Impairment of Bone Healing by Insulin Receptor Substrate-1 Deficiency
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m312525200
ISSN1083-351X
AutoresTakashi Shimoaka, Satoru Kamekura, Hirotaka Chikuda, Kazuto Hoshi, Ung‐il Chung, Toru Akune, Zenjiro Maruyama, Toshihisa Komori, Michihiro Matsumoto, Wataru Ogawa, Yasuo Terauchi, Takashi Kadowaki, Kozo Nakamura, Hiroshi Kawaguchi,
Tópico(s)TGF-β signaling in diseases
ResumoInsulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1-/- mice). Among 15 IRS-1-/- mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1-/- fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1-/- chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1-/- culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine. Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1-/- mice). Among 15 IRS-1-/- mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1-/- fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1-/- chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1-/- culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine. In efforts to develop more advanced skeletal regenerative medicine through genetic manipulation, we have been attempting to identify genes implicated in bone and cartilage formation in vivo. Healing of bone fracture is composed of complex multistep processes involving a variety of cellular events for bone and cartilage regeneration (1Bolander M.E. Proc. Soc. Exp. Biol. Med. 1992; 200: 165-170Crossref PubMed Scopus (544) Google Scholar, 2Kawaguchi H. Kurokawa T. Hanada K. Hiyama Y. Tamura M. Ogata E. Matsumoto T. Endocrinology. 1994; 135: 774-781Crossref PubMed Scopus (273) Google Scholar). Under the periosteum adjacent to the fracture gap, undifferentiated mesenchymal cells start differentiation directly to cells of osteoblastic lineage for the membranous ossification, whereas in granulation tissue inside the fracture gap, these mesenchymal cells undergo endochondral bone formation; they differentiate first into chondrocytes to form cartilage which is subsequently replaced by calcified tissues. The size and quality of fracture callus that determine the mechanical property of the fracture site are mostly dependent on the latter process. Because the endochondral bone formation also takes place in embryonic development and in skeletal growth after birth, understanding the molecular mechanism of fracture healing may not only help treat non-union and delayed union of fracture itself but also help advance bone regenerative medicine.Insulin-like growth factor-I (IGF-I) 1The abbreviations used are: IGF, insulin-like growth factor; IRS, insulin receptor substrate; WT, wild-type; BMC, bone mineral content; HE, hematoxylin-eosin; PCNA, proliferating cell nuclear antigen; PBS, phosphate-buffered saline; TUNEL, terminal transferase dUTP nick end labeling; AxLacZ, adenovirus vector carrying β-galactosidase gene; DMEM, Dulbecco's modified Eagle's medium; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; IGF-IR, insulin-like growth factor-I receptor; SHC, Src homology collagen; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; FBS, fetal bovine serum; TdR, [3H]thymidine; PI3K, phosphatidylinositol 3-kinase. 1The abbreviations used are: IGF, insulin-like growth factor; IRS, insulin receptor substrate; WT, wild-type; BMC, bone mineral content; HE, hematoxylin-eosin; PCNA, proliferating cell nuclear antigen; PBS, phosphate-buffered saline; TUNEL, terminal transferase dUTP nick end labeling; AxLacZ, adenovirus vector carrying β-galactosidase gene; DMEM, Dulbecco's modified Eagle's medium; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; IGF-IR, insulin-like growth factor-I receptor; SHC, Src homology collagen; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; FBS, fetal bovine serum; TdR, [3H]thymidine; PI3K, phosphatidylinositol 3-kinase. plays important roles in the anabolic regulation of bone and cartilage metabolism (3Canalis E. Bone (NY). 1993; 14: 273-276Crossref PubMed Scopus (94) Google Scholar). Osteoblasts and chondrocytes produce this growth factor, express its receptor, and respond to it (3Canalis E. Bone (NY). 1993; 14: 273-276Crossref PubMed Scopus (94) Google Scholar, 4Laron Z. Mol. Pathol. 2001; 54: 311-316Crossref PubMed Scopus (338) Google Scholar). IGF-I appears essential for normal bone development because deletion of IGF-I or its receptor leads to a reduction in bone size at birth (5Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2553) Google Scholar, 6Powell-Braxton L. Hollingshead L.P. Warburton C. Dowd M. Pitts-Meek S. Dalton D. Gillett N. Stewart T.A. Genes Dev. 1993; 7: 2609-2617Crossref PubMed Scopus (670) Google Scholar). Clinically, patients with Laron syndrome caused by IGF-I deficiency exhibit growth retardation and osteoporosis (7Laron Z. Klinger B. Silbergeld A. J. Bone Miner. Res. 1999; 14: 156-157Crossref PubMed Scopus (14) Google Scholar). IGF-I is also reported to be expressed during fracture healing and to stimulate it, suggesting a role as an autocrine/paracrine factor potentiating bone regeneration (8Trippel S.B. Clin. Orthop. Relat. Res. 1998; 355: S301-S313Crossref PubMed Scopus (84) Google Scholar, 9Schmidmaier G. Wildemann B. Heeger J. Gabelein T. Flyvbjerg A. Bail H.J. Raschke M. Bone (NY). 2002; 31: 165-172Crossref PubMed Scopus (99) Google Scholar). Insulin also plays important roles in the anabolic regulation of bone and cartilage metabolism (10Thomas D.M. Hards D.K. Rogers S.D. Ng K.W. Best J.D. Endocrinol. Metab. Clin North Am. 1997; 4: 5-17Google Scholar). Although the anabolic effect of insulin on bone may be primarily related to its ability to stimulate osteoblast proliferation, that on cartilage may involve the acceleration of chondrocyte differentiation (11Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar, 12Kato Y. Gospodarowicz D. J. Cell Physiol. 1984; 120: 354-363Crossref PubMed Scopus (74) Google Scholar). Patients with insulin deficiency as exemplified by type 1 diabetes mellitus are associated with osteoporosis (13Krakauer J.C. McKenna M.J. Rao D.S. Whitehouse F.W. Diabetes Care. 1997; 20: 1339-1340Crossref PubMed Scopus (18) Google Scholar, 14Piepkorn B. Kann P. Forst T. Andreas J. Pfutzner A. Beyer J. Horm. Metab. Res. 1997; 29: 584-591Crossref PubMed Scopus (118) Google Scholar). Diabetes has also been shown to impair fracture healing, which is restored by treatment with insulin in both humans and animals (2Kawaguchi H. Kurokawa T. Hanada K. Hiyama Y. Tamura M. Ogata E. Matsumoto T. Endocrinology. 1994; 135: 774-781Crossref PubMed Scopus (273) Google Scholar, 15Macey L.R. Kana S.M. Jingushi S. Terek R.M. Borretos J. Bolander M.E. J. Bone Jt. Surg. Am. 1989; 71: 722-733Crossref PubMed Scopus (183) Google Scholar, 16Loder R.T. Clin. Orthop. 1988; 232: 210-216PubMed Google Scholar).Both IGF-I and insulin initiate cellular responses by binding to distinct cell-surface receptor tyrosine kinases that regulate a variety of signaling pathways controlling metabolism, growth, and survival. Insulin receptor substrates (IRSs) are essential substrates of the receptor tyrosine kinases, which integrate the pleiotropic effects of IGF-I and insulin on cellular function (17Burks D.J. White M.F. Diabetes. 2001; 50: 140-145Crossref PubMed Google Scholar, 18Kadowaki T. Tobe K. Honda-Yamamoto R. Tamemoto H. Kaburagi Y. Momomura K. Ueki K. Takahashi Y. Yamauchi T. Akanuma Y. Yazaki Y. Endocr. J. 1996; 43 (suppl.): 33-41Crossref PubMed Google Scholar). 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 reported previously that IRS-1 and IRS-2 are expressed in bone (19Ogata N. Chikazu D. Kubota N. Terauchi Y. Tobe K. Azuma Y. Ohta T. Kadowaki T. Nakamura K. Kawaguchi H. J. Clin. Investig. 2000; 105: 935-943Crossref PubMed Scopus (223) Google Scholar, 20Akune T. Ogata N. Hoshi K. Kubota N. Terauchi Y. Tobe K. Azuma Y. Kadowaki T. Nakamura K. Kawaguchi H. J. Cell Biol. 2002; 159: 147-156Crossref PubMed Scopus (102) Google Scholar). Our further studies on bone metabolism of mice lacking the IRS-1 gene (IRS-1-/- mice) or the IRS-2 gene (IRS-2-/- mice) revealed that IRS-1 is important for maintaining bone turnover (19Ogata N. Chikazu D. Kubota N. Terauchi Y. Tobe K. Azuma Y. Ohta T. Kadowaki T. Nakamura K. Kawaguchi H. J. Clin. Investig. 2000; 105: 935-943Crossref PubMed Scopus (223) Google Scholar), whereas IRS-2 is important for maintaining predominance of anabolic function over catabolic function of osteoblasts (20Akune T. Ogata N. Hoshi K. Kubota N. Terauchi Y. Tobe K. Azuma Y. Kadowaki T. Nakamura K. Kawaguchi H. J. Cell Biol. 2002; 159: 147-156Crossref PubMed Scopus (102) Google Scholar). Regarding the role of these molecules on bone growth, IRS-1, but not IRS-2, seems to play an important role in the growth plate function, because IRS-1-/- mice were about 20-30% shorter in limbs and trunk, whereas IRS-2-/- mice were normal in size as compared with wild-type (WT) littermates (19Ogata N. Chikazu D. Kubota N. Terauchi Y. Tobe K. Azuma Y. Ohta T. Kadowaki T. Nakamura K. Kawaguchi H. J. Clin. Investig. 2000; 105: 935-943Crossref PubMed Scopus (223) Google Scholar, 20Akune T. Ogata N. Hoshi K. Kubota N. Terauchi Y. Tobe K. Azuma Y. Kadowaki T. Nakamura K. Kawaguchi H. J. Cell Biol. 2002; 159: 147-156Crossref PubMed Scopus (102) Google Scholar, 21Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka Y. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar, 22Kubota N. Tobe K. Terauchi Y. Eto K. Yamauchi T. Suzuki R. Tsubamoto Y. Komeda K. Nakano R. Miki H. Satoh S. Sekihara H. Sciacchitano S. Lesniak M. Aizawa S. Nagai R. Kimura S. Akanuma Y. Taylor S.I. Kadowaki T. Diabetes. 2000; 49: 1880-1889Crossref PubMed Scopus (423) Google Scholar). These data raise an interesting possibility that IRS-1 may be essential for endochondral ossification. To assess this possibility, the present study investigated the role of IRS-1 in bone healing and its mechanism by an in vivo fracture model and an in vitro culture system.EXPERIMENTAL PROCEDURESAnimals—Mice in a C57BL6/CBA hybrid background were generated and maintained as reported previously (21Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka Y. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar). WT and IRS-1-/- male littermates generated from the intercross between heterozygous IRS-1+/- mice were compared. All experiments were performed according to the protocol approved by the Animal Care and Use Committee of the University of Tokyo.Fracture Model—Twenty five male mice at 8 weeks of age were used in each group. Under general anesthesia with pentobarbiturate (0.5 mg/10 g body weight, Sigma), the bilateral hind limbs were shaved and sterilized. A 15-mm incision was made longitudinally over the right leg, and a blunt dissection of the muscle was made to expose the tibia. The middle point of the tibia was marked with a surgical marker, and a transverse osteotomy was performed using a bone saw (Volvere GX, NSK Nakanishi Inc., Tochigi, Japan). The fracture was repositioned, and then the full-length of the bone marrow cavity was internally stabilized with an intramedullary nail using the inner pin of a spinal needle of 22- or 23-gauge diameter depending on the size of the cavity. After irrigation with saline, the skin was sutured. The left tibia (unfractured side) was sham-operated and an intramedullary nail of the same size as the control was inserted. No external fixation was used, and the animals were allowed unrestricted activity as well as diet and water ad libitum. For histological analyses, animals were killed at 1 (n = 4/group), 3 (n = 3/group), and 6 weeks (n = 3/group) after the operation by diethyl ether, and bilateral tibiae were excised. After extracting the intramedullary nail gently so as not to injure the fracture site, the soft tissue surrounding the tibiae, except for the soft callus around the fracture site, was removed.Radiological Analysis—X-ray pictures of the right tibiae of WT and IRS-1-/- mice (n = 15 each) were taken at 0 (immediately after the operation), 1-3, 6, and 10 weeks after the operation under general anesthesia using a soft x-ray apparatus (CMB-2; Softex Co., Tokyo, Japan). To determine whether there was bone union, bony bridging on radiographs was evaluated by individuals who were blinded with regard to the genotype of mice.Measurement of Callus Area and Bone Mineral Content (BMC)—Area and BMC of the entire bilateral tibia were measured by a single energy x-ray absorptiometry utilizing a bone mineral analyzer for small animals (PIXIMUS, Lunar Co., Ltd., WI) at 0 (immediately after the operation), 1-4, and 6 weeks after the operation. A preliminary experiment revealed that the intramedullary nail did not affect the BMC value. The gain of area and the % gain of BMC during observation periods as compared with those at time 0 were calculated for both fractured and unfractured sides, and the differences were compared between WT and IRS-1-/-.Histological Analysis—Specimens of the harvested tibiae were fixed with 4% paraformaldehyde in 0.1 mol/liter phosphate buffer, pH 7.4, at 4 °C overnight. After decalcification with 4.13% EDTA at 4 °C for 14 days, the tibiae were dehydrated with an increasing concentration of ethanol, embedded in paraffin, and cut into 4-μm-thick sections. The sections were stained with hematoxylin-eosin (HE) or toluidine blue.Immunohistochemistry—Immunohistochemical localizations of IRS-1, IRS-2, type X collagen, and proliferating cell nuclear antigen (PCNA) were examined in 4-μm dewaxed paraffin sections. After dehydration, the sections were treated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min at room temperature. After blocking by PBS containing 1% bovine serum albumin (Sigma) for 1 h at room temperature, the sections were incubated in polyclonal rabbit antibody against IRS-1, IRS-2, or type X collagen (Santa Cruz Biotechnology) or monoclonal mouse antibody against PCNA (PC10, Sigma) (23Smink J.J. Gresnigt M.G. Hamers N. Koedam J.A. Berger R. Van Buul-Offers S.C. J. Endocrinol. 2003; 177: 381-388Crossref PubMed Scopus (52) Google Scholar), at a dilution of 1:100 for 24 h at 4 °C. As negative controls, we used non-immune rabbit IgG and mouse IgG of the same dilution instead of the primary antibodies. Then the sections were rinsed in PBS and incubated with the horseradish peroxidase-conjugated goat antibody against rabbit IgG (Dakopatts, Glostrup, Denmark) for immunohistochemistry of IRS-1, IRS-2, and type X collagen, and with the horseradish peroxidase-conjugated goat antibody against mouse IgG (EY Laboratories, Inc., San Mateo, CA) for that of PCNA, respectively, for 1 h at room temperature. After washing with PBS, the sections were immersed in a diaminobenzidine solution for 10 min at room temperature to visualize immunoreactivity. Terminal transferase dUTP nick-end labeling (TUNEL) staining was performed using an Apoptosis in Situ Detection kit (Wako Pure Chemical Co., Ltd., Osaka, Japan) according to the manufacturer's instructions.Generation of Adenoviruses and Gene Transfer—The recombinant adenovirus vector carrying human IRS-1 gene engineered to express hemagglutinin tag at its N terminus was constructed using an Adenovirus Expression Vector kit (Takara Shuzo Co., Ltd., Shiga, Japan) following the manufacturer's protocol. The adenovirus vector carrying β-galactosidase gene (AxLacZ) was kindly provided by Dr. I. Saito (University of Tokyo). Two days after the operation, a 1 × 1012 plaque-forming units suspension of AxIRS-1 or AxLacZ was injected into the fracture site of IRS-1-/- mice as described previously (24van Griensven M. Lobenhoffer P. Barke A. Tschernig T. Lindenmaier W. Krettek C. Gerich T.G. Lab. Anim. 2002; 36: 455-461Crossref PubMed Scopus (16) Google Scholar, 25Uusitalo H. Hiltunen A. Ahonen M. Gao T.J. Lefebvre V. Harley V. Kahari V.M. Vuorio E. J. Bone Miner. Res. 2001; 16: 1837-1845Crossref PubMed Scopus (62) Google Scholar). The same dose of AxLacZ was also injected to WT mice as a positive control. Animals were sacrificed at 1 week (n = 3/group) and 3 weeks (n = 3/group) after the injection. To confirm the infection efficiency, expression of lacZ was examined by histochemical staining by X-gal staining buffer (1 mg/ml X-gal, 5 mm potassium ferrocyanide, and 5 mm potassium ferricyanide) (Wako).Isolation and Culture of Mouse Growth Plate Chondrocytes—Chondrocytes were isolated from epiphyseal growth plates of WT and IRS-1-/- mice at 3.5 weeks of age. Mice were sacrificed, and tibiae were harvested and cleaned of perichondrium in an aseptic manner. Tibiae were pretreated with 0.3% collagenase in serum-free Dulbecco's modified Eagle's medium (DMEM; Sigma) at 37 °C for 30 min to remove residual tissue. By washing the tibiae with PBS, all soft tissues were detached. Growth plates were dissected microscopically by inserting a 26-gauge needle. Subsequently, tibiae were digested with 0.3% collagenase in serum-free DMEM at 37 °C for 5 h, and matrix debris was removed by filtering through a 70-μm cell strainer (BD Biosciences). Chondrocytes were pelleted by centrifugation and washed twice with PBS. Cells were plated in 6-multiwell dishes at a density of 5,000 cells/cm2 and grown to confluence in DMEM containing 10% FBS and antibiotics in a humidified CO2 incubator.X-gal Staining of Chondrocytes Isolated from Transgenic Mice with Type I Collagen Promoter or Type II Collagen Promoter Driving the lacZ Gene—To confirm the purity of chondrocytes isolated by the method above, transgenic mice expressing osteoblast- or chondrocyte-specific marker gene construct (the 2.3-kb fragment of the α1(I) collagen gene promoter or the 1.0-kb fragment of α1(II) collagen promoter and 0.6-kb enhancer) linked to the Escherichia coli lacZ gene were used (26Liu W. Toyosawa S. Furuichi T. Kanatani N. Yoshida C. Liu Y. Himeno M. Narai S. Yamaguchi A. Komori T. J. Cell Biol. 2001; 155: 157-166Crossref PubMed Scopus (380) Google Scholar, 27Ueta C. Iwamoto M. Kanatani N. Yoshida C. Liu Y. Enomoto-Iwamoto M. Ohmori T. Enomoto H. Nakata K. Takada K. Kurisu K. Komori T. J. Cell Biol. 2001; 153: 87-100Crossref PubMed Scopus (325) Google Scholar). Expression of lacZ was examined by histochemical staining with X-gal. Cells were isolated from the growth plates of transgenic mice by the method above and were cultured for 2 days. They were rinsed in PBS twice and fixed with 0.25% glutaraldehyde in PBS on ice for 10 min. After fixed samples were washed in PBS, staining was carried out by the X-gal staining buffer described above at 37 °C overnight.Western Blot Analysis—To examine the IRS-1 and IRS-2 protein levels, chondrocytes isolated from WT and IRS-1-/- growth plates described above were plated in 6-multiwell plates at a density of 105 cells/well and incubated in DMEM containing 10% FBS for 24 h. For comparison, we also examined the protein levels in primary osteoblasts that were isolated from neonatal mouse calvariae and cultured in α-minimum Eagle's medium containing 10% FBS as described previously (28Shimoaka T. Ogasawara T. Yonamine A. Chikazu D. Kawano H. Nakamura K. Itoh N. Kawaguchi H. J. Biol. Chem. 2002; 277: 7493-7500Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To investigate the signaling pathways through phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs), primary chondrocytes were pre-incubated in DMEM containing 10% FBS for 24 h, and treated with IGF-I (10 nm) in the presence and absence of LY294002 (10 μm), PD98059 (10 μm), and SB203580 (10 μm) (all from Calbiochem-Novabiochem) for 30 min. Cells were lysed with TNE buffer (10 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 10 mm NaF, 2 mm Na3VO4, 1 mm aminoethylbenzenesulfonyl fluoride, and 10 μg/ml aprotinin), and the protein concentration in the cell lysate was measured using a Protein Assay Kit II (Bio-Rad). Equivalent amounts (20 μg) of cell lysates were electrophoresed by 8% SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% bovine serum albumin, the membrane was incubated with polyclonal rabbit antibodies against IRS-1 and IRS-2 as described above, Akt, phospho-Akt, extracellular signal-regulated kinase (ERK), phospho-ERK, p38, phospho-p38 MAPK (all from Cell Signaling Technology, Inc. Beverly, MA) and against actin (Sigma). Immunoreactive bands were visualized using the ECL chemiluminescence reaction (Amersham Biosciences) following the manufacturer's instructions. Signals were quantified by densitometry (Bio-Rad).DNA Synthesis and Proliferation Assays—DNA synthesis and proliferation of isolated chondrocytes were determined by the [3H]thymidine (TdR) uptake and the growth curve, respectively. For the former assay, primary chondrocytes from WT or IRS-1-/- mice were inoculated at a density of 5 × 104 cells/well in a 24-multiwell plate and cultured to confluency in DMEM, 10% FBS for 2 days. Serum was withheld for 12 h before adding the experimental medium with or without IGF-I (10 nm), LY294002 (1, 3, and 10 μm), PD98059 (1, 3, and 10 μm), and SB203580 (1, 3, and 10 μm). Uptake of [3H]TdR (1 μCi/ml in the medium) added for the final 2 h was measured at 18 h. For the growth curve assay, primary chondrocytes from WT or IRS-1-/- mice were inoculated at a density of 105 cells/well in 6-multiwell plates in DMEM, 10% FBS and cultured with or without IGF-I (10 nm). The number of cells/well was counted 1, 3, 5, 7, and 9 days after the seeding.Statistical Analysis—Means of groups were compared by analysis of variance, and significance of differences was determined by post-hoc testing using Bonferroni's method.RESULTSRadiological Findings—Fig. 1A shows an x-ray feature of the fracture model that we used in this study. This model was confirmed to show the bone healing process similar to that in humans in a definite temporal sequence by the time course of x-ray examination in WT mice (Fig. 1B, upper panel). Callus formation could be detected at 1 week, and bony bridging at the fracture site was completed 2 or 3 weeks after the fracture. After the callus size and density reached maximum around 3 weeks, they decreased gradually due to bone remodeling up to 10 weeks. In IRS-1-/- mice, however, neither callus formation nor bridging between the fracture stumps was seen at the early stage, and the fracture site became atrophic without bone union at 10 weeks. Fig. 1C shows x-ray features of the fracture site of all 15 mice in each of WT and IRS-1-/- groups at 3 weeks after fracture. In WT mice, bone union with substantial hard callus formation was observed in all 15 animals. In IRS-1-/- mice, however, fracture healing was extremely impaired, and 12 out of 15 mice showed no bone union (the 12 panels at left). Although bone union was seen in 3 IRS-1-/- mice (the 3 panels at right), the callus looked much smaller and fainter than that of WT mice.The time course of the number of animals with fracture union determined by bony bridging on x-ray revealed that 4 WT mice achieved bone union at 2 weeks after the operation, and all 15 mice did so at 3 weeks. However, only 3 IRS-1-/- mice showed bone union at 3 weeks, but the other 12 animals remained in a non-union state even at 10 weeks (Fig. 2A).Fig. 2Time course of the number of mice with bone union (A), the gain of area (B), and the % gain of BMC (C) at the fracture sites of WT and IRS-1-/- mice. To determine the bone union, the bony bridging at the fracture site on x-rays was judged by individuals blinded with regard to the genotype. The gain of area and the % gain of BMC during observation periods as compared with those at time 0 were calculated for the entire tibiae of fractured and unfractured sides, and the differences were compared between WT and IRS-1-/- callus. Because the fracture site became too displaced to measure these parameters correctly at the later stage in IRS-1-/- mice, the number of mice was decreased as shown in parentheses. Data in B and C are expressed as the mean (symbols) ± S.E. (error bars) for 15 WT mice, and the number shown in parentheses for IRS-1-/- mice. #, p < 0.05; *, p < 0.01 versus WT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Callus Area and BMC—To quantify the callus formation, differences in the gain of area and the % gain of BMC between the fractured and unfractured tibiae were measured by a bone densitometer (Fig. 2, B and C). Significant differences between WT and IRS-1-/- mice were seen from 1 to 4 weeks in the callus area, and from 2 weeks to the end in the BMC. In WT mice, both parameters were increased at the early stage of healing due to the acceleration of the modeling process and decreased thereafter due to remodeling. In IRS-1-/- mice, these parameters remained at low levels throughout the observation period. At the later stage in the IRS-1-/- mice, the fracture site became displaced due to non-union, and at 10 weeks more than half the IRS-1-/- mice showed severe displacement which was beyond evaluation. To exclude the possibility of other parts of tibiae outside the fracture site affecting BMC, BMC at the bilateral femurs and distal third tibiae was measured at 3 and 6 weeks. Because there were no differences between the fractured and unfractured sides (data not shown), the BMC decrease in IRS-1-/- mice was caused by the decrease in that of the callus itself.Histological Findings—To assess the involvement of IRS-1 and IRS-2 in bone healing, we examined the localizations of these proteins at the fracture site in WT and IRS-1-/- mice at 3 weeks after fracture (Fig. 3A). Immunohistochemical analysis of WT callus revealed that IRS-1 was localized at various cells including chondrocytes and fibroblasts, although IRS-2 immunoactivity was very faint. In IRS-1-/- mice, both IRS-1 and IRS-2 proteins were barely detectable at the fracture site, suggesting that there was no compensatory up-regulation of IRS-2 by IRS-1 deficiency.Fig. 3Immunolocalization of IRS-1 and IRS-2 (A) and time course of histological findings (B) at the fracture sites of WT and IRS-1-/- mice. A, immunohistochemical stainings with an anti-IRS-1 antibody (α-IRS-1, left) and an anti-IRS-2 antibody (α-IRS-2, right) were performed on the fracture callus of WT and IRS-1-/- mice at 3 weeks. Positive and specific stainings by α-IRS-1 shown in brown are seen in fibroblasts and chondrocytes of the WT callus. No immunostaining was observed by the respective non-immune rabbit IgGs as negative controls (data not shown). Bar, 100 μm. B, 1, 3, and 6 weeks after the fracture, specimens of the harvested tibiae were stained with HE. At 1 week after fracture, the soft callus outlined by arrowheads is much larger in the WT fracture site than in IRS-1-/-. At 3 weeks, the WT callus was mineralized as outlined by arrowheads, which was hardly seen in the IRS-1-/- specimen. Bar, 1 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3B shows the temporal comparison of histology between WT and IRS-1-/- fracture sites. At 1 week after fra
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