Insulin Receptor Substrate-1 Is Required for Bone Anabolic Function of Parathyroid Hormone in Mice
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内分泌学杂志 2005年第6期
Departments of Sensory and Motor System Medicine (M.Y., N.O., Y.S., T.A., S.K., K.H., U.-I.C., K.N., H.K.) and Metabolic Diseases (Y.T., T.K.), Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Hiroshi Kawaguchi, M.D., Ph.D., Department of Sensory and Motor System Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: kawaguchi-ort@h.u-tokyo.ac.jp.
Abstract
Bone anabolic action of PTH has been suggested to be mediated by induction of IGF-I in osteoblasts; however, little is known about the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation. This study initially confirmed in mouse osteoblast cultures that PTH treatment increased IGF-I mRNA and protein levels and alkaline phosphatase activity, which were accompanied by phosphorylations of IGF-I receptor, insulin receptor substrate (IRS)-1 and IRS-2, essential adaptor molecules for the IGF-I signaling. To learn the involvement of IRS-1 and IRS-2 in the bone anabolic action of PTH in vivo, IRS-1–/– and IRS-2–/– mice and their respective wild-type littermates were given daily injections of PTH (80 μg/kg) or vehicle for 4 wk. In the wild-type mice, the PTH injection increased bone mineral densities of the femur, tibia, and vertebrae by 10–20% without altering the serum IGF-I level. These stimulations were similarly seen in IRS-2–/– mice; however, they were markedly suppressed in IRS-1–/– mice. Although the PTH anabolic effects were stronger on trabecular bones than on cortical bones, the stimulations on both bones were blocked in IRS-1–/– mice but not in IRS-2–/– mice. Histomorphometric and biochemical analyses showed an increased bone turnover by PTH, which was also blunted by the IRS-1 deficiency, though not by the IRS-2 deficiency. These results indicate that the PTH bone anabolic action is mediated by the activation of IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally as an autocrine/paracrine factor.
Introduction
ANABOLIC EFFECTS of PTH on bone have attracted considerable clinical attention and led to the approval of PTH for osteoporosis treatment (1, 2, 3). Although it has been well established that intermittent administration of PTH exerts potent anabolic effects on bone in animals and humans (4), the underlying mechanism is still controversial and unclear. PTH is reported to increase production of osteoprogenitors and differentiation of osteoblasts from an existing pool of osteoprogenitors and to decrease apoptosis of preexisting osteoblasts (5, 6, 7). Accumulated evidence has shown that IGF-I is an attractive candidate as a mediator for some or all of the anabolic actions of PTH on bone, in that PTH stimulates IGF-I production by osteoblastic cells (8, 9) and IGF-1 can reproduce the effects of PTH on osteoblast proliferation, differentiation, and survival (10). From in vitro studies, IGF-I-blocking antibodies inhibited collagen synthesis and alkaline phosphatase (ALP) activity, as well as the expression of osteocalcin mRNA induced by PTH stimulation on osteoblasts (11, 12). Furthermore, PTH anabolic actions were suppressed when administered to IGF-I-deficient mice (13, 14), suggesting the importance of the IGF-I signaling in vivo.
IGF-I initiates cellular responses by binding to its cell-surface receptor tyrosine kinase IGF-I receptor, which then activates essential adaptor molecule insulin receptor substrates (IRS’s) followed by downstream signaling pathways like phosphatidylinositol-3 kinase (PI3K)/Akt and MAPKs (15). The mammalian IRS family contains at least four members: ubiquitous IRS-1 and IRS-2, adipose tissue-predominant IRS-3, and IRS-4 which is expressed in the thymus, brain, and kidney. We previously reported that IRS-1 and IRS-2 are expressed in bone (16, 17). Our further studies on mice lacking the IRS-1 gene (IRS-1–/– mice) or the IRS-2 gene (IRS-2–/– mice) revealed that these knockout mice exhibited severe osteopenia with distinct mechanisms: IRS-1–/– mice showed a low bone turnover in which both bone formation and resorption were decreased (16), whereas IRS-2–/– mice showed an uncoupling status with decreased bone formation and increased bone resorption (17). It therefore seems that under physiological conditions, IRS-1 is important for maintaining bone turnover, whereas IRS-2 is important for retaining the predominance of anabolic function over catabolic function of osteoblasts.
To learn the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation, the present study investigated the role of IRS-1 and IRS-2 in mediating the anabolic effects of recombinant human PTH(1–34) on bone. We first studied the effects of PTH on the IGF-I related molecules in cultured mouse osteoblasts and examined skeletal responses to PTH in IRS-1–/– and IRS-2–/– mice.
Materials and Methods
Animals
Mice with the original C57BL6/CBA hybrid background were generated and maintained as reported previously (16, 17). In each experiment, homozygous wild-type (WT) and IRS-1–/– male mice, as well as homozygous WT and IRS-2–/– male mice, that were littermates generated from the intercross between heterozygous mice were compared. All mice were kept in plastic cages under standard laboratory conditions with a 12-h dark, 12-h light cycle, a constant temperature of 23 C, and humidity of 48%. The mice were fed a standard rodent diet (CE-2; CLEA Japan, Inc., Tokyo, Japan) containing 25.2% protein, 4.6% fat, 4.4% fiber, 6.5% ash, 3.44 kcal/g, 2.5 IU vitamin D3/g, 1.09% calcium, and 0.93% phosphorus with water ad libitum. All animal experiments were reviewed and approved by the University of Tokyo, Faculty of Medical Animal Care and Use Committee, before the study.
Osteoblast cultures
Osteoblasts were isolated from calvariae of neonatal WT, IRS-1–/–, and IRS-2–/– littermates. Calvariae were digested for 10 min, 5 times, at 37 C in an enzyme solution containing 0.1% collagenase and 0.2% dispase. Cells isolated by the last four digestions were combined as an osteoblast population and cultured in MEM (Invitrogen, Carlsbad, CA) containing 10% FBS (HyClone Laboratories, Inc., Logan, UT) and 50 μg/ml ascorbic acid (Sigma-Aldrich Corp., St. Louis, MO).
For real-time quantitative RT-PCR analysis, primary osteoblasts were inoculated at a density of 1 x 104 cells/well in a 24-multiwell plate, and cultured in the medium above, with or without 100 nM recombinant human PTH(1–34) (Sigma-Aldrich Corp.) for 14 d. Total RNA was extracted with an ISOGEN kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), according to the manufacturer’s instructions. One microgram of RNA was reverse-transcribed using a Takara RNA PCR Kit, version 2.1 (Takara Shuzo Co., Shiga, Japan), to make single-stranded cDNA. The ABI Prism Sequence Detection System 7000 and Primer Express Software (Applied Biosystems, Foster City, CA) were used for PCR amplification and quantitative analysis, respectively. For the IGF-I gene, a set of primers was designed using sequences obtained from the GenBank as follows: 5'GACAGATACARRCTGTGCTCA-3' and 5'-CTGAAGCTTGCTAACATCGC-3'. The PCR consisted of QuantiTect SYBR Green Master Mix (QIAGEN, Tokyo, Japan), 0.3 μM specific primers, and 20 ng cDNA.
For the IGF-I protein level measurement, primary osteoblasts were cultured, as described above, for 14 d, and the free IGF-I concentration in the culture media was measured with a Non-Extraction IGF-I ELISA kit (Diagnostic Systems Laboratories, Inc., Sparks, MD).
For ALP activity measurement, primary osteoblasts were cultured in the medium above with or without 10 ng/ml recombinant mouse IGF-I (Sigma-Aldrich Corp.) and 5 nM antimouse IGF-I antibody (Sigma-Aldrich Corp.). At 14 d of culture, cells were sonicated in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl2 and 0.5% Triton X-100, and ALP activity in the lysate was measured using an ALP assay kit (Wako Pure Chemical Industries Ltd.). The protein content was determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).
Immunoprecipitation and immunoblotting
After stimulation by 100 nM PTH for the indicated time, cultured osteoblasts were lysed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, and 10 μg/ml aprotinin). A part of the cell lysates (100 μg) was immunoprecipitated with an antiphosphotyrosine antibody, an antimouse IGF-I receptor antibody, an antimouse insulin receptor antibody, an antimouse IRS-1 antibody, or an antimouse IRS-2 antibody (all from Upstate Biotechnology, Inc., Waltham, MA) conjugated to protein G-Sepharose (Invitrogen) for 4 h at 4 C. The cell lysates with or without the immunoprecipitation that contained an equivalent amount of protein (20 μg) were electrophoresed by 8% SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% BSA solution, they were incubated with the antibodies above, and the immunoreactive bands were stained using the ECL chemiluminescence reaction (Amersham, Arlington Heights, IL). The intensity of each band was measured by densitometry (Bio-Rad Laboratories, Inc., Richmond, CA) and was expressed as the mean value of five independent experiments.
PTH treatment on mice
IRS-1–/– mice and their littermates and IRS-2–/– mice and their littermates (males, all n = 10) received either PTH (80 μg/kg body weight) or vehicle (PBS) by sc injection every day for 4 wk beginning at 10 wk of age. Blood samples were collected by heart puncture under nembutal (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) anesthesia before being killed. For radiological and histological analyses, animals were killed after 4 wk of PTH treatment by diethylether. The right femurs and tibiae were obtained for bone densitometry, and the left femurs and tibiae for peripheral quantitative computerized tomography (pQCT) and histological analyses, respectively. Lumbar vertebral bodies from L2–L5 were also obtained for bone densitometry.
Bone densitometry and pQCT
Bone mineral density (BMD; milligrams per square centimeter) of the right femur, tibiae, and L2–L5 vertebral bodies was determined using dual-energy x-ray absorptiometry (PIXImus Mouse Densitometer; Lunar Corp., Madison, WI) according to the manufacturer’s instructions. Computerized tomography was performed with a pQCT analyzer (XCT Research SA+; Stratec Medizintechnik GmbH, Pforzheim, Germany) operating at a resolution of 80 μm. Metaphyseal pQCT scans of the left femurs were performed to measure the trabecular volumetric BMD. The scan was positioned in the metaphysis at 1.2 mm proximal from the distal growth plate. Because this area contains trabecular and cortical bones, the trabecular bone region was defined by setting the threshold to 395 mg/cm3. Middiaphyseal pQCT scans of the left femurs were performed to determine the cortical thickness. The middiaphyseal region of femurs in mice contains mostly cortical bone. The cortical bone region was defined by setting the threshold to 690 mg/cm3. The interassay coefficients of variation for the pQCT measurements were less than 2%.
Histological analyses
For the assessment of dynamic histomorphometric indices, mice were injected with calcein (16 mg/kg body weight) sc at 10 d and 3 d before being killed, after which the left tibiae were excised and fixed with ethanol, and the undecalcified bones were embedded in glycolmethacrylate. Three-micrometer sagittal sections from the proximal parts of tibiae were stained with toluidine blue and were visualized under fluorescent light microscopy for calcein labeling. The specimens were subjected to histomorphometric analyses using a semiautomated system (Osteoplan II; Carl Zeiss, Oberkochen, Germany), and measurements were made at x400 magnification. Parameters for the trabecular bone were measured in an area 1.2 mm in length, from 250 μm below the growth plate at the proximal metaphysis of the tibiae. Nomenclature, symbols, and units are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research (18).
Serum biochemical assays
For serum IGF-I levels, acid ethanol extraction was used to remove the IGF-binding proteins, and the extracted samples were assayed for IGF-I with a RIA kit from Nichols Institute Diagnostics (San Juan Capistrano, CA). Serum osteocalcin levels were determined by using the competitive RIA kit (Biomedical Technologies, Stoughton, MA). The sensitivity of the assay was 19 ng/ml, and the interassay and intraassay coefficients of variation were less than 10%. Serum ALP activity was determined by liquitech ALP kit (Roche Diagnostics, Basel, Switzerland) with an autoanalyzer (type 7170; Hitachi High-Technologies Corporation, Tokyo, Japan). ALP activity of the blood samples was expressed as nanomoles per minute and per milligram of protein.
Statistical analysis
Means of groups were compared by ANOVA, and significance of differences was determined by post hoc testing using Bonferroni‘s method.
Results
Effects of PTH on cultured osteoblasts
We first examined the effects of recombinant human PTH(1–34) in the cultures of primary osteoblasts derived from mouse calvariae. IGF-I mRNA level determined by real-time RT-PCR, IGF-I protein level in the cultured medium, and ALP activity in the cell lysate were all increased about 2-fold with PTH (100 nM) treatment compared with the control cultures (Fig. 1A). A neutralizing antibody against IGF-I significantly, although not completely, suppressed the PTH stimulation of ALP activity. Furthermore, addition of a recombinant mouse IGF-1 at a concentration similar to that of endogenous IGF-I (10 mg/ml) stimulated by PTH increased the ALP activity to a level similar to that by PTH. These lines of results confirm that the PTH anabolic action is, at least partly, mediated by the IGF-I production in osteoblasts, as previously reported (8, 11, 12).
FIG. 1. Effects of PTH on cultured osteoblasts isolated from neonatal mouse calvariae. A, IGF-I mRNA level determined by real-time quantitative RT-PCR (left), IGF-I protein level in the culture medium (middle), and ALP activity in the cell lysate (right) in the primary calvarial osteoblast culture in the presence and absence of PTH (100 nM), an antibody against IGF-I (-IGF-I, 5 nM), and recombinant mouse IGF-I (rmIGF-I, 10 ng/ml) for 2 wk. Data are expressed as means (bars) ± SEM (error bars) for eight wells per group. *, Significant increase compared with the control culture, P < 0.01; #, significant inhibition by -IGF-I, P < 0.05. B, Protein levels by immunoprecipitation (IP) and immunoblotting (IB) of IGF-I receptor, insulin receptor, IRS-1, and IRS-2 with or without phosphorylation in osteoblasts cultured with PTH (100 nM) for the indicated times. Some of the cell lysates were immunoprecipitated with an antiphosphotyrosine antibody (-PY), and the cell lysates with or without (–) the immunoprecipitation were immunoblotted with an antimouse IGF-I receptor (-IGF-IR), an antimouse insulin receptor (-IR), an antimouse IRS-1 (-IRS-1), or an antimouse IRS-2 antibody (-IRS-2). Some of the cell lysates were reciprocally immunoprecipitated with -IGFR or -IR and immunoblotted with -PY. Blottings with an anti-?-actin (-actin) were used as loading controls. Similar results were obtained in five independent experiments. The graph below shows the mean values of the band intensities of phosphorylated proteins normalized to -actin quantified using densitometry in five independent experiments. Data are expressed as the ratio of the value at time zero. Although the data of proteins without phosphorylation are not shown in the graph, they were not significantly affected by PTH during the observation period (the ratio values were from 0.8–1.2). Data are expressed as means (bars) ± SEM (error bars) of five independent experiments. *, Significant difference from that at time zero, P < 0.01.
To provide some insights into signaling pathways that are involved in the PTH action on primary osteoblasts, we examined the phosphorylations of IGF-I receptor, insulin receptor, IRS-1, and IRS-2 in five independent experiments (Fig. 1B). Immunoprecipitation and immunoblotting analyses revealed that phosphorylations of IGF-I receptor and IRS-1 were clearly induced at 1 min and reached maximum at 5 min. IRS-2 was also phosphorylated by PTH, although not as strongly as IGF-I receptor and IRS-1. Insulin receptor was hardly phosphorylated by PTH. None of the protein levels of IGF-I receptor, insulin receptor, IRS-1, or IRS-2 were altered by PTH during the observation period up to 60 min, suggesting that PTH does not show transcriptional or translational regulation of these signaling molecules. These results indicate that IGF-I production followed by the activation of its intracellular signaling pathways may be related to the PTH action in osteoblasts.
Effects of PTH on bones in IRS-1–/– and IRS-2–/– mice
To learn the roles of the IRS-1 and IRS-2 in the PTH action on bone in vivo, we analyzed the PTH effects on the knockout mice by comparing them with those of respective WT littermates using radiological and histological analyses. Both knockout mice were healthy, with no abnormality in major organs except that IRS-1–/– mice alone showed about 20% shorter limbs and trunk, whereas IRS-2–/– mice were normal in size compared with WT littermates (19, 20, 21). The mice (10 wk old, males) were given daily sc injections of PTH (80 μg/kg) or vehicle for 4 wk, after which their femurs, tibiae, and lumbar vertebrae underwent radiological and histological analyses. As we previously reported for bone phenotypes under physiological conditions (16, 17), both knockout mice showed osteopenia when injected with vehicle: BMDs of femur, tibia, and lumbar vertebra in IRS-1–/– and those of femur and tibia in IRS-2–/– were significantly lower than respective WT littermates (Fig. 2). The PTH injection increased BMDs of these bones 10–20% in WT; however, this increase was hardly seen in the IRS-1–/– bones (Fig. 2A). The stimulations by PTH, on the contrary, seen in the IRS-2–/– bones were similar to those of the WT littermates (Fig. 2B). These results suggest that IRS-1, but not IRS-2, is needed for the bone anabolic action of PTH.
FIG. 2. Effects of PTH treatment on bone densities in IRS-1–/– mice (A) and IRS-2–/– mice (B), compared with respective WT littermates. IRS-1–/– mice and the littermates and IRS-2–/– mice and the littermates (males, 10 wk old, n = 10/group) received daily sc injections of either PTH (80 μg/kg body weight) or vehicle for 4 wk. Mice were killed; and the right femurs, tibiae, and L2–L5 vertebral bodies were excised. BMD values of the entire femurs, tibiae, and vertebral bodies were determined using dual-energy x-ray absorptiometry. Data are expressed as means (bars) ± SEM (error bars) of 10 bones per group. *, Significant effect of PTH, P < 0.01; #, significant difference from WT, P < 0.01.
We further examined trabecular and cortical bones separately in the femurs using pQCT (Fig. 3). In trabecular bones at the distal metaphysis of femurs, both IRS-1–/– and IRS-2–/– mice showed lower bone density (Fig. 3, A and C). PTH injection increased the trabecular bone density about 60% in WT. Here again, this PTH effect was abolished by the IRS-1 deficiency but was not altered by the IRS-2 deficiency. In the cortical bones at the midshaft of the femurs, although the PTH effects on cortical thickness were milder than those on the trabecular density, this was blocked by the IRS-1 deficiency, although not by the IRS-2 deficiency (Fig. 3, B and D).
FIG. 3. Effects of PTH treatment on trabecular and cortical bones in IRS-1–/– mice (A and B) and IRS-2–/– mice (C and D), compared with respective WT littermates. After daily injections of either PTH (80 μg/kg body weight) or vehicle for 4 wk, mice were killed, and the distal metaphysis (A and C) and the middiaphysis (B and D) of the excised left femurs underwent pQCT analysis. The color gradient indicating bone density is shown in the right bars. The trabecular density at the metaphysis and the cortical thickness at the middiaphysis are shown in the graphs below. Data in all graphs are expressed as means (bars) ± SEMs (error bars) of 10 bones per group. *, Significant effect of PTH, P < 0.01; #, significant difference from WT, P < 0.01.
Histological features of the proximal tibiae showed decreases of trabecular bones in vehicle-treated IRS-1–/– and IRS-2–/– mice compared with the respective WT littermates (Fig. 4). After PTH treatment for 4 wk, increases of these bones were observed in IRS-2–/– and WT littermates to a similar extent (Fig. 4B), whereas no increase was observed in the IRS-1–/– trabeculae (Fig. 4A). Bone histomorphometric measurements in this area confirmed that the PTH injection augmented the bone volume (trabecular bone volume expressed as a percent of total tissue volume) of WT mice by 50–60%, with the increases of both bone formation parameters (percent of bone surface covered by cuboidal osteoclasts, and bone formation rate) and resorption parameters (percent of bone surface covered by mature osteoclasts, and percent of eroded surface), indicating a high bone turnover state (Table 1). IRS-1–/– and IRS-2–/– mice showed 30–40% lower bone volume than respective WT littermates when injected with vehicle. As we previously reported (16, 17), IRS-1–/– exhibited a low bone turnover, with decreases in both bone formation and resorption parameters, whereas IRS-2–/– mice showed an uncoupling status of bone turnover, with decreased bone formation and increased bone resorption. The PTH injection affected neither the bone volume nor the bone turnover in the IRS-1–/– mice; however, in the IRS-2–/– mice, PTH increased bone volume mainly through the up-regulation of bone formation rather than bone resorption.
FIG. 4. Effects of PTH treatment on histological features of the proximal metaphysis of tibiae in IRS-1–/– mice (A) and IRS-2–/– mice (B), compared with respective WT littermates. After death, the left tibiae were excised, fixed, and embedded in GMA without decalcification, and the sagittal sections were stained by toluidine blue. Representative samples are shown from mice of each genotype given either PTH or vehicle. Data of histomorphometric analyses are shown in Table 1. Bar, 100 μm.
TABLE 1. Histomorphometry of trabecular bones in proximal tibiae
Effects of PTH on blood chemistries in IRS-1–/– and IRS-2–/– mice
The serum markers osteocalcin and ALP supported the increase of bone formation by the PTH injection in WT mice (Fig. 5). Here again, these stimulations were not seen in IRS-1–/– mice but were maintained in IRS-2–/– mice. Because the serum IGF-I levels were not different between PTH- and vehicle-treated mice in all genotypes, IGF-I that is induced by PTH, as shown in Fig. 1A, seemed not to act as a systemic factor but to act locally in bone as an autocrine/paracrine factor.
FIG. 5. Effects of PTH treatment on serum osteocalcin, ALP, and IGF-I levels in IRS-1–/– mice and IRS-2–/– mice, compared with respective WT littermates. Mice received either PTH or vehicle for 4 wk as described above, and blood samples were collected by heart puncture before death. The levels were measured as described in Materials and Methods. *, Significant effect of PTH, P < 0.05; #, significant difference from WT, P < 0.05.
Discussion
The present study demonstrated that the bone anabolic function of PTH is mediated by the activation of IGF-1R and IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally in bone. Although IRS-1 and IRS-2 are known to be essential for intracellular signaling of IGF-I and insulin, these two adaptor molecules have distinct biological roles and are differentially expressed in a variety of cells. Regarding glucose homeostasis, IRS-1 plays an important role in the metabolic actions of insulin, mainly in skeletal muscle and adipose tissue, whereas IRS-2 does so in the liver (22). Our previous studies revealed that only IRS-1, but not IRS-2, was expressed in the cartilage of the growth plate or the fracture callus, so that skeletal growth and fracture healing were impaired in IRS-1–/– mice, whereas they were normal in IRS-2–/– mice (21, 23). In bone, IRS-1 is expressed solely in cells of osteoblast lineage, whereas IRS-2 is expressed in cells of both osteoblast and osteoclast lineages (16, 17). As described above, our previous studies on bones of these two knockout mice disclosed that IRS-1 is important for maintaining bone turnover, and IRS-2 for maintaining predominance of anabolic function over catabolic function of osteoblasts (16, 17). In the meantime, previous and present studies have shown that PTH treatment increases bone turnover in animals and humans (24, 25). The fact that the suppression of bone turnover by IRS-1 deficiency suppressed the bone anabolic action of PTH suggests the importance of elevated turnover for the PTH function. This is consistent with the results of clinical studies showing that the concurrent use of alendronate, a potent bisphosphonate that markedly suppresses bone turnover, reduced the bone anabolic action of PTH in male and female osteoporosis patients (26, 27).
Because it is unlikely that PTH directly activates IGF-I receptor and IRS-1, there seem to be two possible molecular mechanisms underlying the suppression of PTH action by the IRS-1 deficiency: 1) PTH induces IGF-I production, causing IGF-I receptor and IRS-1 activation; and 2) IRS-1 signaling affects the intracellular signaling lying downstream of the PTH/PTH-related protein receptor after PTH binds to it. The quick phosphorylation of IGF-I receptor and IRS-1 after PTH treatment in primary osteoblast culture in the present study (Fig. 1B) supports the former mechanism. Hormones like PTH and prostaglandin E2 that increase cAMP synthesis and PKA activation are reported to induce the transcription of IGF-I by way of a C/EBP (CCAAT/enhancer-binding protein)-sensitive element in exon 1 of the IGF-I gene (28, 29). However, the latter possibility cannot be denied, because the inhibition of the PTH stimulation on ALP activity by a neutralizing antibody against IGF-I was not complete, but partial, in the primary osteoblast culture (Fig. 1A). In fact, IRS-1 and PTH/PPR (PTH/PTH-related protein receptor) are known to share several common signaling pathways. The main pathways lying downstream of IRS-1 are PI3K/Akt and MAPKs, which are important regulators of cell growth and differentiation. PTH has been shown to directly activate the p42/p44 MAPK by protein kinase C-dependent, but Ras-independent, signaling in rat osteoblasts (30) and to up-regulate PI3K/Akt activity, which contributes to the MAPK activation in rat enterocytes (31). In addition, both PTH and IGF-I have been shown to be involved in the activation of c-fos expression (32, 33) and cyclin-dependent kinase expression in osteoblasts (34, 35). These lines of evidence indicate the PTH signaling pathway may possibly be affected by the IRS-1 signaling at several points, and the absence of an IRS-1 signaling pathway may result in the failure of PTH to stimulate key target molecules necessary for its anabolic action.
Another potential explanation for the lack of PTH response in IRS-1–/– mice could be the impairment of proliferation or differentiation ability of osteoprogenitor cells, so that they are insensitive not only to PTH but also to other stimulations. In fact, our present and previous studies demonstrated the decreases in histomorphometric parameters and serum markers for bone formation in IRS-1–/– mice under physiological conditions (16). In this regard, however, our previous study has shown that proliferation and differentiation of primary calvarial cells were stimulated, responding to fibroblast growth factor-2 and bone morphogenetic protein-2, respectively, similarly to those of the WT cells, indicating that functions of IRS-1–/– cells are normal as long as adequate signals other than IGF-I signal were applied (16). Hence, the decreased bone formation under physiological conditions in IRS-1–/– mice is likely to be due to the deficit of anabolic signaling of endogenous IGF-I. Interestingly, PTH increased the IGF-I protein level in the culture medium of primary osteoblasts (Fig. 1A) but did not affect the serum IGF-I level in vivo (Fig. 5), indicating the importance of local action of IGF-I as an autocrine/paracrine factor for bone formation rather than its systemic action as a hormone. This result is consistent with previous reports that disruption of IGF-I genes, specifically in liver, decreased serum IGF-I by 80% but caused no skeletal abnormality (36, 37). Because the remaining 20% IGF-I in serum is probably derived from several tissues, including bone, it is not surprising that PTH treatment did not increase circulating levels of IGF-I. The findings that PTH anabolic effects can be suppressed by an IGF-I-neutralizing antibody in osteoblast cultures in the present and previous studies (11, 12) also support the idea that the PTH-induced bone formation involves increased local production, but not increased circulating levels, of IGF-I.
The requirement of IGF-I/IRS-1 for mediating the anabolic effects of bone regulatory hormones may not be unique to PTH, because previous findings have revealed that many of the major hormones exert significant effects on IGF-I expression. GH is a well-known stimulus of IGF-I production in a variety of tissues, including bone, and exerts its effects on bone mainly through IGF-I mediation (38). Estradiol, another important regulator of skeletal metabolism, has been shown to increase IGF-I production, and IGF-I receptor-blocking antibodies inhibited the proliferative effect of estradiol in rat osteoblasts (39, 40). Similarly, other hormones with potent effects on bone, such as thyroid hormone and androgens, alter IGF-I levels in bone in a manner consistent with IGF-I playing a role in the actions of these hormones on bone (41, 42). Although these studies did not examine the involvement of IRS-1 or IRS-2 in their anabolic actions, it is possible that the IGF/IRS pathway might be a common signaling for actions of these major hormones in bone. Further understanding of the molecular mechanism by which the hormones induce IGF-I and the intracellular signaling that lies downstream of IGF-I/IRS in osteoblasts will greatly help to elucidate the complex network of bone formation under systemic regulations.
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Address all correspondence and requests for reprints to: Hiroshi Kawaguchi, M.D., Ph.D., Department of Sensory and Motor System Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: kawaguchi-ort@h.u-tokyo.ac.jp.
Abstract
Bone anabolic action of PTH has been suggested to be mediated by induction of IGF-I in osteoblasts; however, little is known about the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation. This study initially confirmed in mouse osteoblast cultures that PTH treatment increased IGF-I mRNA and protein levels and alkaline phosphatase activity, which were accompanied by phosphorylations of IGF-I receptor, insulin receptor substrate (IRS)-1 and IRS-2, essential adaptor molecules for the IGF-I signaling. To learn the involvement of IRS-1 and IRS-2 in the bone anabolic action of PTH in vivo, IRS-1–/– and IRS-2–/– mice and their respective wild-type littermates were given daily injections of PTH (80 μg/kg) or vehicle for 4 wk. In the wild-type mice, the PTH injection increased bone mineral densities of the femur, tibia, and vertebrae by 10–20% without altering the serum IGF-I level. These stimulations were similarly seen in IRS-2–/– mice; however, they were markedly suppressed in IRS-1–/– mice. Although the PTH anabolic effects were stronger on trabecular bones than on cortical bones, the stimulations on both bones were blocked in IRS-1–/– mice but not in IRS-2–/– mice. Histomorphometric and biochemical analyses showed an increased bone turnover by PTH, which was also blunted by the IRS-1 deficiency, though not by the IRS-2 deficiency. These results indicate that the PTH bone anabolic action is mediated by the activation of IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally as an autocrine/paracrine factor.
Introduction
ANABOLIC EFFECTS of PTH on bone have attracted considerable clinical attention and led to the approval of PTH for osteoporosis treatment (1, 2, 3). Although it has been well established that intermittent administration of PTH exerts potent anabolic effects on bone in animals and humans (4), the underlying mechanism is still controversial and unclear. PTH is reported to increase production of osteoprogenitors and differentiation of osteoblasts from an existing pool of osteoprogenitors and to decrease apoptosis of preexisting osteoblasts (5, 6, 7). Accumulated evidence has shown that IGF-I is an attractive candidate as a mediator for some or all of the anabolic actions of PTH on bone, in that PTH stimulates IGF-I production by osteoblastic cells (8, 9) and IGF-1 can reproduce the effects of PTH on osteoblast proliferation, differentiation, and survival (10). From in vitro studies, IGF-I-blocking antibodies inhibited collagen synthesis and alkaline phosphatase (ALP) activity, as well as the expression of osteocalcin mRNA induced by PTH stimulation on osteoblasts (11, 12). Furthermore, PTH anabolic actions were suppressed when administered to IGF-I-deficient mice (13, 14), suggesting the importance of the IGF-I signaling in vivo.
IGF-I initiates cellular responses by binding to its cell-surface receptor tyrosine kinase IGF-I receptor, which then activates essential adaptor molecule insulin receptor substrates (IRS’s) followed by downstream signaling pathways like phosphatidylinositol-3 kinase (PI3K)/Akt and MAPKs (15). The mammalian IRS family contains at least four members: ubiquitous IRS-1 and IRS-2, adipose tissue-predominant IRS-3, and IRS-4 which is expressed in the thymus, brain, and kidney. We previously reported that IRS-1 and IRS-2 are expressed in bone (16, 17). Our further studies on mice lacking the IRS-1 gene (IRS-1–/– mice) or the IRS-2 gene (IRS-2–/– mice) revealed that these knockout mice exhibited severe osteopenia with distinct mechanisms: IRS-1–/– mice showed a low bone turnover in which both bone formation and resorption were decreased (16), whereas IRS-2–/– mice showed an uncoupling status with decreased bone formation and increased bone resorption (17). It therefore seems that under physiological conditions, IRS-1 is important for maintaining bone turnover, whereas IRS-2 is important for retaining the predominance of anabolic function over catabolic function of osteoblasts.
To learn the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation, the present study investigated the role of IRS-1 and IRS-2 in mediating the anabolic effects of recombinant human PTH(1–34) on bone. We first studied the effects of PTH on the IGF-I related molecules in cultured mouse osteoblasts and examined skeletal responses to PTH in IRS-1–/– and IRS-2–/– mice.
Materials and Methods
Animals
Mice with the original C57BL6/CBA hybrid background were generated and maintained as reported previously (16, 17). In each experiment, homozygous wild-type (WT) and IRS-1–/– male mice, as well as homozygous WT and IRS-2–/– male mice, that were littermates generated from the intercross between heterozygous mice were compared. All mice were kept in plastic cages under standard laboratory conditions with a 12-h dark, 12-h light cycle, a constant temperature of 23 C, and humidity of 48%. The mice were fed a standard rodent diet (CE-2; CLEA Japan, Inc., Tokyo, Japan) containing 25.2% protein, 4.6% fat, 4.4% fiber, 6.5% ash, 3.44 kcal/g, 2.5 IU vitamin D3/g, 1.09% calcium, and 0.93% phosphorus with water ad libitum. All animal experiments were reviewed and approved by the University of Tokyo, Faculty of Medical Animal Care and Use Committee, before the study.
Osteoblast cultures
Osteoblasts were isolated from calvariae of neonatal WT, IRS-1–/–, and IRS-2–/– littermates. Calvariae were digested for 10 min, 5 times, at 37 C in an enzyme solution containing 0.1% collagenase and 0.2% dispase. Cells isolated by the last four digestions were combined as an osteoblast population and cultured in MEM (Invitrogen, Carlsbad, CA) containing 10% FBS (HyClone Laboratories, Inc., Logan, UT) and 50 μg/ml ascorbic acid (Sigma-Aldrich Corp., St. Louis, MO).
For real-time quantitative RT-PCR analysis, primary osteoblasts were inoculated at a density of 1 x 104 cells/well in a 24-multiwell plate, and cultured in the medium above, with or without 100 nM recombinant human PTH(1–34) (Sigma-Aldrich Corp.) for 14 d. Total RNA was extracted with an ISOGEN kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), according to the manufacturer’s instructions. One microgram of RNA was reverse-transcribed using a Takara RNA PCR Kit, version 2.1 (Takara Shuzo Co., Shiga, Japan), to make single-stranded cDNA. The ABI Prism Sequence Detection System 7000 and Primer Express Software (Applied Biosystems, Foster City, CA) were used for PCR amplification and quantitative analysis, respectively. For the IGF-I gene, a set of primers was designed using sequences obtained from the GenBank as follows: 5'GACAGATACARRCTGTGCTCA-3' and 5'-CTGAAGCTTGCTAACATCGC-3'. The PCR consisted of QuantiTect SYBR Green Master Mix (QIAGEN, Tokyo, Japan), 0.3 μM specific primers, and 20 ng cDNA.
For the IGF-I protein level measurement, primary osteoblasts were cultured, as described above, for 14 d, and the free IGF-I concentration in the culture media was measured with a Non-Extraction IGF-I ELISA kit (Diagnostic Systems Laboratories, Inc., Sparks, MD).
For ALP activity measurement, primary osteoblasts were cultured in the medium above with or without 10 ng/ml recombinant mouse IGF-I (Sigma-Aldrich Corp.) and 5 nM antimouse IGF-I antibody (Sigma-Aldrich Corp.). At 14 d of culture, cells were sonicated in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl2 and 0.5% Triton X-100, and ALP activity in the lysate was measured using an ALP assay kit (Wako Pure Chemical Industries Ltd.). The protein content was determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).
Immunoprecipitation and immunoblotting
After stimulation by 100 nM PTH for the indicated time, cultured osteoblasts were lysed with TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, and 10 μg/ml aprotinin). A part of the cell lysates (100 μg) was immunoprecipitated with an antiphosphotyrosine antibody, an antimouse IGF-I receptor antibody, an antimouse insulin receptor antibody, an antimouse IRS-1 antibody, or an antimouse IRS-2 antibody (all from Upstate Biotechnology, Inc., Waltham, MA) conjugated to protein G-Sepharose (Invitrogen) for 4 h at 4 C. The cell lysates with or without the immunoprecipitation that contained an equivalent amount of protein (20 μg) were electrophoresed by 8% SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% BSA solution, they were incubated with the antibodies above, and the immunoreactive bands were stained using the ECL chemiluminescence reaction (Amersham, Arlington Heights, IL). The intensity of each band was measured by densitometry (Bio-Rad Laboratories, Inc., Richmond, CA) and was expressed as the mean value of five independent experiments.
PTH treatment on mice
IRS-1–/– mice and their littermates and IRS-2–/– mice and their littermates (males, all n = 10) received either PTH (80 μg/kg body weight) or vehicle (PBS) by sc injection every day for 4 wk beginning at 10 wk of age. Blood samples were collected by heart puncture under nembutal (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) anesthesia before being killed. For radiological and histological analyses, animals were killed after 4 wk of PTH treatment by diethylether. The right femurs and tibiae were obtained for bone densitometry, and the left femurs and tibiae for peripheral quantitative computerized tomography (pQCT) and histological analyses, respectively. Lumbar vertebral bodies from L2–L5 were also obtained for bone densitometry.
Bone densitometry and pQCT
Bone mineral density (BMD; milligrams per square centimeter) of the right femur, tibiae, and L2–L5 vertebral bodies was determined using dual-energy x-ray absorptiometry (PIXImus Mouse Densitometer; Lunar Corp., Madison, WI) according to the manufacturer’s instructions. Computerized tomography was performed with a pQCT analyzer (XCT Research SA+; Stratec Medizintechnik GmbH, Pforzheim, Germany) operating at a resolution of 80 μm. Metaphyseal pQCT scans of the left femurs were performed to measure the trabecular volumetric BMD. The scan was positioned in the metaphysis at 1.2 mm proximal from the distal growth plate. Because this area contains trabecular and cortical bones, the trabecular bone region was defined by setting the threshold to 395 mg/cm3. Middiaphyseal pQCT scans of the left femurs were performed to determine the cortical thickness. The middiaphyseal region of femurs in mice contains mostly cortical bone. The cortical bone region was defined by setting the threshold to 690 mg/cm3. The interassay coefficients of variation for the pQCT measurements were less than 2%.
Histological analyses
For the assessment of dynamic histomorphometric indices, mice were injected with calcein (16 mg/kg body weight) sc at 10 d and 3 d before being killed, after which the left tibiae were excised and fixed with ethanol, and the undecalcified bones were embedded in glycolmethacrylate. Three-micrometer sagittal sections from the proximal parts of tibiae were stained with toluidine blue and were visualized under fluorescent light microscopy for calcein labeling. The specimens were subjected to histomorphometric analyses using a semiautomated system (Osteoplan II; Carl Zeiss, Oberkochen, Germany), and measurements were made at x400 magnification. Parameters for the trabecular bone were measured in an area 1.2 mm in length, from 250 μm below the growth plate at the proximal metaphysis of the tibiae. Nomenclature, symbols, and units are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research (18).
Serum biochemical assays
For serum IGF-I levels, acid ethanol extraction was used to remove the IGF-binding proteins, and the extracted samples were assayed for IGF-I with a RIA kit from Nichols Institute Diagnostics (San Juan Capistrano, CA). Serum osteocalcin levels were determined by using the competitive RIA kit (Biomedical Technologies, Stoughton, MA). The sensitivity of the assay was 19 ng/ml, and the interassay and intraassay coefficients of variation were less than 10%. Serum ALP activity was determined by liquitech ALP kit (Roche Diagnostics, Basel, Switzerland) with an autoanalyzer (type 7170; Hitachi High-Technologies Corporation, Tokyo, Japan). ALP activity of the blood samples was expressed as nanomoles per minute and per milligram of protein.
Statistical analysis
Means of groups were compared by ANOVA, and significance of differences was determined by post hoc testing using Bonferroni‘s method.
Results
Effects of PTH on cultured osteoblasts
We first examined the effects of recombinant human PTH(1–34) in the cultures of primary osteoblasts derived from mouse calvariae. IGF-I mRNA level determined by real-time RT-PCR, IGF-I protein level in the cultured medium, and ALP activity in the cell lysate were all increased about 2-fold with PTH (100 nM) treatment compared with the control cultures (Fig. 1A). A neutralizing antibody against IGF-I significantly, although not completely, suppressed the PTH stimulation of ALP activity. Furthermore, addition of a recombinant mouse IGF-1 at a concentration similar to that of endogenous IGF-I (10 mg/ml) stimulated by PTH increased the ALP activity to a level similar to that by PTH. These lines of results confirm that the PTH anabolic action is, at least partly, mediated by the IGF-I production in osteoblasts, as previously reported (8, 11, 12).
FIG. 1. Effects of PTH on cultured osteoblasts isolated from neonatal mouse calvariae. A, IGF-I mRNA level determined by real-time quantitative RT-PCR (left), IGF-I protein level in the culture medium (middle), and ALP activity in the cell lysate (right) in the primary calvarial osteoblast culture in the presence and absence of PTH (100 nM), an antibody against IGF-I (-IGF-I, 5 nM), and recombinant mouse IGF-I (rmIGF-I, 10 ng/ml) for 2 wk. Data are expressed as means (bars) ± SEM (error bars) for eight wells per group. *, Significant increase compared with the control culture, P < 0.01; #, significant inhibition by -IGF-I, P < 0.05. B, Protein levels by immunoprecipitation (IP) and immunoblotting (IB) of IGF-I receptor, insulin receptor, IRS-1, and IRS-2 with or without phosphorylation in osteoblasts cultured with PTH (100 nM) for the indicated times. Some of the cell lysates were immunoprecipitated with an antiphosphotyrosine antibody (-PY), and the cell lysates with or without (–) the immunoprecipitation were immunoblotted with an antimouse IGF-I receptor (-IGF-IR), an antimouse insulin receptor (-IR), an antimouse IRS-1 (-IRS-1), or an antimouse IRS-2 antibody (-IRS-2). Some of the cell lysates were reciprocally immunoprecipitated with -IGFR or -IR and immunoblotted with -PY. Blottings with an anti-?-actin (-actin) were used as loading controls. Similar results were obtained in five independent experiments. The graph below shows the mean values of the band intensities of phosphorylated proteins normalized to -actin quantified using densitometry in five independent experiments. Data are expressed as the ratio of the value at time zero. Although the data of proteins without phosphorylation are not shown in the graph, they were not significantly affected by PTH during the observation period (the ratio values were from 0.8–1.2). Data are expressed as means (bars) ± SEM (error bars) of five independent experiments. *, Significant difference from that at time zero, P < 0.01.
To provide some insights into signaling pathways that are involved in the PTH action on primary osteoblasts, we examined the phosphorylations of IGF-I receptor, insulin receptor, IRS-1, and IRS-2 in five independent experiments (Fig. 1B). Immunoprecipitation and immunoblotting analyses revealed that phosphorylations of IGF-I receptor and IRS-1 were clearly induced at 1 min and reached maximum at 5 min. IRS-2 was also phosphorylated by PTH, although not as strongly as IGF-I receptor and IRS-1. Insulin receptor was hardly phosphorylated by PTH. None of the protein levels of IGF-I receptor, insulin receptor, IRS-1, or IRS-2 were altered by PTH during the observation period up to 60 min, suggesting that PTH does not show transcriptional or translational regulation of these signaling molecules. These results indicate that IGF-I production followed by the activation of its intracellular signaling pathways may be related to the PTH action in osteoblasts.
Effects of PTH on bones in IRS-1–/– and IRS-2–/– mice
To learn the roles of the IRS-1 and IRS-2 in the PTH action on bone in vivo, we analyzed the PTH effects on the knockout mice by comparing them with those of respective WT littermates using radiological and histological analyses. Both knockout mice were healthy, with no abnormality in major organs except that IRS-1–/– mice alone showed about 20% shorter limbs and trunk, whereas IRS-2–/– mice were normal in size compared with WT littermates (19, 20, 21). The mice (10 wk old, males) were given daily sc injections of PTH (80 μg/kg) or vehicle for 4 wk, after which their femurs, tibiae, and lumbar vertebrae underwent radiological and histological analyses. As we previously reported for bone phenotypes under physiological conditions (16, 17), both knockout mice showed osteopenia when injected with vehicle: BMDs of femur, tibia, and lumbar vertebra in IRS-1–/– and those of femur and tibia in IRS-2–/– were significantly lower than respective WT littermates (Fig. 2). The PTH injection increased BMDs of these bones 10–20% in WT; however, this increase was hardly seen in the IRS-1–/– bones (Fig. 2A). The stimulations by PTH, on the contrary, seen in the IRS-2–/– bones were similar to those of the WT littermates (Fig. 2B). These results suggest that IRS-1, but not IRS-2, is needed for the bone anabolic action of PTH.
FIG. 2. Effects of PTH treatment on bone densities in IRS-1–/– mice (A) and IRS-2–/– mice (B), compared with respective WT littermates. IRS-1–/– mice and the littermates and IRS-2–/– mice and the littermates (males, 10 wk old, n = 10/group) received daily sc injections of either PTH (80 μg/kg body weight) or vehicle for 4 wk. Mice were killed; and the right femurs, tibiae, and L2–L5 vertebral bodies were excised. BMD values of the entire femurs, tibiae, and vertebral bodies were determined using dual-energy x-ray absorptiometry. Data are expressed as means (bars) ± SEM (error bars) of 10 bones per group. *, Significant effect of PTH, P < 0.01; #, significant difference from WT, P < 0.01.
We further examined trabecular and cortical bones separately in the femurs using pQCT (Fig. 3). In trabecular bones at the distal metaphysis of femurs, both IRS-1–/– and IRS-2–/– mice showed lower bone density (Fig. 3, A and C). PTH injection increased the trabecular bone density about 60% in WT. Here again, this PTH effect was abolished by the IRS-1 deficiency but was not altered by the IRS-2 deficiency. In the cortical bones at the midshaft of the femurs, although the PTH effects on cortical thickness were milder than those on the trabecular density, this was blocked by the IRS-1 deficiency, although not by the IRS-2 deficiency (Fig. 3, B and D).
FIG. 3. Effects of PTH treatment on trabecular and cortical bones in IRS-1–/– mice (A and B) and IRS-2–/– mice (C and D), compared with respective WT littermates. After daily injections of either PTH (80 μg/kg body weight) or vehicle for 4 wk, mice were killed, and the distal metaphysis (A and C) and the middiaphysis (B and D) of the excised left femurs underwent pQCT analysis. The color gradient indicating bone density is shown in the right bars. The trabecular density at the metaphysis and the cortical thickness at the middiaphysis are shown in the graphs below. Data in all graphs are expressed as means (bars) ± SEMs (error bars) of 10 bones per group. *, Significant effect of PTH, P < 0.01; #, significant difference from WT, P < 0.01.
Histological features of the proximal tibiae showed decreases of trabecular bones in vehicle-treated IRS-1–/– and IRS-2–/– mice compared with the respective WT littermates (Fig. 4). After PTH treatment for 4 wk, increases of these bones were observed in IRS-2–/– and WT littermates to a similar extent (Fig. 4B), whereas no increase was observed in the IRS-1–/– trabeculae (Fig. 4A). Bone histomorphometric measurements in this area confirmed that the PTH injection augmented the bone volume (trabecular bone volume expressed as a percent of total tissue volume) of WT mice by 50–60%, with the increases of both bone formation parameters (percent of bone surface covered by cuboidal osteoclasts, and bone formation rate) and resorption parameters (percent of bone surface covered by mature osteoclasts, and percent of eroded surface), indicating a high bone turnover state (Table 1). IRS-1–/– and IRS-2–/– mice showed 30–40% lower bone volume than respective WT littermates when injected with vehicle. As we previously reported (16, 17), IRS-1–/– exhibited a low bone turnover, with decreases in both bone formation and resorption parameters, whereas IRS-2–/– mice showed an uncoupling status of bone turnover, with decreased bone formation and increased bone resorption. The PTH injection affected neither the bone volume nor the bone turnover in the IRS-1–/– mice; however, in the IRS-2–/– mice, PTH increased bone volume mainly through the up-regulation of bone formation rather than bone resorption.
FIG. 4. Effects of PTH treatment on histological features of the proximal metaphysis of tibiae in IRS-1–/– mice (A) and IRS-2–/– mice (B), compared with respective WT littermates. After death, the left tibiae were excised, fixed, and embedded in GMA without decalcification, and the sagittal sections were stained by toluidine blue. Representative samples are shown from mice of each genotype given either PTH or vehicle. Data of histomorphometric analyses are shown in Table 1. Bar, 100 μm.
TABLE 1. Histomorphometry of trabecular bones in proximal tibiae
Effects of PTH on blood chemistries in IRS-1–/– and IRS-2–/– mice
The serum markers osteocalcin and ALP supported the increase of bone formation by the PTH injection in WT mice (Fig. 5). Here again, these stimulations were not seen in IRS-1–/– mice but were maintained in IRS-2–/– mice. Because the serum IGF-I levels were not different between PTH- and vehicle-treated mice in all genotypes, IGF-I that is induced by PTH, as shown in Fig. 1A, seemed not to act as a systemic factor but to act locally in bone as an autocrine/paracrine factor.
FIG. 5. Effects of PTH treatment on serum osteocalcin, ALP, and IGF-I levels in IRS-1–/– mice and IRS-2–/– mice, compared with respective WT littermates. Mice received either PTH or vehicle for 4 wk as described above, and blood samples were collected by heart puncture before death. The levels were measured as described in Materials and Methods. *, Significant effect of PTH, P < 0.05; #, significant difference from WT, P < 0.05.
Discussion
The present study demonstrated that the bone anabolic function of PTH is mediated by the activation of IGF-1R and IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally in bone. Although IRS-1 and IRS-2 are known to be essential for intracellular signaling of IGF-I and insulin, these two adaptor molecules have distinct biological roles and are differentially expressed in a variety of cells. Regarding glucose homeostasis, IRS-1 plays an important role in the metabolic actions of insulin, mainly in skeletal muscle and adipose tissue, whereas IRS-2 does so in the liver (22). Our previous studies revealed that only IRS-1, but not IRS-2, was expressed in the cartilage of the growth plate or the fracture callus, so that skeletal growth and fracture healing were impaired in IRS-1–/– mice, whereas they were normal in IRS-2–/– mice (21, 23). In bone, IRS-1 is expressed solely in cells of osteoblast lineage, whereas IRS-2 is expressed in cells of both osteoblast and osteoclast lineages (16, 17). As described above, our previous studies on bones of these two knockout mice disclosed that IRS-1 is important for maintaining bone turnover, and IRS-2 for maintaining predominance of anabolic function over catabolic function of osteoblasts (16, 17). In the meantime, previous and present studies have shown that PTH treatment increases bone turnover in animals and humans (24, 25). The fact that the suppression of bone turnover by IRS-1 deficiency suppressed the bone anabolic action of PTH suggests the importance of elevated turnover for the PTH function. This is consistent with the results of clinical studies showing that the concurrent use of alendronate, a potent bisphosphonate that markedly suppresses bone turnover, reduced the bone anabolic action of PTH in male and female osteoporosis patients (26, 27).
Because it is unlikely that PTH directly activates IGF-I receptor and IRS-1, there seem to be two possible molecular mechanisms underlying the suppression of PTH action by the IRS-1 deficiency: 1) PTH induces IGF-I production, causing IGF-I receptor and IRS-1 activation; and 2) IRS-1 signaling affects the intracellular signaling lying downstream of the PTH/PTH-related protein receptor after PTH binds to it. The quick phosphorylation of IGF-I receptor and IRS-1 after PTH treatment in primary osteoblast culture in the present study (Fig. 1B) supports the former mechanism. Hormones like PTH and prostaglandin E2 that increase cAMP synthesis and PKA activation are reported to induce the transcription of IGF-I by way of a C/EBP (CCAAT/enhancer-binding protein)-sensitive element in exon 1 of the IGF-I gene (28, 29). However, the latter possibility cannot be denied, because the inhibition of the PTH stimulation on ALP activity by a neutralizing antibody against IGF-I was not complete, but partial, in the primary osteoblast culture (Fig. 1A). In fact, IRS-1 and PTH/PPR (PTH/PTH-related protein receptor) are known to share several common signaling pathways. The main pathways lying downstream of IRS-1 are PI3K/Akt and MAPKs, which are important regulators of cell growth and differentiation. PTH has been shown to directly activate the p42/p44 MAPK by protein kinase C-dependent, but Ras-independent, signaling in rat osteoblasts (30) and to up-regulate PI3K/Akt activity, which contributes to the MAPK activation in rat enterocytes (31). In addition, both PTH and IGF-I have been shown to be involved in the activation of c-fos expression (32, 33) and cyclin-dependent kinase expression in osteoblasts (34, 35). These lines of evidence indicate the PTH signaling pathway may possibly be affected by the IRS-1 signaling at several points, and the absence of an IRS-1 signaling pathway may result in the failure of PTH to stimulate key target molecules necessary for its anabolic action.
Another potential explanation for the lack of PTH response in IRS-1–/– mice could be the impairment of proliferation or differentiation ability of osteoprogenitor cells, so that they are insensitive not only to PTH but also to other stimulations. In fact, our present and previous studies demonstrated the decreases in histomorphometric parameters and serum markers for bone formation in IRS-1–/– mice under physiological conditions (16). In this regard, however, our previous study has shown that proliferation and differentiation of primary calvarial cells were stimulated, responding to fibroblast growth factor-2 and bone morphogenetic protein-2, respectively, similarly to those of the WT cells, indicating that functions of IRS-1–/– cells are normal as long as adequate signals other than IGF-I signal were applied (16). Hence, the decreased bone formation under physiological conditions in IRS-1–/– mice is likely to be due to the deficit of anabolic signaling of endogenous IGF-I. Interestingly, PTH increased the IGF-I protein level in the culture medium of primary osteoblasts (Fig. 1A) but did not affect the serum IGF-I level in vivo (Fig. 5), indicating the importance of local action of IGF-I as an autocrine/paracrine factor for bone formation rather than its systemic action as a hormone. This result is consistent with previous reports that disruption of IGF-I genes, specifically in liver, decreased serum IGF-I by 80% but caused no skeletal abnormality (36, 37). Because the remaining 20% IGF-I in serum is probably derived from several tissues, including bone, it is not surprising that PTH treatment did not increase circulating levels of IGF-I. The findings that PTH anabolic effects can be suppressed by an IGF-I-neutralizing antibody in osteoblast cultures in the present and previous studies (11, 12) also support the idea that the PTH-induced bone formation involves increased local production, but not increased circulating levels, of IGF-I.
The requirement of IGF-I/IRS-1 for mediating the anabolic effects of bone regulatory hormones may not be unique to PTH, because previous findings have revealed that many of the major hormones exert significant effects on IGF-I expression. GH is a well-known stimulus of IGF-I production in a variety of tissues, including bone, and exerts its effects on bone mainly through IGF-I mediation (38). Estradiol, another important regulator of skeletal metabolism, has been shown to increase IGF-I production, and IGF-I receptor-blocking antibodies inhibited the proliferative effect of estradiol in rat osteoblasts (39, 40). Similarly, other hormones with potent effects on bone, such as thyroid hormone and androgens, alter IGF-I levels in bone in a manner consistent with IGF-I playing a role in the actions of these hormones on bone (41, 42). Although these studies did not examine the involvement of IRS-1 or IRS-2 in their anabolic actions, it is possible that the IGF/IRS pathway might be a common signaling for actions of these major hormones in bone. Further understanding of the molecular mechanism by which the hormones induce IGF-I and the intracellular signaling that lies downstream of IGF-I/IRS in osteoblasts will greatly help to elucidate the complex network of bone formation under systemic regulations.
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