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Endocrinology, doi:10.1210/en.2007-1349
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Endocrinology Vol. 149, No. 3 1302-1313
Copyright © 2008 by The Endocrine Society

Abnormalities of Insulin-Like Growth Factor-I Signaling and Impaired Cell Proliferation in Osteoblasts from Subjects with Osteoporosis

Sebastio Perrini, Annalisa Natalicchio, Luigi Laviola, Angelo Cignarelli, Mariangela Melchiorre, Francesca De Stefano, Cristina Caccioppoli, Anna Leonardini, Sabina Martemucci, Gaetana Belsanti, Sara Miccoli, Anna Ciampolillo, Ada Corrado, Francesco Paolo Cantatore, Riccardo Giorgino and Francesco Giorgino

Department of Emergency and Organ Transplantation (S.P., A.N., L.L., A.Cig., M.M., F.D.S., C.C., A.L., S.Ma., G.B., S.Mi., A.Cia., R.G., F.G.), Section of Internal Medicine, Endocrinology, and Metabolic Diseases, University of Bari School of Medicine, I-70124 Bari, Italy; and Department of Rheumatology (A.Co., F.P.C.), University of Foggia School of Medicine, 71100 Foggia, Italy

Address all correspondence and requests for reprints to: Francesco Giorgino, M.D., Ph.D., Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology, and Metabolic Diseases, University of Bari, Piazza Giulio Cesare, 11, I-70124 Bari, Italy. E-mail: f.giorgino{at}endo.uniba.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE REMODELING REQUIRES continually balanced rates of bone formation by osteoblasts and resorption by osteoclasts (1). These two metabolic processes are closely coupled and are regulated by multiple systemic and local factors, including GH, PTH, estrogen, insulin, and IGF-I (1). An imbalance between osteoclast and osteoblast activities arising from hormonal changes or perturbations in inflammatory cytokines and growth factors can result in skeletal abnormalities characterized by a net loss (osteoporosis) or gain (osteopetrosis) of bone mass (2, 3, 4).

Insulin and IGF-I initiate cellular responses by binding to distinct cell-surface receptors containing a tyrosine kinase intrinsic to the receptor intracellular portion that regulate a variety of signaling pathways controlling metabolism, growth, and survival (5, 6). The major substrates of both insulin and IGF-I receptor tyrosine kinases are known to be closely related high-molecular-weight proteins, the insulin receptor substrate (IRS)-1 and IRS-2, which become rapidly phosphorylated on multiple tyrosine residues after receptor engagement by the specific ligand (7). In bone cells, IRS-1 and IRS-2 are both expressed (8). Even though IRS-1 and IRS-2 display substantial amino acid similarity, they possess significant structural differences that are associated with distinct expression levels and signaling properties in the various cell types (9). Consistent with this concept, it has recently been proposed that IRS-2 is needed to maintain the predominance of bone formation over bone resorption, whereas IRS-1 maintains bone turnover, and that the integration of these two signals mediates a potent anabolic response to IGF-I in the bone (10, 11). The IRS proteins are a family of docking molecules that connect insulin and IGF-I receptors to downstream kinase cascades, involving phosphatidylinositol 3-kinase and Erk-1/2, which mediate the pleiotropic effects of insulin, IGF-I, and other cytokines on cellular functions (12, 13, 14). Erk-1 and Erk-2, which belong to the family of MAPKs, are protein serine/threonine kinases involved in the control of cell proliferation. The role of Erk in IGF-I mitogenic signaling in normal and transformed bone cells has been also investigated (15, 16). These studies show that IGF-I-stimulated cell proliferation is dependent on sustained activation of Erk-1 and Erk-2, whereas other MAPK family members, such as c-Jun N-terminal kinase and p38, seem to be less involved (16, 17).

Individuals with insulin deficiency, as exemplified by type 1 diabetic patients, are susceptible to develop osteoporosis (18). Patients with Laron’s syndrome caused by IGF-I deficiency are also prone to exhibit this condition (19). A reduction in IGF-I levels is implicated as an important factor in the etiology of evolutional osteoporosis, especially age-related bone loss (20, 21, 22). Interestingly, whereas it has been shown that recombinant human IGF-I administration increases osteoblast function in healthy females in whom IGF-I levels and bone turnover were decreased by short-term caloric deprivation (23), recombinant human IGF-I was found to be ineffective in increasing bone mineral density in women with postmenopausal osteoporosis (24). Whether abnormalities in IGF-I signaling may explain altered IGF-I action on human osteoblasts under specific conditions of net bone loss has not been determined.

The objective of this study was to investigate IGF-I action and signaling in primary cultures of human osteoblasts and to analyze cells from osteoporotic and control donors comparatively. Here we show for the first time that IGF-I-induced activation of the IGF-I receptor, IRS proteins, and Erk occurs differently in osteoblasts from subjects with osteoporosis, compared with controls. Unlike control cells, osteoporotic osteoblasts show increased basal phosphorylation of IRS-2 and Erk-1/2, which is minimally augmented by IGF-I stimulation. Furthermore, desensitization of the IRS-2/Erk signaling pathway in osteoporotic cells is linked to reduced rates of DNA synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Primary osteoblastic cell cultures of normal human bone (OB/C) were obtained from small specimens of trabecular bone of subjects undergoing surgery for traumatic fractures of the femoral neck (three women and two men; aged 55.8 ± 17.6 yr).

Osteoporotic osteoblasts (OB/OP) were isolated from bone specimens derived from patients with a hip fracture secondary to osteoporotic disease undergoing prosthetic hip replacement (eight women; aged 76.0 ± 5.6 yr; P = 0.067 vs. OB/C). All cultures were established within 6–12 h from the occurrence of fractures. Donors were considered to be affected by osteoporosis when their bone mineral density was less than 2.5 SD below the mean for young adults and in addition had suffered hip fracture. Bone mineral density was measured using dual-energy x-ray absorptiometry. In addition, the inclusion into the osteoporosis group was based on the macroscopic analysis of the femoral head at surgery (25). The donors selected for the study had no metabolic or systemic diseases, and they had taken no drugs affecting bone metabolism, such as corticosteroids, bisphosphonates, or estrogens. All investigations were conducted in accordance with the guidelines in the Declaration of Helsinki and were formally approved by the institutional review committee. Informed consent was obtained from all subjects.

Cell culture
Human osteoblasts were obtained as previously described (26). Briefly, the soft connective tissue and the periosteal and corticoendosteal bone were removed, and the trabecular bone was minced into small fragments. The bone explants were cultured in MEM supplemented with glutamine (292 mg/liter), antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), and 10% fetal calf serum and were incubated in a humidified (95%) atmosphere with 5% CO2 at 37 C. The cells were subcultured using 0.003% actinase E (Kaken Pharmacological Co., Tokyo, Japan) in PBS. When confluence was obtained, cells were subcultured using 0.5 g/liter trypsin in calcium- and magnesium-free PBS and were plated at the density required for the experimental procedures. All assays were carried out on second- or third-passage cell cultures. The cells in culture were confirmed to be osteoblasts by analyzing their morphology, ability to produce alkaline phosphatase and osteocalcin, mineralization, and expression of functional PTH receptors, as described below. The osteoblastic cultures from each individual subject were analyzed on multiple occasions by repeating experiments one or more times, as stated in the figure legends, with consistent findings.

Alkaline phosphatase and osteocalcin assays
Cells at first passage were grown to confluence and then treated with 10 nmol/liter 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] for 48 h, as described previously (26), or left untreated. For determination of alkaline phosphatase (ALP), the cell monolayer was solubilized with 0.1% (vol/vol) Triton X-100. ALP activity was determined by colorimetry using p-nitrophenylphosphate as a substrate (Metra Bap EIA kit; Quidel Corp., San Diego, CA; within-assay variability 5.2%; between-assay variability 5%) and reading the OD at 405 nm. The protein content of each well was determined by the Bradford method (Bio-Rad protein assay, Bio-Rad Laboratories, Hercules, CA). ALP activity was expressed as production of nanomoles of p-nitrophenylphosphate per microgram of cell protein per minute. In parallel, the cell medium was removed and stored at –80 C until assayed for osteocalcin. Osteocalcin release into the culture medium was measured using an intact human osteocalcin ELISA (Biomedical Technologies Inc., Stoughton, MA; within-assay variability 7%; between-assay variability 10.5%). Osteocalcin secretion rates are expressed as nanogram per microgram of cell protein per 48 h.

Homo sapiens PTH receptor (hPTH-R)-1 mRNA expression
Total RNA was isolated from control and osteoporotic osteoblasts using RNeasy minikit (QIAGEN, Hilden, Germany) and analyzed by quantitative real-time PCR using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA). First-strand cDNA was synthesized from 1 µg of total RNA with QuantiTect reverse transcription kit (QIAGEN). Real-time PCR was performed in a 25-µl reaction volume containing 25 ng cDNA, 300 nM of each primer, and Power SYBR Green PCR master mix (Applied Biosystems). The primers for real-time PCR are as follows: hPTH-R1, forward, 5'-CGTGAACGGGAGGTGTTTG-3' and reverse, 5'-CAGTGCAGCCGCCTAAAGTAG-3'; 18S, forward, 5'-CGAACGTCTGCCCTATCAACTT-3' and reverse, 5'-ACCCGTGGTCACCATGGTA-3'. The thermocycler parameters were 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. The mRNA level of hPTH-R1 gene was normalized using 18S as internal control.

cAMP generation
Control and osteoporotic osteoblasts were plated in 6-well plates and allowed to grow until 80–90% confluence. After 30 min of preincubation in serum-free medium containing 1 mM 3-isobutyl-1-methylxanthine, cells were incubated for an additional 15 min in the presence or absence of 200 ng/ml human PTH (1–34) (Sigma-Aldrich, St. Louis, MO) at 37 C. At the end of the incubation, the medium was rapidly removed, the plates were immediately frozen in liquid nitrogen for 1 min, and the frozen cells were subsequently lysed directly into 0.1 M HCl. Total cellular cAMP in the acidic extracts was assessed using a commercial ELISA kit (Cayman Chemical, Ann Arbor, MI) (27). cAMP production was expressed as log (picomoles per milliliter) per microgram of cell protein.

Mineralization assay
To promote mineralization in vitro, OB/C and OB/OP cells were seeded into 12-well plates and cultured in standard medium (as above) supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich) and 0.1 µM [SCAP];l-ascorbic acid-2-phosphate (Sigma-Aldrich). The osteogenic induction medium was renewed every second day for 3 wk, and the degree of mineralization was determined using von Kossa and Alizarin Red staining methods, respectively (28). Both staining methods were used for each culture of control or osteoporotic osteoblasts, in triplicate. For the von Kossa method, cells were fixed with 3% paraformaldehyde for 10 min, stained with AgNO3, rinsed with distilled water, exposed for 1 h to bright light, and finally observed. For the Alizarin Red method, cells were fixed with ice-cold 70% (vol/vol) ethanol for 1 h and then stained with 40 mmol/liter Alizarin Red in demineralized water (adjusted to pH 4.2) for 10 min at room temperature. After visual identification of the Alizarin Red-positive nodules in each well under light microscopy, the cells were destained with 10% cetylpyridinium chloride in 10 mmol/liter sodium phosphate (pH 7.0). The extracted stain was transferred to a 96-well plate, and the absorbance at 570 nm was measured using a microplate spectrophotometer (28).

Measurement of IGF-I secretion
Osteoblasts from control and osteoporotic donors were starved for 16 h in serum-free MEM containing 0.2% BSA. IGF-I was evaluated in the culture medium by immunoradiometric analysis using a specific assay kit (DRG Instruments GmbH, Marbung, Germany; within assay variability 3.0%; between assay variability 1.5%) and expressed as nanogram per milliliter of culture medium per microgram of cell protein.

Immunoprecipitation and immunoblotting
For time-course studies, osteoblasts were grown in serum-free MEM for 16 h and then incubated in the absence or presence of 100 nmol/liter IGF-I for the indicated times at 37 C. At the end of incubation, the cells were rapidly washed with Ca2+/Mg2+-free PBS and then mechanically detached in ice-cold lysis buffer containing 50 mmol/liter HEPES (pH 7.5), 150 mmol/liter NaCl, 1 mmol/liter MgCl2, 1 mmol/liter CaCl2, 10% glycerol, 10 mmol/liter sodium pyrophosphate, 10 mmol/liter sodium fluoride, 2 mmol/liter EDTA, 2 mmol/liter phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 mmol/liter sodium orthovanadate, and 1% Nonidet P-40. After incubation for 45 min at 4 C, the preparation was centrifuged at 12,000 x g for 10 min at 4 C. The resulting supernatant was assayed for determination of protein concentration using the Bradford method and subjected overnight to immunoprecipitation at 4 C with antibodies against the IGF-I receptor, IRS-1, or IRS-2, as indicated. The resulting immune complexes were adsorbed to protein A-Sepharose beads for 2 h at 4 C, and the pelleted beads were washed three times in lysis buffer and then incubated in Laemmli buffer for 5 min at 100 C. Protein samples were resolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, as appropriate, directly or after immunoprecipitation, and subjected to immunoblotting with the appropriate antibodies, as described previously (29). The proteins were visualized by enhanced chemiluminescence using horseradish peroxidase-labeled antirabbit or antimouse IgG (Amersham Biosciences, Piscataway, NJ), and quantified by densitometric analysis using Quantity One image analysis software (Bio-Rad Laboratories).

Antibodies and specialized reagents
Polyclonal anti-IGF-I receptor β-subunit, anti-glyceraldehyde-3-phosphate dehydrogenase, and monoclonal antiphosphotyrosine antibodies (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-Akt, antiphospho-Akt (Thr308), antiphospho-Akt (Ser473), antiphospho-p42/44 MAPK (Erk-1/2; Thr202/Tyr204), and antiphospho-glycogen synthase kinase (GSK)-3{alpha}/β (Ser21/Ser9) were obtained from New England Biolabs (Beverly, MA). Polyclonal antibodies against IRS-1, IRS-2, or GSK-3 were purchased from Upstate Millipore (Chicago, IL). Anti-MAPK (Erk-1/2) antibodies were obtained from Zymed Laboratories (San Francisco, CA). Polyclonal anti-p38 and antiphospho-p38 (Thr180/Tyr182) were obtained from Cell Signaling Technology (Danvers, MA). The MAPK kinase (MEK) inhibitor PD098059 was purchased from Calbiochem (La Jolla, CA). SDS-polyacrylamide gels were obtained from Bio-Rad. Unless otherwise specified, all chemical reagents were purchased from Sigma-Aldrich. The tissue culture media were obtained from Invitrogen (Carlsbad, CA).

DNA synthesis
Osteoblasts were plated at an identical initial density in 6-well plates, and their proliferative capacity was measured by determining the incorporation of [3H]thymidine into cellular DNA, as previously described (30). For each condition, experiments were carried out in triplicate. For inhibition of MEK, PD098059 was added to serum-free medium to a final concentration of 20 µmol/liter 20 min before addition of IGF-I.

Statistical analyses
For the evaluation of changes in the phosphorylation levels of the proteins assessed, one-way ANOVA was used to examine statistical significance, and Fisher’s post hoc tests were used to identify the time points responsible for the statistical significance revealed by ANOVA tests. Comparisons between OB/C and OB/OP sample groups were performed with the use of the unpaired Student’s t test. All data are expressed as mean ± SE. P < 0.05 was considered to represent statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of osteoblastic cultures
Cultures of primary human osteoblasts were obtained from specimens of trabecular bone of normal controls and subjects with osteoporosis, as described in Materials and Methods. To characterize their osteoblastic phenotype, cells were studied under basal conditions and after stimulation with 10 nmol/liter 1,25(OH)2D3. As shown in Table 1Go, basal ALP activity was not significantly different in OB/C and OB/OP donors, even though OB/OP showed a tendency to have lower levels (P = 0.075; Table 1Go). In addition, 1,25(OH)2D3 treatment resulted in similar increases in ALP activity in OB/C and OB/OP (191 and 196% of basal, respectively; P = 0.001 and P < 0.001 vs. basal in OB/C and OB/OP, respectively; Table 1Go). In line with these results, cellular osteocalcin production in the basal state was similar in OB/C and OB/OP (P = 0.365), and it was similarly increased in response to 1,25(OH)2D3 in the two cell groups (177 and 174% of basal, respectively; P < 0.001 and P = 0.005 vs. basal in OB/C and OB/OP, respectively; Table 1Go). Furthermore, mRNA expression of hPTH-R1 and PTH-induced cAMP production were measured in the bone cells under investigation. hPTH-R1 was readily amplified and expressed at similar levels in control and osteoporotic osteoblasts (P = 0.81), whereas it was almost undetectable in the unrelated human hepatocellular liver carcinoma cell line (HepG2) (P < 0.001 vs. both OB/C and OB/OP) (Fig. 1AGo). In addition, cellular cAMP content in the basal state was similar in OB/C and OB/OP (P = 0.38), and it was similarly increased after exposure to hPTH in both cell populations (180 and 186% of basal, respectively; P < 0.005 and P = 0.01 vs. basal in OB/C and OB/OP, respectively; P = 0.21, OB/C vs. OB/OP; Fig. 1BGo). Finally, the osteoblast differentiation potential was assessed by evaluating the mineralization of the cell cultures in the presence of osteogenic induction medium. No mineralization was evident in cells grown in control medium. By contrast, after 21 d of exposure to the osteogenic induction medium, both OB/C and OB/OP showed marked and diffuse mineralization, as assessed by two independent staining methods (Fig. 1Go, C and D). A severalfold increase in the extent of mineralization was also demonstrated by spectrophotometric reading (P = 0.001 vs. cells grown in control medium). No significant differences in mineralization were observed between OB/C and OB/OP (P = 0.82; Fig. 1Go, C–E).


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  		TABLE 1. Cellular ALP and secretion of osteocalcin and IGF-I by human osteoblasts from OB/C and OB/OP donors

 

Figure 1
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  		FIG. 1. Assessment of the osteoblastic cell phenotype. A, hPTH-R1gene expression in human control and osteoporotic osteoblasts. hPTH-R1 mRNA levels were measured by quantitative real-time RT-PCR in control osteoblasts (OB/N, n = 5), OB/OP (n = 8), and HepG2 cells as negative control. mRNA levels were normalized to 18S ribosomal RNA. *, P < 0.05 vs. HepG2 cells. B, Cellular cAMP production in response to (1–34) hPTH. Cells were incubated with 200 ng/ml hPTH for 15 min or left untreated. Results are expressed as mean ± SE for OB/C (n = 5) and OB/OP (n = 8), assessed in triplicate (*, P < 0.05 vs. basal). C–E, Mineralization properties of bone-derived cells. OB/C and OB/OP osteoblasts were grown to confluence and treated with or without 10 mmol/liter β-glycerophosphate and 0.1 µmol/liter L-ascorbic acid-2-phosphate [osteogenic induction medium, (OM)] for 21 d. Cells were then stained by the Alizarin red (C) or von Kossa (D) methods, as described in Materials and Methods. Arabic numbers indicate cell cultures from independent control and osteoporotic subjects, respectively. E, Cell cultures stained with Alizarin red were destained, and the extent of mineralization was quantified by spectrophotometric reading at 570 nm (*, P < 0.05, compared with cells grown in control medium). Results are expressed as mean ± SE for OB/C (n = 5) and OB/OP (n = 8), assessed in triplicate. F, Akt and Erk-1/2 phosphorylation. Immunoblotting with antibodies against the phosphorylated forms of Akt and Erk-1/2 was performed as described in Materials and Methods. The top immunoblot shows phosphorylation of Akt on Ser473, the middle immunoblot phosphory-lation of Erk-1/2 on Thr202/Tyr204, and the bottom immunoblot protein content of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control in OB/C from two independent subjects (subject 1, 44 yr old, male; subject 2, 66 yr old, female). G, IGF-I-induced Akt phosphorylation on Ser473 in young (filled circles) and old (open circles) OB/C. Data are expressed as mean ± SE of young OB/C (n = 2) and old OB/C (n = 3), analyzed in triplicate. *, P < 0.05 vs. respective basal.

 
As noted above, IGF-I plays an important role in osteogenesis by promoting anabolic responses in osteoblastic cells. To assess the feasibility of studying the IGF-I signal transduction system in the human osteoblastic cell cultures, OB/C were incubated in the presence of various concentrations of IGF-I for 10 min, and the phosphorylation levels of Akt and Erk, two proteins implicated in the metabolic, growth, and survival signals induced by this growth factor (16), were measured by immunoblotting. Cells challenged for 10 min with different IGF-I concentrations clearly showed a dose-dependent stimulation of the phosphorylation levels of both Akt and Erk-1/2 (Fig. 1FGo), which continued to increase up to 100 nmol/liter of IGF-I (P < 0.05 for both kinases; Fig. 1FGo). To assess whether aging could affect IGF-I signaling responses, Akt phosphorylation was compared in control subjects, respectively, younger or older than 60 yr (38.0 ± 1.4 vs. 68.0 ± 1.2 yr; P < 0.05). Both basal and IGF-I-induced phosphorylation of Akt on Ser473 were similar in osteoblasts from younger and older subjects at all time points analyzed (P = 0.94) (Fig. 1GGo). Similar results were obtained for phosphorylation of Akt on Thr308, GSK-3{alpha} and -β, and Erk-1 and -2 (see supplemental Table 1 and Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). Thus, IGF-I signaling responses were not affected by the difference in age of the subjects investigated.

IGF-I receptor and IRS proteins
The proximal steps in IGF-I signaling, i.e. tyrosine phosphorylation of IGF-I receptors and IRS proteins, were analyzed next in OB/C and OB/OP (Fig. 2Go and Figure 2Go, SD, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). As shown in Fig. 2AGo, basal tyrosine phosphorylation of the IGF-I receptor was increased approximately 2-fold in OB/OP, compared with OB/C (P = 0.032). The levels of IGF-I receptor protein, determined by immunoblotting of the immunoprecipitates with anti-IGF-I receptor antibodies, were slightly and not significantly increased in OB/OP, compared with OB/C (P = 0.205; Fig. 2AGo). Therefore, when normalized per amount of IGF-I receptor protein, basal tyrosine phosphorylation of the IGF-I receptor remained significantly increased in OB/OP, compared with OB/C (180 ± 17% of OB/C, P = 0.025). This suggests that higher levels of receptor tyrosine phosphorylation in OB/OP were not caused by increased receptor protein. In addition, whereas exposure to IGF-I produced a marked 6-fold increase in IGF-I receptor tyrosine phosphorylation in OB/C (P = 0.001 vs. basal), this response was stimulated only 1.7-fold in OB/OP (P = 0.034 vs. basal), and it was reduced, compared with OB/C (P = 0.009 vs. OB/C; Fig. 2AGo).


Figure 2
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  		FIG. 2. IGF-I receptor, IRS-1, and IRS-2 protein content and tyrosine phosphorylation in osteoblasts from control (OB/C) and osteoporotic (OB/OP) subjects. OB/C (black bars/filled circles) and OB/OP (white bars/empty squares) were studied in the basal state and after IGF-I stimulation for 2 min. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to the IGF-I receptor, IRS-1 or IRS-2 followed by immunoblotting (IB) with antibodies to the IGF-I receptor, IRS-1, or IRS-2, or phosphotyrosine (PY), as indicated. In each panel, representative immunoblots (left panel) and the quantitation of results from all experimental subjects (right panel) are shown. The top blots and left graphs show tyrosine phosphorylation, and the bottom blots and right graphs show total content of each protein. A, Tyrosine phosphorylation and protein content of IGF-I receptors (IGF-I R). *, P < 0.05 vs. respective basal; #, P < 0.05 vs. basal OB/C; §, P < 0.05 vs. IGF-I OB/C. B, Tyrosine phosphorylation and protein content of IRS-1. *, P < 0.05 vs. respective basal; §, P < 0.05 vs. IGF-I OB/C. C, Tyrosine phosphorylation and protein content of IRS-2. *, P < 0.05 vs. respective basal; #, P < 0.05 vs. basal OB/C; {dagger} and §, P < 0.05 vs. IGF-I OB/C. Results are expressed as mean ± SE for OB/C (n = 5) and OB/OP (n = 5), assessed in duplicate. For the evaluation of total protein content, values of basal and IGF-I-stimulated samples were pooled.

 
To determine whether increased IGF-I receptor phosphorylation in OB/OP could be due to higher production and release of IGF-I by these compared with control cells, IGF-I levels were measured in the culture medium from the experimental osteoblasts. As shown in Table 1Go, IGF-I concentrations were similar in OB/C and OB/OP (P = 0.95).

IRS-1 and IRS-2 represent the immediate substrates for the IGF-I receptor tyrosine kinase and act as scaffold molecules to mediate downstream IGF-I signaling (7). In OB/C, the levels of IRS-1 and IRS-2 tyrosine phosphorylation were low in the basal state and were increased 6.0- and 3.2-fold, respectively, upon IGF-I stimulation (P = 0.002 and P = 0.001 vs. basal for IRS-1 and IRS-2, respectively; Fig. 2Go, B and C; and supplemental Fig. 2). In OB/OP, basal tyrosine phosphorylation of IRS-1 was low, and it was significantly increased by IGF-I (P = 0.002 vs. basal), although to a lesser extent when compared with OB/C (P = 0.024 vs. OB/C; Fig. 2BGo). In contrast to IRS-1, basal tyrosine phosphorylation of IRS-2 was increased approximately 5-fold in OB/OP, compared with OB/C (P < 0.001; Fig. 2CGo). It is of note that the mean levels of basal IRS-2 tyrosine phosphorylation in OB/OP were even higher than those in IGF-I-stimulated OB/C (P = 0.040; Fig. 2CGo). In addition, exposure of OB/OP to IGF-I did not result in a further increase in IRS-2 tyrosine phosphorylation (P = 0.190 vs. respective basal). The observed changes in tyrosine phosphorylation of IRS proteins could not be explained by different IRS-1 or IRS-2 protein levels, which were similar in OB/C and OB/OP (P = 0.81 and P = 0.91 for IRS-1 and IRS-2, respectively; Fig. 2Go, B and C). Therefore, osteoblasts from patients with osteoporosis display constitutively increased IRS-2 tyrosine phosphorylation, which is not responsive to IGF-I stimulation, and impaired IGF-I-induced tyrosine phosphorylation of IRS-1.

Akt and GSK-3
The Akt and GSK-3 serine/threonine-kinases act downstream of IRS proteins and are sequentially activated by IGF-I. Because coordinate phosphorylation of Akt on Thr308 and Ser473 is required for full activation of the enzyme (31), the levels of Akt phosphorylation on each amino acid residue were determined by immunoblotting with phospho-specific anti-Akt antibodies. Under basal conditions, phosphorylation of Akt on Thr308 was not significantly different in OB/C and OB/OP (P = 0.16; Fig. 3AGo). In OB/C, stimulation with IGF-I resulted in a significant and time-dependent increase in Akt phosphorylation on Thr308 (P = 0.001 vs. basal). IGF-I rapidly stimulated this response 20-fold after 2 min, with a subsequent decrease at 30 min (Fig. 3Go, A and B). In OB/OP, IGF-I-stimulated phosphorylation of Akt on Thr308 was apparently increased at all time points, although significantly only at 2 and 5 min (P = 0.035 vs. basal) and less than in OB/C (P = 0.025 vs. OB/OP at 30 min; Fig. 3Go, A and B). In the absence of IGF-I, basal Akt phosphorylation on Ser473 was not different in OB/C and OB/OP (P = 0.30; Fig. 3AGo). In OB/C, IGF-I rapidly stimulated Akt phosphorylation on Ser473 5- to 7-fold at 2–5 min, and thereafter the phosphorylation of the kinase remained significantly elevated up to 30 min (P < 0.001 vs. basal). In OB/OP, the levels of Akt phosphorylation on Ser473 were rapidly and markedly stimulated on exposure to IGF-I (P = 0.004 vs. basal). However, 30 min after IGF-I stimulation the level of Akt phosphorylation on Ser473 was reduced in OB/OP, compared with OB/C (P = 0.012 vs. OB/C; Fig. 3Go, A and B). The total amount of Akt protein was similar in the two groups of cells examined (P = 0.963; Fig. 3Go, A and C).


Figure 3
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  		FIG. 3. IGF-I stimulation of Akt phosphorylation in control (OB/C) and osteoporotic (OB/OP) osteoblastic cultures. OB/C (black bars/filled circles) and OB/OP (white bars/empty squares) were studied in the absence or presence of 100 nmol/liter IGF-I for the indicated times. Akt protein content and phosphorylation on Thr308 and Ser473, respectively, were evaluated by immunoblotting with specific antibodies, as described in Materials and Methods. Representative immunoblots (A) and the quantitation of multiple experiments (B) are shown. In both OB/C and OB/OP, levels of phosphorylated Akt were normalized to respective basal. C, Total Akt protein levels in the experimental cells (values of basal and IGF-I-stimulated samples were normalized to the mean value of control subjects and pooled). Data are expressed as mean ± SE of OB/C (n = 5) and OB/OP (n = 5), analyzed in duplicate. *, P < 0.05 vs. respective basal; #, P < 0.05 vs. IGF-I OB/C.

 
GSK-3 is directly phosphorylated by Akt on Ser21 ({alpha}-isoform) and Ser9 (β-isoform) and is inactivated after its phosphorylation (31). To assess whether the observed temporal profiles of Akt activation in OB/C and OB/OP were associated with changes in Akt activity, the phosphorylation of the endogenous Akt substrate GSK-3 was next examined by immunoblotting with phospho-specific antibodies. In the absence of IGF-I, GSK-3{alpha} phosphorylation was similar in OB/C and OB/OP (P = 0.20; Fig. 4AGo). With IGF-I stimulation, the levels of GSK-3{alpha} phosphorylation on Ser21 were rapidly increased 10-fold with a 10-min peak, and this response was significantly maintained up to 30 min in OB/C (P = 0.002 vs. basal; Fig. 4Go, A and B). In contrast, GSK-3{alpha} phosphorylation was modestly and not significantly increased by IGF-I in OB/OP (P = 0.093 vs. basal; Fig. 4Go, A and B), and this response was reduced, compared with OB/C (P = 0.001 vs. OB/C; Fig. 4Go, A and B). Basal GSK-3β phosphorylation on Ser9 was also not significantly different in OB/C and OB/OP (P = 0.46; Fig. 4AGo). Phosphorylation of GSK-3β was significantly increased by IGF-I in both groups of cells (P = 0.001 and P = 0.001 vs. respective basal; Fig. 4Go, A and B). However, in OB/OP the levels of GSK-3β phosphorylation after IGF-I stimulation were significantly lower at all time points, compared with OB/C (P < 0.005; Fig. 4Go, A and B). Total levels of GSK-3{alpha} and GSK-3β proteins were similar in OB/C and OB/OP (P = 0.684 and P = 0.396, respectively; Fig. 5CGo).


Figure 4
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  		FIG. 4. IGF-I stimulation of GSK-3 phosphorylation in control (OB/C, black bars/filled circles) and osteoporotic (OB/OP, white bars/empty squares) osteoblasts. GSK-3{alpha}/β phosphorylation and protein content were evaluated by immunoblotting with antibodies specific to total or phosphorylated (Ser21/Ser9) forms of GSK-3{alpha}/β, as described in Materials and Methods. A, Representative immunoblots of GSK-3{alpha}/β phosphorylation (top panels) and protein content (bottom panels). B, Quantitation of GSK-3{alpha} and GSK-3β phosphorylation in multiple experiments. Levels of both phosphorylated GSK isoforms were normalized to respective basal. C, Quantitation of total GSK-3{alpha} and GSK-3β protein content (values of basal and IGF-I-stimulated samples were normalized to the mean value of control subjects and pooled). Data are expressed as mean ± SE of OB/C (n = 5) and OB/OP (n = 5), analyzed in duplicate. *, P < 0.05 vs. basal; #, P < 0.05 vs. IGF-I OB/C.

 

Figure 5
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  		FIG. 5. Activation of Erk in control osteoblasts (OB/C) and osteoblasts from subjects with osteoporosis (OB/OP). OB/C (black bars/filled circles) and OB/OP (white bars/empty squares) were studied in the absence or presence of 100 nmol/liter IGF-I for the indicated times. Protein lysates were subjected to immunoblotting with antibodies specific to total or phosphorylated forms of Erk-1 and Erk-2. A, Representative immunoblots of Erk-1/2 phosphorylation (top panels) and protein content (bottom panels) in OB/C and OB/OP. Levels of both phosphorylated Erk isoforms were normalized to respective basal. B, Quantitation of the phosphorylation levels of each isoform in the basal state (left) and after stimulation with IGF-I (right). C, Quantitation of total Erk-1/2 protein content (values of basal and IGF-I-stimulated samples were normalized to the mean value of control subjects and pooled together). Data are expressed as mean ± SE of five experiments. *, P < 0.05 vs. basal; #, P < 0.05 vs. OB/C.

 
Erk-1/2
Activation of Erk-1/2 by IGF-I plays an essential role in the proliferation and expression of bone-specific proteins of human osteoblasts (15, 16). Erk-1/2 phosphorylation in the basal state was found to be increased approximately 5-fold in OB/OP, compared with OB/C (P = 0.002 and P < 0.001 for Erk-1 and Erk-2, respectively; Fig. 5Go, A and B). In OB/C, IGF-I stimulation resulted in rapid and prominent activation of both Erk-1 and Erk-2 proteins (P < 0.001 vs. basal; Fig. 5Go, A and B) with peak levels occurring 10 min after IGF-I stimulation. However, in comparison with OB/C, phosphorylation of Erk-1/2 exhibited markedly lower stimulation by IGF-I in OB/OP because it was augmented only 1.5-fold, compared with the 5-fold increase seen in OB/C (P = 0.002 vs. OB/C at the 10 min time point; Fig. 5Go, A and B). No differences in total protein levels of Erk-1 and Erk-2 were observed in the two groups of experimental cells (P = 0.436 and P = 0.480 for Erk-1 and Erk-2, respectively; Fig. 5CGo), demonstrating that the increased basal phosphorylation of Erk in osteoporosis was not due to elevated Erk protein levels.

Effect of IGF-I on osteoblast proliferation
Because Erk-1/2 activation was found to be altered in OB/OP, the effects of IGF-I on DNA synthesis in the experimental cells were investigated. In OB/C, IGF-I stimulation for 15 h resulted in a 2.2-fold increase in DNA synthesis, measured by determining the rates of [3H]thymidine incorporation into DNA (P < 0.001 vs. basal; Fig. 6AGo). By contrast, DNA synthesis was reduced in the basal state in OB/OP (P = 0.005 vs. OB/C), and IGF-I stimulated this response 1.8-fold (P = 0.007 vs. basal) in these cells, significantly less than in OB/C (P = 0.003; Fig. 6AGo). To assess whether exaggerated activation of Erk in the basal state could potentially contribute to reduced cell proliferation in OB/OP, measurements of [3H]thymidine incorporation were carried out in the presence of PD098059, a specific inhibitor of MEK, the Erk-activating kinase (30). The MEK inhibitor reduced the levels of basal Erk-1/2 phosphorylation in OB/OP to those observed in OB/C (P = 0.12 vs. OB/C; Fig. 6BGo), and this resulted in a significant increase in basal DNA synthesis (P = 0.001 vs. untreated OB/OP; Fig. 6AGo). However, when OB/OP were challenged with IGF-I after pretreatment with PD098059, no increase in Erk-1/2 phosphorylation was observed (Fig. 6BGo), and DNA synthesis was minimally stimulated (P = 0.065 vs. basal; Fig. 6AGo). In OB/C, the MEK inhibitor did not affect basal Erk phosphorylation and DNA synthesis, but fully abrogated IGF-I stimulation of both of these responses (Fig. 6Go, A and B), in line with previous studies (16). Altogether these results establish a link between constitutive and dysregulated activation of Erk-1/2 and impaired DNA synthesis in OB/OP.


Figure 6
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  		FIG. 6. Basal and IGF-I-induced DNA synthesis in control (OB/C) and osteoporotic (OB/OP) osteoblastic cell cultures and effects of Erk inhibition. A, Basal (white bars) and IGF-I-stimulated (black bars) [3H]thymidine incorporation into DNA in OB/C and OB/OP. Studies were carried out in the absence or presence of 20 µmol/liter PD098059. B, Representative immunoblot and quantitation of the effects of PD098059 on Erk-1/2 phosphorylation, assessed with phospho-specific antibodies. Results from five independent experiments determined in quadruplicate. *, P < 0.05 vs. respective basal; #, P < 0.05 vs. basal OB/C; §, P < 0.05 vs. IGF-I OB/C; {dagger}, P < 0.05 vs. basal OB/OP in the absence of PD098059; {ddagger}, P < 0.05 vs. IGF-I OB/OP in the absence of PD098059.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the IGF-I signaling system in osteoblasts from patients with osteoporosis exhibited multiple abnormalities, including increased tyrosine phosphorylation of the IGF-I receptor, IRS-2, and Erk-1/2 in the basal state, and blunted stimulation of these proteins by IGF-I. In addition, although phosphorylation of IRS-1, Akt, and GSK-3 was markedly increased by IGF-I in both osteoporotic and control cells, the effect of IGF-I was significantly lower in osteoporosis. These signaling abnormalities could not be accounted for by different age of the experimental subjects because IGF-I induced the phosphorylation of Akt, GSK-3, and Erk similarly in osteoblasts from younger and older control subjects (Fig. 1Go and supplemental data Table 1 and Fig. 1Go).

The observed signaling defects may be primarily responsible for the osteoporotic phenotype or may be secondary to other, yet unknown, cellular or biochemical abnormalities associated with osteoporosis. The osteoblast clones were selected in the subculturing procedure, potentially not reflecting the complexity of the osteoporotic bone in vivo. However, it is important to note that the reduced proliferative capacity of the osteoblastic cultures from osteoporotic women in this study correlates with histomorphometric indices of reduced bone formation in bioptic fragments ex vivo (32). The persistence of the cellular abnormalities in vitro suggests that they may represent intrinsic or epigenetic characteristics of the osteoporotic osteoblasts.

The mechanisms responsible for the enhanced activation of the IGF-I receptor under basal conditions in OB/OP are unclear. One possibility is that this abnormality may result from impaired protein tyrosine phosphatase activity and/or integrin protein levels. Recent studies in smooth muscle cells have shown that the ability of IGF-I to initiate intracellular signals is regulated by not only its binding to the cell-surface receptor but also other transmembrane proteins, such as {alpha}Vβ3 integrin, which recruit other signaling proteins such as SH2 domain-containing protein-tyrosine phosphatase (SHP-2) and Src-homology-2 and collagen-homology domains (Shc) (33, 34, 35). The integrin-mediated recruitment of SHP-2 is essential for regulation of overall IGF-I receptor phosphorylation levels (35) and propagation of downstream signaling (36). In support of this concept, both a catalytically inactive form of SHP-2 and echistatin, an inhibitor of the {alpha}Vβ3 integrin receptor, lead to increased IGF-I receptor tyrosine phosphorylation in the absence of hormone stimulation (37). Changes in both protein tyrosine phosphatase and integrins may influence intensity and/or duration of IGF-I receptor tyrosine phosphorylation under basal conditions and upon hormone stimulation and thus deserve further investigation as putative mechanisms regulating IGF-I receptor activation in osteoblasts from subjects with osteoporosis. The role of integrin in acquired osteoporosis was recently evaluated in osteoclasts. It was shown that soluble ligands, which compete for {alpha}Vβ3, block bone resorption by osteoclasts and augment bone mineral density (38, 39), supporting an important role for integrins in the pathophysiology of bone cells.

In OB/OP, IGF-I failed to stimulate tyrosine phosphorylation of IRS-2, which was already elevated in the basal state, whereas it did stimulate tyrosine phosphorylation of IRS-1. Several potential factors may explain this phenomenon. First, differential phosphorylation of the two IRS proteins may reflect a different capability of the IGF-I receptor to engage binding with IRS-2, compared with IRS-1, also in the absence of IGF-I stimulation. It is known that IRS-2 interacts with the IGF-I receptor via the phosphotyrosine binding as well as via a central domain, which is absent in IRS-1 (40, 41). The presence of this domain in IRS-2, but not IRS-1, may result in differential kinetics/affinity of interaction between the receptor and the two IRS proteins, such that IRS-2 phosphorylation may follow receptor activation more tightly, compared with IRS-1. Second, in vitro studies have shown that it is possible to selectively impair IRS-1 vs. IRS-2 tyrosine phosphorylation by modifying the insulin or IGF-I receptor primary structure (42, 43). For example, a mutant insulin receptor, in which Arg1152 was replaced by Gln, induced constitutive phosphorylation of IRS-2 not further responding to the insulin stimulus, whereas basal and hormone-stimulated tyrosine phosphorylation of IRS-1 was unaltered (43). Third, IRS-1 and IRS-2 may differ in their intracellular compartmentalization and may be coupled to distinct receptor pools. According to this hypothesis, one IGF-I receptor pool with low activity in the basal state and normal (or subnormal) IGF-I responsiveness could be coupled to IRS-1, whereas a second pool of IGF-I receptors with augmented basal activity could be phosphorylating IRS-2. At present, no studies are available that directly support this concept. However, it was shown that IRS-1 is 2-fold more concentrated in the intracellular membrane compartment than in the cytosol, in marked contrast to IRS-2, which shows opposite localization, in 3T3-L1 adipocytes (44).

Osteoblasts from patients with osteoporosis showed changes in basal and IGF-I-stimulated Erk activation that correlated with tyrosine phosphorylation levels of IRS-2 but not with those of IRS-1. Distinct abnormalities in IRS-1 and IRS-2 proteins occur in experimental and human diseases. In earlier studies, we demonstrated that IRS-1 is reduced, whereas IRS-2 is unchanged in the skeletal muscle and myocardium of streptozotocin-diabetic rats (45, 46). More recently we found that the placentas of intrauterine growth restricted fetuses are characterized by reduced protein content of IRS-2, with no changes in IRS-1 (47). Differential regulation of IRS-1 vs. IRS-2 in disease states may result in selective changes of specific downstream signaling pathways. Relevant to this concept, it was found recently in L6 rat myotubes (9) that 70% loss of endogenous IRS-1 exerted only minimal impairment of Erk activation, whereas it led to markedly blunted insulin-stimulated Ser473 phosphorylation of Akt. On the other hand, small interfering RNA-mediated reduction of IRS-2 reduced insulin-stimulated Erk phosphorylation 85%, suggesting that IRS-2 may have a more prominent role than IRS-1 in mediating insulin-stimulated mitogenic signaling via the Erk pathway in these cells. Most relevant to the tissue context investigated in this study, Shimoaka et al. (48) have recently shown that chondrocytes display significant impairment of the phosphatidylinositol 3-kinase/Akt pathway but not the Erk pathway in mice lacking the IRS-1 gene. The potential impact of altered activation of IRS-1 and IRS-2 proteins on bone growth and metabolism is underscored by studies in genetically manipulated mice. In these experimental systems, the lack of the IRS-1 gene resulted in decreased bone formation and bone resorption, indicating the development of a low-turnover osteopenia (11), whereas IRS-2 deficiency resulted in an uncoupling state with decreased bone formation and increased bone resorption (10).

Another finding of this study is that whereas Erk-1/2 phosphorylation was low in the basal state and increased markedly with IGF-I stimulation in control osteoblasts, Erk-1/2 phosphorylation was already elevated in the absence of IGF-I in osteoporotic osteoblasts and did not increase further on IGF-I stimulation. These results are similar to those reported recently by Rodriguez et al. (49) in osteoblasts differentiated by mesenchymal stem cells. Under basal conditions, the phosphorylation levels of Erk-1/2 were 2.5-fold higher in mesenchymal stem cells obtained from osteoporotic compared with control women. Constitutively active Erk-1/2 has been shown to impair S-phase entry in fibroblasts and other cell types (50), and this may explain the reduced DNA synthesis under basal conditions and in response to IGF-I in osteoblasts from osteoporotic subjects in this study. In fact, the analysis of multiple differentiation markers demonstrated no difference between OB/C and OB/OP (Fig. 1Go and Table 1Go), suggesting that the defective bone formation in osteoporosis is associated with impaired proliferative capacity of the osteoblasts rather than from faulty osteoblast differentiation, in line with earlier reports (51). Importantly, when the constitutively elevated levels of Erk-1/2 phosphorylation in OB/OP were reduced by pretreatment with a specific MEK inhibitor, the rates of DNA synthesis were significantly improved, underscoring the contribution of altered Erk activation to the proliferation abnormality. On the other hand, the impaired ability of IGF-I to stimulate DNA synthesis in the osteoporotic cells could be attributable to the its almost absent stimulation of Erk-1/2 phosphorylation as well as reduced activation of the IRS-1/Akt pathway. IGF-I-mediated tyrosine phosphorylation of IRS-1 and activation of Akt have also been associated with cell growth and proliferation (52, 53). In this study, phosphorylation of Akt on Ser473 and Thr308 by IGF-I stimulation was significantly reduced in OB/OP, compared with control, and this led to decreased GSK-3 phosphorylation, suggesting that decreased Akt signals may also contribute to the impaired IGF-I effects on DNA synthesis in OB/OP.

Blocking the IGF-I receptor with a specific monoclonal antibody impairs cell proliferation and DNA synthesis in response to mechanical strain in human osteoblasts (54). Thus, inefficient signaling through the IGF-I receptor, as demonstrated in this paper, may also contribute to the reduced ability of osteoporotic bones to withstand functional loading without damage.

In conclusion, in the osteoblasts from osteoporotic subjects, the IGF-I receptor is activated under basal conditions and shows blunted stimulation by IGF-I. This is likely responsible for increased basal tyrosine phosphorylation of IRS-2 and activation of Erk-1/2, which are also poorly responsive to IGF-I stimulation. In addition, the IRS-1/Akt pathway, which does not show apparent abnormalities in the basal state, exhibits suboptimal activation by IGF-I. The distinct abnormalities in IGF-I signaling may contribute to the reduced DNA synthesis in osteoporotic osteoblasts, representing novel mechanisms for the impaired cell proliferation and decreased bone formation that occur in human osteoporosis.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: ALP, Alkaline phosphatase; GSK, glycogen synthase kinase; hPTH-R, Homo sapiens PTH receptor; IRS, insulin receptor substrate; MEK, MAPK kinase; OB/C, osteoblasts from control donors; OB/OP, osteoblasts from osteoporotic donors; 1,25(OH)2D3, 1,25 dihydroxyvitamin D3; SHP-2, SH2 domain-containing protein-tyrosine phosphatase.

This work was supported by grants from the Ministero dell’Università e Ricerca (Italy), the Cofinlab 2000-Centro di Eccellenza "Genomica comparata: geni coinvolti in processi fisiopatologici in campo biomedico e agrario" (Italy), and an educational grant from Pfizer Italia srl (ARADO Program) (to F.G.).

Disclosure Statement: The authors have nothing to disclose.

Received October 1, 2007.

Accepted for publication November 30, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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