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Transforming Growth Factor-β-induced Osteoblast Elongation Regulates Osteoclastic Bone Resorption through a p38 Mitogen-activated Protein Kinase- and Matrix Metalloproteinase-dependent Pathway

2001; Elsevier BV; Volume: 276; Issue: 42 Linguagem: Inglês

10.1074/jbc.m008738200

1083-351X

M.A. Karsdal, Marianne Scheel Fjording, Niels T. Foged, Jean‐Marie Delaissé, André Lochter,

Bone health and treatments

Transforming growth factor-β (TGF-β) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-β-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-β1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-β1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-β1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-β1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-β on bone metabolism. Transforming growth factor-β (TGF-β) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-β-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-β1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-β1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-β1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-β1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-β on bone metabolism. parathyroid hormone transforming growth factor-β TGF-β-activated kinase-1 mitogen-activated protein matrix metalloproteinase α-minimum essential medium c-jun-N-terminal kinase extracellular signal-regulated kinase focal adhesion kinase phosphate-buffered saline MC3T3-E1 cells basic fibroblast growth factor basic multicellular unit The skeletons of developing and adult mammals undergo constant remodeling, i.e. old bone is regularly removed and new bone is regularly laid down (1Parfitt A.M. J. Cell. Biochem. 1994; 55: 273-286Crossref PubMed Scopus (764) Google Scholar, 2Erlebacher A. Filvaroff E.H. Gitelman S.E. Derynck R. Cell. 1995; 80: 371-378Abstract Full Text PDF PubMed Scopus (617) Google Scholar). The major players in the remodeling of bone are two specialized and functionally coupled cell types: osteoblasts, which deposit organic and inorganic matrix, and osteoclasts, which remove bone matrix. Osteoclast function is controlled by both systemic and local factors, most of which act through or are produced by osteoblasts. Similarly, the ability of osteoblasts to deposit bone matrix is stimulated by factors that are produced by osteoclasts or released from bone in the wake of matrix dissolution. Thus, in living bone, osteoblasts play a paramount role in determining the functional state of osteoclasts, and osteoclasts are of supreme importance for osteoblast function. Most of the arguments concerned with the quest for identification of key mechanisms that produce bone resorbing osteoclasts,i.e. those mechanisms involved in osteoclast precursor proliferation and commitment, and osteoclast migration, fusion, and resorption focus on the direct effect of molecules found in the extracellular space or on the cell surface of osteoblasts or other stromal cells on osteoclast precursors and maturing osteoclasts (3Suda T. Udagawa N. Takahashi N. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996: 87-102Google Scholar). However, based on the observation that cells of the osteoblast lineage, the bone lining cells, display a cobblestone morphology and cover the bone surface in an epithelium-like manner, an additional mode of how osteoblasts may modulate the ability of osteoclasts to resorb bone has been proposed (4Rodan G.A. Martin T.J. Calcif. Tissue Int. 1981; 33: 349-351Crossref PubMed Scopus (780) Google Scholar, 5Zambonin Z.A. Teti A. Primavera M.V. Cell Tissue Res. 1984; 235: 561-564PubMed Google Scholar). In this model, bone lining cells mechanically hinder the access of osteoclasts to the bone surface. Consequently, in order to allow osteoclasts to degrade bone matrix, bone lining cells must retreat from part of the bone surface. One of the few examples in the literature that illustrates how this may take place is parathyroid hormone (PTH)1-induced osteoblast retraction (6Tram K.K. Spencer M.J. Murray S.S. Lee D.B. Tidball J.G. Murray E.J. Biochem. Mol. Biol. Int. 1993; 29: 981-987PubMed Google Scholar, 7Miller S.S. Wolf A.M. Arnaud C.D. Science. 1976; 192: 1340-1343Crossref PubMed Scopus (94) Google Scholar). In response to PTH, osteoblasts in culture adopt a stellate morphology and expose the underlying substratum. Osteoblast retraction occurs within 1 h, is reversible, and involves cyclic AMP signaling and activation of intracellular proteases. However, whether or not the retraction of osteoblasts triggered by PTH is indeed able to increase recruitment of osteoclasts to the bone surface and, hence, to promote bone resorption has never been investigated. Transforming growth factor-β (TGF-β) is another growth factor that profoundly alters osteoblast shape (8Hurley M.M. Abreu C. Gronowicz G. Kawaguchi H. Lorenzo J. J. Biol. Chem. 1994; 269: 9392-9396Abstract Full Text PDF PubMed Google Scholar). It is one of the most abundant growth factors in skeletal tissues (9Bonewald, L. F., and Mundy, G. R. (1990) Clin. Orthop. 261–276.Google Scholar) and, in mammals, comprises three isoforms, TGF-β1, TGF-β2, and TGF-β3, all of which are expressed by bone cells (10Horner A. Kemp P. Summers C. Bord S. Bishop N.J. Kelsall A.W. Coleman N. Compston J.E. Bone. 1998; 23: 95-102Crossref PubMed Scopus (114) Google Scholar) and interact with the known TGF-β receptors types I, II, and III (betaglycan) (11Hartsough M.T. Mulder K.M. Pharmacol. Ther. 1997; 75: 21-41Crossref PubMed Scopus (110) Google Scholar). All TGF-β isoforms display similar biological activities (12Bonewald L. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996: 647-659Google Scholar), although one isoform may be more potent than another in a given assay (13Joyce M.E. Roberts A.B. Sporn M.B. Bolander M.E. J. Cell Biol. 1990; 110: 2195-2207Crossref PubMed Scopus (683) Google Scholar). Whereas the role of TGF-β-induced changes in osteoblast morphology is largely unknown, numerous studies suggest that TGF-β has multiple functions in bone metabolism. In vivo studies involving the application of exogenously administered recombinant TGF-β show that TGF-β can increase bone formation and promote fracture healing (12Bonewald L. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996: 647-659Google Scholar,14Rosier, R. N., O'Keefe, R. J., and Hicks, D. G. (1998)Clin. Orthop. S294-S300.Google Scholar). TGF-β1 knock-out mice display an about 30% decrease in tibial length and a reduction in bone mineral content (15Geiser A.G. Zeng Q.Q. Sato M. Helvering L.M. Hirano T. Turner C.H. Bone. 1998; 23: 87-93Crossref PubMed Scopus (121) Google Scholar), consistent with the idea that TGF-β functions as a bone-forming agent. On the other hand, overexpression of TGF-β2 controlled by the osteoblast-specific osteocalcin promoter leads to bone loss (16Erlebacher A. Derynck R. J. Cell Biol. 1996; 132: 195-210Crossref PubMed Scopus (307) Google Scholar), and mice expressing a dominant-negative TGF-β type II receptor have no obvious skeletal defects apart from joint abnormalities that are probably due do chondrocyte malfunction (17Serra R. Johnson M. Filvaroff E.H. LaBorde J. Sheehan D.M. Derynck R. Moses H.L. J. Cell Biol. 1997; 139: 541-552Crossref PubMed Scopus (414) Google Scholar). These apparently conflicting results are also reflected in numerous studies in cell and tissue cultures, where TGF-β, in dependence on particular experimental parameters, modulates various bone cell activities in opposite ways. For instance, both increases and decreases in osteoclast formation, bone resorption, osteoblast proliferation, and osteoblast differentiation have been reported (12Bonewald L. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996: 647-659Google Scholar, 18Centrella M. Horowitz M.C. Wozney J.M. McCarthy T.L. Endocr. Rev. 1994; 15: 27-39PubMed Google Scholar). Like the functional aspects of TGF-β action on bone cells, the intracellular signals triggered by binding of TGF-β to its receptor are numerous and complex. They include phosphorylation and activation of Smad transcription factors, regulation of Ras, Rho, and other small G-proteins, activation of protein kinases such as TGF-β-activated kinase-1 (TAK1), mitogen-activated protein (MAP) kinases, and Src, recruitment of adaptor proteins and regulation of ion channels (19Visser J.A. Themmen A.P. Mol. Cell. Endocrinol. 1998; 146: 7-17Crossref PubMed Scopus (32) Google Scholar). Not surprisingly then, TGF-β regulates expression of a spectrum of genes with potential significance for bone cell function, such as those coding for various extracellular matrix molecules and their receptors, proteinases and proteinase inhibitors, cell-cell adhesion molecules, and growth factors. The fact that some of the signaling pathways downstream from the TGF-β receptor are also common to and/or modulated by a variety of other growth factors and extracellular matrix molecules would also explain the seemingly contradictory results obtained with TGF-β in various functional assays as outlined above, i.e. the precise nature of the cellular microenvironment may determine the outcome of TGF-β action. Promotion of osteoclast recruitment through morphological transformation of osteoblasts as a result of local release and/or activation of TGF-β may represent an attractive model to explain spatiotemporal regulation of bone resorption and the functional coupling of osteoblast and osteoclasts. In this study, we set out to better define the effect of TGF-β on cell shape and to investigate the mechanism by which TGF-β induces osteoblast elongation. We provide evidence that two classes of molecules that are regulated by TGF-β1, MAP kinases and matrix metalloproteinases (MMPs), play a pivotal role in TGF-β1-induced alterations in osteoblast shape and that osteoblast elongation promotes osteoclastic bone resorption. The mouse osteoblast cell lines MC3T3-E1 (MC) (20Sudo H. Kodama H.A. Amagai Y. Yamamoto S. Kasai S. J. Cell Biol. 1983; 96: 191-198Crossref PubMed Scopus (1499) Google Scholar) was routinely maintained and passaged in growth medium consisting of αMEM (Life Technologies, Inc.) containing 5% fetal bovine serum (Life Technologies), 100 units/ml penicillin and 100 µg/ml streptomycin. For experiments, MC cells were plated at a density of 30,000 cells/cm2 into 96-well tissue culture plates (Costar) or onto glass coverslips and maintained under serum-free conditions in α-MEM (Life Technologies, Inc.) for 24 h unless otherwise indicated. Substrata were either left untreated or coated with 30 µg/ml type I collagen (Nitta Collagen). Unless otherwise indicated, growth factors and inhibitors were added at the time of plating at the following concentrations: TGF-β1 2.5 ng/ml, bFGF 2 ng/ml, and PTH 10 nm (R&D Systems); GM6001 10 µm(AM Scientific); PD169316 10 µm, SB203580 10 µm, PD98059 10 µm, genistein 2 µm, LY294002 1 µm, chelerythrine 1 µm, aprotinin 10 µm, pepstatin 10 µm, and E-64 10 µm (Calbiochem). To analyze osteoclastic bone resorption in the presence of a bone lining cell layer, MC cells were seeded at a density of 80,000 cells/cm2 onto slices of bovine femur cortical bone (6 mm in diameter, 0.2 mm thick) placed in 96-well plates, and cultured under serum-free conditions for 24 h, after which time they had formed a confluent layer of bone lining cells. MC cells were then washed once with serum-free medium, and 200 µl of cell suspension containing ∼500 osteoclasts in α-MEM with 2% fetal bovine serum were added on top of the bone lining cell layer. After a settling period of 90 min, nonadherent cells were removed by replacing the medium with αMEM containing 0.5% fetal bovine serum. The culture was then continued for a further 48 h. To analyze osteoclastic bone resorption in the absence of a bone lining cell layer, MC cells were mixed with osteoclasts at the time of plating. In one set of experiments, bone lining cells were fixed with 96% ethanol for 20 min at −20 °C and washed three times with medium before osteoclasts were seeded on top of the bone lining cells. Osteoclasts were obtained as unfractionated bone cells according to the method described by Tezuka et al. (21Tezuka K. Sato T. Kamioka H. Nijweide P.J. Tanaka K. Matsuo T. Ohta M. Kurihara N. Hakeda Y. Kumegawa M. Biochem. Biophys. Res. Commun. 1992; 186: 911-917Crossref PubMed Scopus (202) Google Scholar) with minor modifications. Briefly, long bones from 10-day-old rabbits were minced in αMEM and gently agitated for 30 s with a vortex mixer. After sedimentation of bone fragments for 1.5 min, the supernatant was harvested and washed twice by centrifugation at 45 × gfor 2 min. Cells were then resuspended and plated as described above. To quantify cell number, the Alamar Blue assay (Trek Diagnostics) was used according to the manufacturer's instructions. MMP-2 expression and activity were determined by gelatinase zymography (22Kleiner D.E. Stetler-Stevenson W.G. Anal. Biochem. 1994; 218: 325-329Crossref PubMed Scopus (817) Google Scholar) using 0.5 mg/ml gelatin (Sigma) as a substrate in 7.5% SDS-polyacrylamide gels. After electrophoresis, gels were washed three times with 2.5% Triton X-100 in water and then incubated overnight at 37 °C in 0.1% Triton X-100, 5 mmCaCl2, 1 mm ZnCl2, 3 mmNaN3, 50 mm Tris, pH 7.4, in a closed container. Gels were then stained for 30 min with 0.25% Coomassie R-250 (Sigma) in 10% acetic acid and 45% methanol and destained for 30 min with 20% acetic acid, 20% methanol, 17% ethanol, 0.6% diethylether. Gels were then dried and scanned for documentation. Western blotting was performed on total cell lysates in radioimmune precipitation buffer (30 mmNaCl, 5 mm ethylenediamine tetraacetic acid, 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS) containing 10 mm NaF and 50 mmNa3VO4. Loading of the gels was normalized to the amount protein, which was proportional to cell numbers as measured by Alamar Blue assay. Samples were resolved on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked overnight at 4 °C with TBST (0.1% Tween 20, 100 mm NaCl, 50 mmTris, pH 7.5) containing 5% milk powder. Membranes were then incubated for 1 h at ambient temperature with antibodies against MMP-2, MMP-13, and MMP-14 (MT-1 MMP) (from Chemicon Int.), p38 MAP kinase, phospho-p38 MAP kinase, ERK1/2 (p44/42 MAP kinase), phospho-ERK1/2, c-jun-N-terminal kinase (JNK), and phospho-JNK (from New England Biolabs), and β-catenin or focal adhesion kinase (FAK) (from Transduction Laboratories). After washing vigorously with TBST for 1 h, membranes were incubated for 1 h at ambient temperature with horseradish peroxidase-conjugated rabbit anti-mouse antibodies (DAKO) and developed with an enhanced chemiluminescence kit (ECLTM, Amersham Pharmacia Biotech), according to the manufacturer's instructions. In case of immunoblotting, to detect FAK, blots were striped for re-blotting in 100 mm2-mercaptoethanol, 2% SDS, 62,5 mm Tris-HCL, pH 6.7, at 50 °C for 30 min followed by blocking and re-probing as described above. To study cell shape, cells were fixed with 5% glutaraldehyde in phosphate-buffered saline (PBS), washed extensively with water, and stained with 0.5% toludine blue (Sigma) in 2.5% Na2CO3. Cell shape changes were quantified with ImagePro software by measuring the areas of the substratum that were covered and not covered by the cell monolayer. To quantify pit formation by osteoclasts at the end of the culture period, cells were scraped gently off of the bone slices with a cotton stick. The bone slices were then washed with water and stained for resorption pits with Mayer's hematoxylin (Sigma). After brief sonication in a water bath and subsequent washing, the resorbed area was measured using CAST-GRID software (Microsoft, Olympus). For detection of FAK by indirect immunofluorescence, cells were maintained on glass coverslips, fixed with formalin, and processed as described (23Lochter A. Galosy S. Muschler J. Freedman N. Werb Z. Bissell M.J. J. Cell Biol. 1997; 139: 1861-1872Crossref PubMed Scopus (537) Google Scholar). FAK expression was visualized using mouse monoclonal antibodies from Transduction Laboratories and rhodamine-conjugated donkey anti-mouse secondary antibodies from Jackson Laboratories. For actin staining, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS for 15 min, and permeabilized with acetone for 5 min at −20 °C. Cells were rinsed several times with PBS and incubated with fluorescein isothiocyanate-conjugated phalloidin (Sigma) for 1 h in PBS. Cells were mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) for visualization of nuclei. Osteoblasts maintained in the presence of serum have repeatedly been shown to transform from a cuboidal or cobblestone to an elongated morphology in response to TGF-β1 (8Hurley M.M. Abreu C. Gronowicz G. Kawaguchi H. Lorenzo J. J. Biol. Chem. 1994; 269: 9392-9396Abstract Full Text PDF PubMed Google Scholar,24Breen E.C. Ignotz R.A. McCabe L. Stein J.L. Stein G.S. Lian J.B. J. Cell. Physiol. 1994; 160: 323-335Crossref PubMed Scopus (133) Google Scholar, 25Ibbotson K.J. Orcutt C.M. Anglin A.M. D'Souza S.M. J. Bone Miner. Res. 1989; 4: 37-45Crossref PubMed Scopus (49) Google Scholar, 26Rosen D.M. Stempien S.A. Thompson A.Y. Seyedin S.M. J. Cell. Physiol. 1988; 134: 337-346Crossref PubMed Scopus (177) Google Scholar). When MC cells that were plated at high cell density on tissue culture plastic in chemically defined medium were exposed to TGF-β1, cell elongation was apparent after 16–24 h in culture but not at earlier time points (Fig.1A and not shown). These shape changes were accompanied by a decrease in cell spreading and, hence, by an increase in the area of cell-free substratum (Fig. 1, Aand B). Furthermore, they occurred regardless of whether TGF-β1 was added at the time of cell plating (Fig.1A) or when cells were allowed to establish a cobblestone morphology prior to TGF-β1 treatment, i.e. when TGF-β1 was added 1 day after cell plating (not shown). The effect of TGF-β1 on cell shape was dose-dependent and maximal at 2.5 ng/ml (Fig. 1B). When cells were plated on a type I collagen substratum, TGF-β1 also induced cell elongation, albeit to a lesser extent than on tissue culture plastic (Fig. 1A), an observation consistent with the reported decrease in expression of the TGF-β1 receptors in cells exposed to type I collagen (27Takeuchi Y. Nakayama K. Matsumoto T. J. Biol. Chem. 1996; 271: 3938-3944Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The effect of TGF-β1 on cell shape was reversible when cells were treated for 1 day with TGF-β1 and subsequently maintained for another 4 days without TGF-β1 (Fig. 1C). Similar results were also obtained with cells maintained in serum-containing medium (Fig.1C). Under serum-free conditions, cell numbers increased only slightly during a 5-day culture period and were unaffected by TGF-β1 (Fig. 1D). In contrast, TGF-β1 reduced the time-dependent increase in cell numbers observed in serum-containing medium (Fig. 1D). These results indicate that the effect of TGF-β1 on cell shape is unrelated to and independent of its effect on osteoblast proliferation. The morphological transformation in response to TGF-β1 suggests that TGF-β1 alters focal adhesion contacts and cytoskeletal organization in MC cells. To address this question, cells were labeled with phalloidin, and antibodies against FAK. As shown in Fig.2, A and B, stress fibers were much more elaborate in osteoblasts treated with TGF-β1, and localization of FAK at focal contacts was increased. Furthermore, expression of FAK protein was increased in response to an 8- and 24-hour treatment with TGF-β1 (Fig. 2C). MAP kinases control cell function and shape through integration of signals from the extracellular matrix and growth factors (28Boudreau N.J. Jones P.L. Biochem. J. 1999; 339: 481-488Crossref PubMed Scopus (519) Google Scholar). Furthermore, the activation of MAP kinases by TGF-β1 has been described in several cell types (19Visser J.A. Themmen A.P. Mol. Cell. Endocrinol. 1998; 146: 7-17Crossref PubMed Scopus (32) Google Scholar). We therefore analyzed the expression and activation of MAP kinases in MC cells stimulated with TGF-β1. Expression levels of the three MAP kinase members, Erk1/2, p38 MAP kinase, and JNK, were not altered by TGF-β1 (Fig.3). However, Erk1/2 and p38 MAP kinase, but not JNK, were activated as determined by phosphorylation state-specific antibodies (Fig. 3). Phosphorylation/activation of Erk1/2 and p38 MAP kinase was obvious between 1 and 9 h after the addition of TGF-β1. At 24 h, Erk1/2 activation was no longer detectable, but p38 MAP kinase still displayed increased phosphorylation (Fig. 3). This suggests that the effect of TGF-β1 on cell shape, which is apparent after 24 h but not after 9 h of TGF-β1 treatment, is mediated by sustained activation of p38 MAP kinase but not by Erk1/2. To determine the consequences of MAP kinase activation for osteoblast morphology, cells were treated with the MEK1/2 inhibitor PD98059, which specifically blocks the Erk1/2 pathway and not the p38 MAP kinase pathway (29Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 30Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 36Hayashi K. Takahashi M. Kimura K. Nishida W. Saga H. Sobue K. J. Cell Biol. 1999; 145: 727-740Crossref PubMed Scopus (167) Google Scholar), and with the kinase inhibitor PD169316 (31Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 36Hayashi K. Takahashi M. Kimura K. Nishida W. Saga H. Sobue K. J. Cell Biol. 1999; 145: 727-740Crossref PubMed Scopus (167) Google Scholar, 73Paine E. Palmantier R. Akiyama S.K. Olden K. Roberts J.D. J. Biol. Chem. 2000; 275: 11284-11290Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 74Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar), which specifically blocks the p38 MAP kinase and not the Erk1/2 pathway. Cells treated with TGF-β1 and PD98059 had a morphology similar to cells maintained with TGF-β1 alone (Fig.4A). In contrast, cells treated with TGF-β1 and PD169316 had morphologies similar to cells not treated with TGF-β1 (Fig. 4A). Furthermore, PD169316 but not PD98059 strongly reduced the increase in cell-free substratum area observed in response to TGF-β1 (Fig. 4B). Results similar to those obtained with PD169316 were also obtained with the related p38 MAP kinase inhibitor SB203580 (not shown). None of the MAP kinase inhibitors tested affected cell shape in the absence of TGF-β1 (not shown). To verify the specificity of the inhibitors used, we tested the effect of SB203580, PD169316, and PD98059 on both the p38 and p44/42 MAP kinase activity. PD98059 did not inhibit the p38 MAP kinase activity but totally abrogated the p44/42 MAP kinase activity as previously reported (30Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar). SB203580 and PD169316 did not inhibit the p44/42 MAP kinase but blocked p38 MAP kinase activity as previously reported (31Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 36Hayashi K. Takahashi M. Kimura K. Nishida W. Saga H. Sobue K. J. Cell Biol. 1999; 145: 727-740Crossref PubMed Scopus (167) Google Scholar, 73Paine E. Palmantier R. Akiyama S.K. Olden K. Roberts J.D. J. Biol. Chem. 2000; 275: 11284-11290Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 74Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) (data not shown). The broad-spectrum tyrosine kinase inhibitor genistein and the phosphatidylinositol-3 kinase inhibitor LY294002 had no significant effect on cell shape in the presence (Fig. 4, C andD) or absence (not shown) of TGF-β1. The protein kinase C inhibitor chelerythrine had a slight but insignificant effect on cell shape and only in the presence of TGF-β1 (Fig. 4D and not shown). Taken together, these data indicate that p38 MAP kinase plays a central role in the TGF-β1-induced conversion of osteoblasts from cuboidal to elongated. It has recently been shown that MMPs can trigger the morphological transformation of epithelial cells from a cobblestone to an elongated and fusiform morphology, both in culture and in vivo (23Lochter A. Galosy S. Muschler J. Freedman N. Werb Z. Bissell M.J. J. Cell Biol. 1997; 139: 1861-1872Crossref PubMed Scopus (537) Google Scholar,32Sternlicht M.D. Lochter A. Sympson C.J. Huey B. Rougier J.P. Gray J.W. Pinkel D. Bissell M.J. Werb Z. Cell. 1999; 98: 137-146Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Thus, to further delineate the mechanism underlying TGF-β1-induced changes in osteoblast morphology, we next examined whether TGF-β1 alters expression of major MMPs produced by osteoblasts, namely MMP-2 (gelatinase A), MMP-13 (collagenase-3), and MMP-14 (membrane-type 1 MMP). As judged by gelatin zymography, MC cells expressed proteolytic activity corresponding to the molecular mass of latent (72 kDa) but not active MMP-2 (67 kDa) (Fig.5). Expression of latent MMP-2, which was found in both cell culture medium and cell lysate, was not altered by TGF-β1 (Fig. 5). Likewise, MMP-14 expression, as visualized by immunoblotting, was unaffected by TGF-β1 (Fig. 5). In contrast, using immunoblot analysis, we found that MMP-13, which was barely detectable in untreated cultures, was strongly up-regulated when cells were incubated with TGF-β1 (Fig. 5). Most of the MMP-13 induced by TGF-β1 was found in the cell culture medium at both 6 and 24 h after the addition of TGF-β1, whereas only a small amount of MMP-13 was found in cell lysates and only 6 h after initiation of TGF-β1 treatment (Fig. 5). Neither expression nor activation of MMP-13 induced by TGF-β1 was influenced by PD169316 (not shown), indicating that p38 MAP kinase-independent signals are involved in the up-regulation of MMP-13 expression by TGF-β1. To investigate whether MMPs contribute to the regulation of morphology by TGF-β1, cells were maintained in the presence of the broad-spectrum hydroxamate MMP inhibitor GM6001 (33Grobelny D. Poncz L. Galardy R.E. Biochemistry. 1992; 31: 7152-7154Crossref PubMed Scopus (254) Google Scholar). GM6001 reduced cell elongation and inhibited the increase in cell-free substratum area in response to TGF-β1 by about half (Fig.6, A and B). Similar results as those with GM6001 were also obtained with the hydroxamate-type MMP inhibitor BB-94 (34Davies B. Brown P.D. East N. Crimmin M.J. Balkwill F.R. Cancer Res. 1993; 53: 2087-2091PubMed Google Scholar) and with the phosphinate-type MMP inhibitor 11A (35Buchardt J. Ferreras M. Krog-Jensen C. Delaissé J.-M. Foged N.T. Meldal M. Chem. Eur. J. 1999; 10: 2877-2884Crossref Scopus (47) Google Scholar) (not shown). This effect of MMP inhibitors was specific, because the serine proteinase inhibitor aprotinin, the cysteine proteinase inhibitor E64, and the aspartyl proteinase inhibitor pepstatin were without effect on the TGF-β1-induced changes in cell shape (Fig. 6, A and B). None of the proteinase inhibitors used affected cell number or cell shape in the absence of TGF-β1 (not shown). GM6001 did not affect the induction by TGF-β of either the p38 or p44/42 MAP kinase (not shown). Thus, MMPs appear to play an important role in the TGF-β1-induced shape changes. To examine whether p38 MAP kinases and MMPs are selectively involved in the regulation of cell shape by TGF-β1, the ability of PD169316 and GM6001 to interfere with the inhibition of proliferation by TGF-β1 was studied in serum-containing medium. Under these conditions, Neither PD169316 nor GM6001 affected cell number in the absence or presence of TGF-β1 (Fig. 7A). The same observatio