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Adult Human Mesenchymal Stem Cell Differentiation to the Osteogenic or Adipogenic Lineage Is Regulated by Mitogen-activated Protein Kinase

2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês

10.1074/jbc.275.13.9645

1083-351X

Rama K. Jaiswal, Neelam Jaiswal, Scott P. Bruder, Gabriel Mbalaviele, Daniel R. Marshak, Mark F. Pittenger,

Osteoarthritis Treatment and Mechanisms

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor γ2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively. Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor γ2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively. human bone marrow-derived mesenchymal stem cells polyacrylamide gel electrophoresis osteogenic supplements mitogen-activated protein extracellular signal-regulated protein kinase MAP kinase/ERK kinase peroxisome proliferator-activated receptor γ c-Jun N-terminal kinase phosphate-buffered saline alkaline phosphatase 4′,6-diamidino-2-phenylindole base pair polymerase chain reaction glutathioneS-transferase myelin basic protein Human bone marrow-derived mesenchymal stem cells (hMSCs)1 are multipotent, capable of differentiating into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under defined in vitro conditions (1.Bruder S.P. Fink D.J. Caplan A.I. J. Cell. Biochem. 1994; 56: 283-294Crossref PubMed Scopus (778) Google Scholar, 2.Mackay A.M. Beck S.C. Murphy J.M. Barry F.P. Chichester C.O. Pittenger M.F. Tissue Eng. 1998; 4: 415-428Crossref PubMed Scopus (1127) Google Scholar, 3.Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.M. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18443) Google Scholar). The hMSCs do not differentiate spontaneously, and their in vitro and in vivoosteogenic potential has been very well characterized by us and others (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar, 5.Lennon, D. P., Haynesworth, S. E., Bruder, S. P., Jaiswal, N., and Caplan, A. I. (1996), In Vitro Cell. Dev. Biol. 32, 602–611Google Scholar, 6.Haynesworth S.E. Goshima J. Goldberg V.M. Caplan A.I. Bone (New York). 1992; 13: 81-88Crossref PubMed Scopus (1118) Google Scholar). When cultured in the presence of the synthetic glucocorticoid dexamethasone, ascorbic acid, and β-glycerophosphate (osteogenic supplements, OS), hMSCs differentiate to the osteogenic lineage, producing bone-like nodules with a mineralized extracellular matrix containing hydroxyapatite (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar). The similar developmental phenomenon has also been described by others (7.Cheng S.-L. Yang J.W. Rifas L. Zhang S.-F. Avioli L.V. Endocrinology. 1994; 134: 277-286Crossref PubMed Scopus (602) Google Scholar, 8.Rickard D.J. Kassem M. Hefferan T.E. Sarkar G. Spelsberg T.C. Riggs B.L. J. Bone Miner. Res. 1996; 11: 312-324Crossref PubMed Scopus (338) Google Scholar) using bone marrow-derived cells. Other than the osteoinductive effect that OS has on MSCs, OS also acts as a mitogen (9.Bruder S.P. Jaiswal N. Haynesworth S.E. J. Cell. Biochem. 1997; 64: 278-294Crossref PubMed Scopus (1312) Google Scholar). Presumably, the osteoinductive and mitogenic effects are due to dexamethasone present in OS because glucocorticoids are potent regulators of cellular growth and differentiation (10.Lukert B.P. Raisz L.G. Ann. Intern. Med. 1990; 112: 352-364Crossref PubMed Scopus (1020) Google Scholar). However, the underlying molecular mechanisms of OS-induced mitogenic and osteogenic differentiation are presently unknown. In order to acquire a new cell phenotype, uncommitted hMSCs must undergo proliferative and differentiative changes, the two most fundamental biological processes in the life cycle of cells. One of the potential signal transduction pathways that might regulate the proliferation and differentiation of hMSCs is the MAP kinase pathway. Activation of the MAP kinase pathway in other cell types such as neuronal cells, adipocytes, T-cells, and muscle cells promotes cell differentiation (11.Lowry D.R. Willumsen B.M. 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Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4300) Google Scholar). ERKs are activated by dual phosphorylation on tyrosine and threonine residues separated by a glutamate residue (TEY) by a single upstream kinase known as MEK (MAP kinase or ERKkinase). Other than growth factors, hormones such as estrogen and parathyroid hormone, which have a profound effect on bone development and remodeling, will also transiently activate ERK1 in MCF-7 cells and ERK2 in the osteosarcoma cell line UMR 106-01 (16.Migliaccio A. Di Domenico M. Castoria G. deFalco A. Bontempo P. Nola E. Auricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (875) Google Scholar, 17.Swarthout J.T. Lemker J.F. Wilhelm D. Dieckmann A. Angel P. Partridge N.C. J. Bone Miner. Res. 1997; 12 (abstr.): 5162Google Scholar). Activation of ERK1/ERK2 has also been demonstrated as an important signaling mechanism in the differentiation of the preadipocyte cell line 3T3-L1 into mature adipocytes (18.Sale E.M. Atkinson P.G.P. Sale G.J. EMBO J. 1995; 14: 674-684Crossref PubMed Scopus (248) Google Scholar). The ERK phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ), which is a key component of transcription machinery of adipogenic differentiation and selectively expressed in adipocytes, in vitro as well asin vivo, results in reduction of PPARγ transcriptional activity (19.Adams M. Reginato M.J. Shao D. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 1997; 272: 5128-5132Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 20.Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (949) Google Scholar). Growth factors and cytokines, such as tumor necrosis factor-α, which activate MAP kinases (21.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1186) Google Scholar, 22.Cobb M. Hepler J.E. Cheng M. Robbins D. Semin. Cancer Biol. 1994; 4: 261-268Google Scholar), are potent inhibitors of adipocyte differentiation (23.Serrero G. Mills D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3912-3916Crossref PubMed Scopus (75) Google Scholar, 24.Torti F.M. Torti S.V. Larrick J.W. Ringold G.M. J. Cell Biol. 1989; 108: 1105-1113Crossref PubMed Scopus (162) Google Scholar, 25.Berg M. Fraker D.L. Alexander H.R. Cytokines. 1994; 6: 425-432Crossref Scopus (67) Google Scholar). The relationship between MAP kinases and adipogenesis is further strengthened by the recent demonstration that leptin, product of the ob gene, induced activation of MAP kinase in mouse embryonic cell line C3H10T1/2 (26.Takahashi Y. Okimura Y. Mizuno I. Iida K. Takahashi T. Kaji H. Abe H. Chihara K. J. Biol. Chem. 1997; 272: 12897-12900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Leptin-induced MAP kinase activation may cause a reduction in the adipogenic differentiation by phosphorylating PPARγ and thus might play an important role in controlling adipogenesis. Recently, two new members of the MAP kinase family have been described as follows: the c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase, and p38-reactivating kinase (p38 RK or simply, p38). These kinases can be activated by a variety of cytokines, environmental stress, as well as ultraviolet and ionizing radiation (27.Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 438-443Abstract Full Text PDF Scopus (1008) Google Scholar). Transforming growth factor-β stimulates ERK (28.Huwiler A. Pfeilschifter J. FEBS Lett. 1994; 354: 255-258Crossref PubMed Scopus (50) Google Scholar) as well as p38 (29.Hannigan M. Zhan L. Huang C.K. Biochem, Biophys. Res. Commun. 1998; 246: 55-58Crossref PubMed Scopus (105) Google Scholar) and acts as a physiological regulator of osteoblast differentiation and bone remodeling by regulating and coordinating the activities of osteoblasts and osteoclasts (30.Erlebacher A. Filvoroff E.H. Ye J.Q. Derynck R. Mol. Biol. Cell. 1998; 7: 1903-1918Crossref Scopus (214) Google Scholar). Cytokines such as tumor necrosis factor-α, interleukin-1, and interleukin-6, which are important regulators of bone resorption, activate JNK and p38 in human and mouse osteoblastic cells (31.Chaudhary L.R. Avioli L.V. Mol. Cell. Biochem. 1998; 78: 59-68Crossref Scopus (53) Google Scholar, 32.Blanque R. Cottereaux C. Gardner C.R. Drug Exp. Clin. Res. 1997; 23: 63-70PubMed Google Scholar). Despite the rapid progress in the elucidation of the signaling pathways involving MAP kinases, the most crucial question of how the specificity and integration of multiple signals are processed in parallel remains unanswered. In the present study, we demonstrate that activation of MAP kinases in hMSCs, cultured in OS medium, resulted in their differentiation into osteocytes and that inhibition of the MAP kinase pathway by PD98059, a specific MEK inhibitor (33.Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), caused these cells to develop into fully mature adipocytes. These results provide a potential mechanism of action for ERK in regulating the lineage commitment of multipotential adult stem cells. These results may also have important clinical implications during the aging process or diseases, such as osteoporosis, where bone marrow stroma is progressively replaced by fat (34.Meunier P. Aaron J. Edouard C. Vignon G. Clin. Orthop. 1971; 80: 147-154Crossref PubMed Scopus (656) Google Scholar). The monoclonal MAP kinase antibody raised against a C-terminal synthetic peptide (amino acids 324–345) was purchased from Zymed Laboratories Inc. Laboratory, San Francisco, CA. Phosphospecific MAP kinase and phosphospecific p38 MAP kinase antibodies were from New England Biolabs, Beverly, MA. MEK1 inhibitor PD98059 was purchased from Calbiochem. [γ-32P]ATP, ECL detection kit, horseradish peroxidase-conjugated mouse and rabbit IgG were from Amersham Pharmacia Biotech. Alkaline Phosphatase Diagnostic Kit 85 and Calcium Diagnostic Kit 587 were purchased from Sigma. All other chemicals were obtained commercially and were of the highest purity available. Bone marrows were obtained from normal human volunteer donors (age 23–40 years) after informed consent. hMSCs were purified from the marrow by Percoll density gradient centrifugation method (2.Mackay A.M. Beck S.C. Murphy J.M. Barry F.P. Chichester C.O. Pittenger M.F. Tissue Eng. 1998; 4: 415-428Crossref PubMed Scopus (1127) Google Scholar, 3.Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.M. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18443) Google Scholar, 6.Haynesworth S.E. Goshima J. Goldberg V.M. Caplan A.I. Bone (New York). 1992; 13: 81-88Crossref PubMed Scopus (1118) Google Scholar). The hMSCs were cultured in 100-mm Petri dishes and treated with osteogenic supplements (OS) as described earlier (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar). To make lysates, cells were washed two times with cold PBS and were suspended in 0.5 ml of lysis buffer (20 mm Tris-HCl, pH 7.5, 1.0 mm EDTA, 1.0 mm EGTA, 1.0 mm dithiothreitol, 150 mm NaCl, 1% Triton X-100, 1.0 mm sodium orthovanadate, 10 mm NaF, 25 mm p-nitrophenyl phosphate, 0.1% SDS, 0.5 mmphenylmethylsulfonyl fluoride, and 2.0 μg/ml aprotinin and leupeptin). The SDS was omitted from lysis buffer in samples prepared for kinase assays. The lysates were sonicated briefly (2 × 20 s) on ice and centrifuged at 100,000 × g for 35 min. Protein concentrations were determined using the bicinchoninic acid assay (Pierce). In each experiment, control and OS-treated cells were processed in parallel. In experiments using the MEK1 inhibitor PD98059, the 50 mm stock solution was prepared in Me2SO. The final concentration of Me2SO never exceeded 0.1%, and the same amount of Me2SO vehicle was added to control wells. Cell lysates containing 20 μg of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The membranes were incubated for 2 h at room temperature in blocking buffer (5% non-fat dry milk in Tris-buffered saline + 0.2% Triton X-100) and then incubated overnight at 4 °C with various antibodies (1:1000). Antigen-antibody complexes were visualized by incubation of the blots in a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit immunoglobulin G and the ECL detection system (Amersham Pharmacia Biotech). Alkaline phosphatase activity was assayed by measuring the formation ofp-nitrophenol from p-nitrophenyl phosphate as described previously (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar). Alkaline phosphatase histochemistry was performed using Sigma Diagnostic Kit 85. Selected specimens were also stained for mineral deposition by the von Kossa method (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar). Accumulated calcium was measured in 0.5 n HCl extracts according to the manufacturer's instructions contained in Sigma Diagnostic Kit 587. Total calcium was calculated from standard solutions prepared in parallel and expressed as μg/mg cellular protein. Cell numbers were determined by using the nuclear dye, crystal violet (35.Westergren-Thorsson G. Onnerevik P.-O. Fransson L.-A. Maelstrom A. J. Cell. Physiol. 1991; 147: 523-530Crossref PubMed Scopus (110) Google Scholar). In-gel kinase assays were performed using whole cell lysates (20 μg of protein) on SDS-PAGE separated proteins that were renatured in the gel according to the procedure of Kamehita and Fugisawa (36.Kameshita I. Fugisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (486) Google Scholar). Polyacrylamide (10%) gels were cast with 0.5 mg of myelin basic protein (MBP) per ml added as substrate to the SDS-PAGE gel solution prior to polymerization. For JNK assays, GST-c-Jun (amino acids 1–79) fusion protein was purified by glutathione-agarose chromatography of Escherichia coli extracts containing the expression plasmid pGEX2T-c-Jun, and 40 μg/ml was co-polymerized with the SDS-PAGE solution. After the denaturation/renaturation procedure, kinase reactions were performed by incubating the gel with kinase buffer (20 mm Tris-HCl, pH 7.2, 20 mmMgCl2, 15 mm β-glycerophosphate, 1.0 mm dithiothreitol, 0.1 mm EGTA, and 0.5 mm sodium vanadate) containing 200 μCi of [γ-32P]ATP. The gel was incubated for 3 h at room temperature, and autoradiograms were developed after washing several times in 5% trichloroacetic acid and 1% sodium pyrophosphate to remove free isotope. Phosphorylated bands were excised from the gel, and incorporated radioactivity was measured by Cerenkov counting. The epitope-tagged (EEEEYMPME, termed “EE”) wild type (MEK-WT), dominant negative (S218A, S222A; MEK-2A), and constitutively active (S218E, S218E; MEK-2E) rat MEK-1 vectors driven by cytomegalovirus promoters (62.Yan M Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar) were obtained from Dennis Templeton (Case Western Reserve University, Cleveland, OH). XL1-Blue supercompetent cells (Stratagene) were transformed with 20 ng of plasmid DNA following the manufacturer's protocol, and large scale plasmid DNA was prepared from overnight cultures (250 ml) using Qiagen Endofree Midi Prep Kit. For transfection, 20 μg of DNA and 2 μg of pCI neo (Promega) was prepared in 150 mm NaCl to a final volume of 50 μl and then added to 3 × 106 hMSCs in 200 μl of Opti-MEM (Life Technologies, Inc.). The cells were electroporated in 0.4-cm gap size cuvettes at 970 microfarads, 200 V with the time constant between 40 and 42 using a Gene Pulser II (Bio-Rad). The cells were then poured into culture dishes containing prewarmed Dulbecco's modified Eagle's medium + 10% fetal bovine serum. The medium was changed after 24 h to remove the dead cells and debris, and the cells were allowed to grow for 48 h. The cells were then treated with OS, and alkaline phosphatase activity and calcium deposition were measured as described above. The expression of epitope-tagged MEK-1 protein was confirmed by Western blotting using anti EE antibody (Berkeley Antibody Co., Berkeley, CA). Human MSCs were treated for 16 days with PD98059 (25 μm) in the presence or absence of OS medium. Total RNA was extracted by Purescript RNA isolation Kit (Gentra System, Minneapolis, MN) and quantified by UV spectroscopy. To prepare RNA for PCR analysis, 1.0 μg of RNA was converted to cDNA using Moloney murine leukemia virus reverse transcriptase and random hexamer primers. PCR reagents were purchased from Perkin-Elmer, and all experiments were performed using a GeneAmp PCR system 9600 (Perkin-Elmer) in MicroAmp reaction tubes (Perkin-Elmer). The following specific oligonucleotide primers were used: PPARγ2 (5′-GCTGTTATGGGTGAAACTCTG, 3′-ATAAGGTGGAGATGCAGGTTC), aP2(5′-TGGTTGATTTTCCATCCCAT, 3′-TACTGGGCCAGGAATTTGAC), lipoprotein lipase (5′-ATGGAGAGCAAAGCCCTGCTC, 3′-TACAGGGCGGCCACAAGTTTT), and osteopontin (5′-CTAGGCATCACCTGTGCCATACC, 3′-CAGTGACCAGTTCATCAGATTCATC). Amplification reactions were carried out through 30 cycles, and the reaction products were subjected to 1.5% agarose gel electrophoresis. The reaction products were 352 (PPARγ2), 114 (aP2), 298 (lipoprotein lipase), and 330 bp (osteopontin), respectively. The accumulation of intracellular triglyceride droplets was visualized by staining with Oil Red O as described previously (37.Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (274) Google Scholar). A fluorescent quantitative method for determining Nile Red-stained fat droplets was employed to determine the extent of adipogenesis (3.Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.M. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18443) Google Scholar). Briefly, cells growing in 6-well plates were fixed for 30 min in 10% neutral buffered formalin at room temperature. The cells were washed twice with PBS, and background fluorescence was measured in the presence of 1.0 ml PBS/well with a Molecular Devices fMAX Fluorescent Microplate Reader using 355/460 and 485/538 nm filter sets. Cells were then incubated for 15 min with 0.2% saponin, 8 μg/ml DAPI (4′,6-diamidino-2-phenylindole), and 1 μg/ml Nile Red in PBS at room temperature. Cells were washed three times with PBS, and fluorescence was measured as above, and background values were subtracted. Cell numbers were normalized by the use of DAPI staining. As previously demonstrated (4.Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.C. J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1857) Google Scholar), hMSCs differentiate into osteoblasts in response to OS over 14–16 days. Therefore, we examined ERK activation over the entire period of 3 weeks during treatment with OS. ERK activity in OS-treated cultures was determined by immunoblot analysis using phospho-specific MAP kinase antibody and by an in-gel kinase assay. As shown in Fig. 1 A, OS did not have an effect on ERK activation up to day 5 of treatment. However, a robust and sustained activation of ERK was observed from day 7 to day 11, and the vast increase in phosphorylation was on ERK2. The maximal activation was observed at day 7, and ERK activity declined to basal levels after day 11. We also tested ERK activation by an in-gel kinase assay using MBP as a substrate (Fig. 1, C andD). The immunoblot and in-gel kinase assays gave consistent results. A densitometric analysis of the autoradiogram from in-gel kinase assays revealed a 6–7-fold increase in kinase activity at day 7 in OS-treated cells as compared with non-treated controls. This increase declined to a 3-fold increase at day 11 and subsequently returned to basal level by day 13. Next, we measured JNK activity by an in-gel kinase assay using a GST-c-Jun (amino acids 1–79) fusion protein as a substrate. JNK2 binds c-Jun and catalyzes the phosphorylation of serine 63 and serine 73 (38.Kallunki T. Su B. Tsigelny I. Sluss H.K. Derijard B. Moore G. Davis R. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (604) Google Scholar). The continuous presence of OS led to a dramatic increase in JNK activity starting at day 13, peaking at day 15, and tapering off after day 17 (Fig. 2 B). A 5-fold increase in JNK2 activation over non-treated controls was observed at day 15 of OS treatment as assessed by radioactivity incorporated into the JNK band after in-gel kinase assay (Fig. 2 C). The third member of the MAP kinase family, p38, appears to have a distinct function in cells due to its substrate specificity that differs from those of ERK and JNK. Therefore, we tested the activation of p38 in OS-treated hMSCs. The p38 activation was measured by an immunoblotting procedure using a phospho-specific p38 antibody. Prolonged treatment with OS stimulates the p38 activation and essentially paralleled ERK activation, starting at day 9 and declining to basal levels after day 13 (Fig. 2 A). Since p38 is known to be activated by environmental stresses such as hyperosmolarity (39.Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), it is important to note that the time window when p38 is activated (9–13 days) is associated with Gla protein and extracellular matrix synthesis, increased alkaline phosphatase activity, and initiation of mineral deposition (40.Stein G.S. Lian J.B. Stein VanWijen J.L. Frenkel B. Montecino M. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996: 69-86Google Scholar) which may collectively cause an increase in intra- or extracellular osmolarity. To ask whether ERK activation is necessary for osteogenic differentiation, PD98059, a selective inhibitor of MEK1, was used to prevent the phosphorylation and activation of the ERKs (41.Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3128) Google Scholar). To quantify osteogenic differentiation, alkaline phosphatase (AP) activity was measured at day 7 of OS-treated hMSCs by both histochemical and biochemical methods. Continuous incubation of hMSCs with OS for 7 days resulted in an 8-fold increase in AP activity over the non-treated control (3.15 nmol of p-nitrophenyl phosphate/106 control versus 25 nmol ofp-nitrophenyl phosphate/106 OS-treated cells,p < 0.01) (Fig. 4 A). Treatment of hMSCs with OS-containing PD98059 produced a concentration-dependent inhibition of AP activity as determined by both histochemical and biochemical methods (Fig.3 A and 4 A). A 50 μm dose of PD98059 inhibited AP activity by 83%, whereas 10 and 25 μm PD98059 produced 71 and 62% inhibition, respectively. To confirm that inhibition of AP activity by PD98059 was due to abolition of MEK1 activity, we measured the activation of ERKs in parallel experiments by Western blotting using an anti-phospho-MAP kinase antibody. The dose-dependent inhibition of AP by PD98059 paralleled the decrease in ERK2 tyrosine phosphorylation (Fig.3 B).Figure 3A, effect of blockade of ERKs activation on alkaline phosphatase staining of hMSCs. Cells were cultured in control medium or OS medium or medium supplemented with PD98059. Alkaline phosphatase staining was performed at day 7 as described under “Experimental Procedures.” Differentiated stem cells positive for alkaline phosphatase are stained red. B, PD98059 inhibits ERK activation in a dose-dependent manner, without altering synthesis. hMSCs were grown for 7 days in control medium or OS containing increasing concentrations of PD98059, and lysates were prepared. Lysates were analyzed by immunoblotting using an anti-phospho-MAP kinase-specific (top panel) or MAP kinase antibody (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also tested the effect of PD98059 on the ability of hMSCs to mineralize the extracellular matrix that they produce when cultured in the presence of OS. hMSCs grown in OS deposited a calcium-rich mineralized matrix by day 15 as measured by a sensitive colorimetric calcium assay (Fig. 4 B). Importantly, the mineralization pattern as judged by the von Kossa staining was distributed throughout the culture rather than localized to a few discrete foci (data not shown). However, when added with OS, PD98059 significantly inhibited calcium deposition in a dose-dependent manner at all concentrations tested. Cells grown in control or OS medium containing 50 μm PD98059 failed to deposit any detectable calcium throughout the culture period. These results demonstrate that OS-induced APase activity and mineral deposition, the hallmarks of bone formation, are associated directly with ERK2 stimulation and, therefore, ERK2 plays an important role in osteogenic differentiation of hMSCs. We attempted to test inhibitors of p38 and JNK for effects on the osteogenic differentiation of the hMSCs. We were unable to acquire an inhibitor of JNK for testing. The inhibitor of p38, compound SB203580, did not inhibit AP activity significantly nor did it inhibit calcium accumulation (data not shown), and therefore its role in osteogenic differentiation is unclear. However, elevated expression of activity for this kinase is likely associated with other cellular processes, and JNK activity occurred at a much later time than ERK2 activation. A striking observation of the present study was that large numbers of adipocytes appeared in cultures that were treated with the MEK1 inhibitor PD98059 together with osteogenic differentiation medium. As early as 6–7 days after the onset of treatment, adipocytes bega