911243.
  • 29
    Ahmad S, Banville D, Zhao Z, Fischer EH, Shen SH 1993 A widely expressed human protein-tyrosine phosphatase containing src homology 2 domains. Proc Natl Acad Sci USA 90:219721201.
  • 30
    Giancotti FG, Ruoslahti E 1999 Integrin signaling. Science 285:10281032.
  • 31
    Zheng B, Clemmons DR 1998 Blocking ligand occupancy of the alphaVbeta3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells. Proc Natl Acad Sci USA 95:1121711222.
  • 32
    Clemmons DR, Horvitz G, Engleman W, Nichols T, Moralez A, Nickols GA 1999 Synthetic alphaVbeta3 antagonists inhibit insulin-like growth factor-I-stimulated smooth muscle cell migration and replication. Endocrinology 140:46164621.
  • 33
    Maile LA, Clemmons DR 2002 The alphaVbeta3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the Src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor. Endocrinology 143:42594264.
  • 34
    Bikle DD, Harris J, Halloran BP, Morey-Holton ER 1994 Skeletal unloading induces resistance to insulin-like growth factor I. J Bone Miner Res 9:17891796.
  • 35
    Kostenuik PJ, Harris J, Halloran BP, Turner RT, Morey-Holton ER, Bikle DD 1999 Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I. J Bone Miner Res 14:2131.
  • 36
    Sakata T, Halloran BP, ElAlieh HZ, Munson SJ, Rudner L, Venton L, Ginzinger D, Rosen CJ, Bikle DD 2003 Skeletal unloading induces resistance to insulin-like growth factor-I on bone formation. Bone 32:669680.
  • 37
    Swarup G, Speeg KV Jr, Cohen S, Garbers DL 1982 Phosphotyrosyl-protein phosphatase of TCRC-2 cells. J Biol Chem 257:72987301.
  • 38
    Kawase T, Orikasa M, Ogata S, Burns DM 1995 Protein tyrosine phosphorylation induced by epidermal growth factor and insulin-like growth factor-I in a rat clonal dental pulp-cell line. Arch Oral Biol 40:921929.
  • 39
    Jones JI, Prevette T, Gockerman A, Clemmons DR 1996 Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor. Proc Natl Acad Sci USA 93:24822487.
  • 40
    Taylor SJ, Shalloway D 1996 Cell cycle-dependent activation of Ras. Curr Biol 6:16211627.
  • 41
    Foschi M, Chari S, Dunn MJ, Sorokin A 1997 Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16:64396451.
  • 42
    Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, Moorman AF 2002 Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307:6369.
  • 43
    Alfonso J, Pollevick GD, Castensson A, Jazin E, Frasch AC 2002 Analysis of gene expression in the rat hippocampus using Real Time PCR reveals high inter-individual variation in mRNA expression levels. J Neurosci Res 67:225234.
  • 44
    Ginzinger DG 2002 Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Exp Hematol 30:503512.
  • 45
    Barou O, Palle S, Vico L, Alexandre C, Lafage-Proust MH 1998 Hindlimb unloading in rat decreases preosteoblast proliferation assessed in vivo with BrdU incorporation. Am J Physiol 274:E108E114.
  • 46
    Joneson T, Bar-Sagi D 1997 Ras effectors and their role in mitogenesis and oncogenesis. J Mol Med 75:587593.
  • 47
    Martin JL, Baxter RC 1999 Oncogenic ras causes resistance to the growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3) in breast cancer cells. J Biol Chem 274:1640716411.
  • 48
    Miao D, Tong XK, Chan GK, Panda D, McPherson PS, Goltzman D 2001 Parathyroid hormone-related peptide stimulates osteogenic cell proliferation through protein kinase C activation of the Ras/mitogen-activated protein kinase signaling pathway. J Biol Chem 276:3220432213.
  • 49
    Meadows KN, Bryant P, Pumiglia K 2001 Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation. J Biol Chem 276:4928949298.
  • 50
    Herrmann C, Nassar N 1996 Ras and its effectors. Prog Biophys Mol Biol 66:141.
  • 51
    Hughes DE, Salter DM, Dedhar S, Simpson R 1993 Integrin expression in human bone. J Bone Miner Res 8:527533.
  • 52
    Gronthos S, Stewart K, Graves SE, Hay S, Simmons PJ 1997 Integrin expression and function on human osteoblast-like cells. J Bone Miner Res 12:11891197.