SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2027254518> ?p ?o ?g. }

Showing items 1 to 100 of ±121 with 100 items per page.

W2027254518 endingPage "19219" @default.
W2027254518 startingPage "19213" @default.
W2027254518 abstract "A possible regulatory mechanism of protein kinase C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been investigated. Inhibition or down-regulation of PKC resulted in the activation of a mitogen-activated protein kinase subtype Erk-1 and the inhibition of chondrogenesis. On the other hand, inhibition of Erk-1 with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of PKC inhibited proliferation of chondrogenic competent cells, and Erk-1 inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the inhibition of Erk-1. Expression of N-cadherin was detected at the early period of chondrogenesis. Inhibition or depletion of PKC induced sustained expression of N-cadherin, and Erk-1 inhibition blocked the effects of PKC modulation. The expression of integrin α5β1 and fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC. Because reduction of the examined cell adhesion molecule expression is a prerequisite for the progression of chondrogenesis after cell condensation, our results indicate that PKC regulates chondrogenesis by modulating expression of these molecules via Erk-1 signaling. A possible regulatory mechanism of protein kinase C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been investigated. Inhibition or down-regulation of PKC resulted in the activation of a mitogen-activated protein kinase subtype Erk-1 and the inhibition of chondrogenesis. On the other hand, inhibition of Erk-1 with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of PKC inhibited proliferation of chondrogenic competent cells, and Erk-1 inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the inhibition of Erk-1. Expression of N-cadherin was detected at the early period of chondrogenesis. Inhibition or depletion of PKC induced sustained expression of N-cadherin, and Erk-1 inhibition blocked the effects of PKC modulation. The expression of integrin α5β1 and fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC. Because reduction of the examined cell adhesion molecule expression is a prerequisite for the progression of chondrogenesis after cell condensation, our results indicate that PKC regulates chondrogenesis by modulating expression of these molecules via Erk-1 signaling. Prechondrogenic mesenchymes differentiate to chondrocytes when they become closely packed in the limb buds of chick embryo or duringin vitro micromass culture of mesenchymes (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 3Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (9958) Google Scholar). Chondrogenesis in vitro requires transient proliferation of cells to increase the number of chondrogenic competent cells, and the increased number of cells undergoes extensive cell-cell interaction such as precartilage condensation, which is one of the earliest morphogenetic events (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 3Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (9958) Google Scholar). Precartilage condensation is regulated by several cell adhesion molecules such as N-cadherin (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar, 5Tsonis P.A. Rio-Tsonis K.D. Millan J.L. Wheelock M.J. Exp. Cell Res. 1994; 213: 433-437Crossref PubMed Scopus (52) Google Scholar, 6Oberlender S.O. Tuan R.S. Development. 1994; 120: 177-187Crossref PubMed Google Scholar) and extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate. 1The abbreviations used are: ECM, extracellular matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate.molecules including fibronectin, type I collagen, laminin, and tenascin (7Mackie E.J. Thesleft I. Chiquet-Ehrismann R. J. Cell Biol. 1987; 105: 2569-2579Crossref PubMed Scopus (291) Google Scholar, 8Maleski M.P. Knudson C.B. Exp. Cell Res. 1996; 225: 55-66Crossref PubMed Scopus (86) Google Scholar, 9Downie S.A. Newman S.A. Dev. Biol. 1994; 162: 195-208Crossref PubMed Scopus (71) Google Scholar, 10Tavella S. Bellese G. Castagnola P. Martin I. Piccini D. Doliana R. Colombatti A. Cancedda R. Tacchetti C. J. Cell Sci. 1997; 110: 2261-2270Crossref PubMed Google Scholar). Thereafter, a large amount of cartilage-specific ECM components such as sulfated proteoglycans and type II collagen are synthesized until individual chondrocytes are surrounded by the ECM (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar,2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar). Several groups of extracellular molecules have been found to regulate chondrogenesis during the stages of cell proliferation and precartilage condensation. For example, a fibroblast growth factor appears to enhance chondrogenesis by increasing the number of chondrogenic competent cells (11Savage M.P. Hart C.E. Riley B.B. Sasse J. Olwin B.B. Falion J.F. Dev. Dyn. 1993; 198: 159-170Crossref PubMed Scopus (148) Google Scholar), whereas transforming growth factor β family members enhance chondrogenic differentiation by promoting precartilage condensation (12Duprez D.M. Coltey M. Amthor H. Brikell P.M. Tickle C. Dev. Biol. 1996; 174: 448-452Crossref PubMed Scopus (115) Google Scholar, 13Roark E.F. Geer K. Dev. Dyn. 1994; 200: 103-116Crossref PubMed Scopus (121) Google Scholar).Although much progress has been made in finding a mechanism governing chondrogenesis at levels of cell proliferation and precartilage condensation, the signal transduction pathways involved in chondrogenesis are poorly understood. Increasing evidence indicates that protein kinase C (PKC) might play a key role in chondrogenic differentiation (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar, 15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar, 16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar), as shown by the fact that PKC activities in both cytosol and particulate membrane fractions of mesenchymes increased during chondrogenesis (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar). The activities of PKC seem to be required for chondrogenic differentiation of mesenchymes because inhibition or down-regulation of PKC blocks chondrogenesis (15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar) and also causes dedifferentiation of rabbit costal and articular chondrocytes (16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar). PKC is a multigene family composed of 11 known isoforms (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar, 19Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2353) Google Scholar, 20Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). Chick limb bud mesenchymes express α, ε, ζ, and λ/ι isoforms, and depletion of PKCα and ε is sufficient to inhibit chondrogenesis (15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar), suggesting that PKCα and/or ε induces chondrogenic differentiation of mesenchymes.Although the requirement of PKC in chondrogenesis has been clearly established, its molecular mechanism in the regulation of chondrogenesis is not evident. To elucidate a possible mechanism involved in PKC action, our effort in this study has been focused on whether the regulatory role of PKC in chondrogenesis is mediated by mitogen-activated protein (MAP) kinase and also on whether PKC and/or MAP kinase regulates chondrogenesis at stages of proliferation of chondrogenic competent cells and precartilage condensation.DISCUSSIONSeveral lines of evidence support the hypothesis that chondrogenic differentiation of mesenchymes is regulated by PKC (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar, 15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar, 16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar). PKC was initially thought to regulate chondrogenesis in a negative way because staurosporine, a potent inhibitor of PKC, enhanced chondrogenesis (30Kulyk W.M. Dev. Biol. 1991; 146: 38-48Crossref PubMed Scopus (38) Google Scholar). However, it was found later that the stimulatory effect of staurosporine was caused by effects other than its ability to inhibit PKC (31Kulyk W.M. Hoffman L.M. Exp. Cell Res. 1996; 223: 290-300Crossref PubMed Scopus (28) Google Scholar). PKC has been implicated in a variety of cellular processes including growth and differentiation of many types of cells (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar). Regulation of cellular processes by PKC has been suggested to be mediated by direct regulation of gene expression and/or regulation of activities of other signaling molecules such as MAP kinase (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar, 19Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2353) Google Scholar, 20Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). It is well known that direct stimulation of PKC with phorbol ester leads to an activation of MAP kinase (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar) which is a common intermediate in intracellular signaling cascades (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar).Studies on PKC regulation of MAP kinase indicated that stimulation of PKC activated MAP kinase signaling (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Unexpectedly, in the present study we found that the inhibition or down-regulation of PKC resulted in the activation of Erk-1 and that the activated Erk-1 was responsible for the inhibitory role of PKC in chondrogenesis. This conclusion was derived from the observations that phosphorylation of Erk-1 was related to the inhibition of chondrogenesis (Fig. 2) and that the inhibition of Erk-1 phosphorylation relieved the inhibition of chondrogenesis induced by PKC (Fig. 4). The signaling pathway leading to the activation of Erk-1 by the inhibition or down-regulation of PKC is not currently known. Because stimulation of PKC is known to activate Erk-1 and -2 by the stimulation of upstream signaling molecules such as Raf (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), it is not likely that activation of Erk-1 is induced by the stimulation of Raf as a consequence of PKC inhibition or depletion. Rather, it is quite possible that inhibition or depletion of PKC inactivates MAP kinase phosphatase, which enhances MAP kinase activation (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar).Subtypes of MAP kinase, Erk-1 and -2, are thought to play a key role in the signaling process of many types of cellular differentiation, acting as an inhibitor or stimulator for cellular differentiation, depending on the types of differentiation. For instance, activation of both Erk-1 and -2 is required for the differentiation of fibroblasts to adipocytes (34Sale E.M. Atkinson P.G. Sale G.J. EMBO J. 1995; 14: 674-684Crossref PubMed Scopus (245) Google Scholar), and maturation of immature T cells also requires MAP kinase activity because expression of catalytically inactive MAP kinase kinase inhibits T cell maturation (35Alberola-Ila J. Forbush K.A. Seger R. Krebs E.G. Perimutter R.M. Nature. 1995; 373: 620-623Crossref PubMed Scopus (369) Google Scholar). In contrast to the above, it has been suggested that inactivation of Erk-2 is required for C2C12 myoblasts to initiate myogenesis (36Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (303) Google Scholar). Inactivation of MAP kinase is also observed during enterocyte differentiation (37Mamajiwalla S.N. Burgess D.R. Oncogene. 1995; 11: 377-386PubMed Google Scholar). We demonstrated in this study that inactivation of Erk-1 is required for the initiation of chondrogenesis.Extracellular molecules known to regulate chondrogenesis exert their effects at the level of cell proliferation and/or precartilage condensation (11Savage M.P. Hart C.E. Riley B.B. Sasse J. Olwin B.B. Falion J.F. Dev. Dyn. 1993; 198: 159-170Crossref PubMed Scopus (148) Google Scholar, 12Duprez D.M. Coltey M. Amthor H. Brikell P.M. Tickle C. Dev. Biol. 1996; 174: 448-452Crossref PubMed Scopus (115) Google Scholar, 13Roark E.F. Geer K. Dev. Dyn. 1994; 200: 103-116Crossref PubMed Scopus (121) Google Scholar). Therefore, we investigated the role of PKC and Erk-1 in the regulation of proliferation of chondrogenic competent cells and the expression of cell adhesion molecules known to be involved in precartilage condensation. Because inhibition or down-regulation of PKC reduces cell proliferation (Fig. 6), PKC activity appeared to be required for the proliferation of chondrogenic competent cells. However, the inhibition of cell proliferation by PKC was eliminated not by inhibiting Erk-1 (Fig. 6), whereas chondrogenesis was recovered significantly (Fig. 4). In addition, the inhibition of Erk-1 in the absence of PKC modulation, a condition that enhanced chondrogenesis, did not affect proliferation of chondrogenic competent cells, and PD98059 enhanced chondrogenesis at low cell density without affecting cell proliferation (Fig. 7). Therefore, we conclude that the stimulation and recovery of chondrogenesis by Erk-1 inhibition were not the result of enhancement of cell proliferation.Precartilage condensation is a process that reduces intercellular spaces and formation of extensive cell-cell contacts between prechondrogenic mesenchymes (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar). The biochemical events leading to cell condensation are not yet understood clearly. However, both cell-cell and cell-ECM interactions have been considered to play a role in precartilage condensation. Therefore, the possibility of whether PKC and MAP kinase regulate chondrogenesis by modulating the expression of cell adhesion molecules and ECM components such as N-cadherin, integrin α5β1, and fibronectin was investigated.N-Cadherin has been known to be expressed in prechondrogenic mesenchymes and during cell condensation (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar, 5Tsonis P.A. Rio-Tsonis K.D. Millan J.L. Wheelock M.J. Exp. Cell Res. 1994; 213: 433-437Crossref PubMed Scopus (52) Google Scholar), and blockage of its function inhibited precartilage condensation (6Oberlender S.O. Tuan R.S. Development. 1994; 120: 177-187Crossref PubMed Google Scholar). However, it was not expressed in chondrocytes that were completely surrounded by cartilage-specific ECM molecules (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar). Our results (Fig. 8) also indicated that N-cadherin was expressed at the early period of micromass culture, and its expression decreased as chondrogenesis proceeded. Inhibition or down-regulation of PKC resulted in sustained expression of N-cadherin, whereas inhibition of Erk-1 enhanced the decrease of N-cadherin expression at the later period of micromass culture. In addition, the effect of PKC on N-cadherin expression was blocked by the inhibition of Erk-1, suggesting that PKC action was mediated by Erk-1. A question of whether the modulation of N-cadherin expression by PKC and Erk-1 is a determinant of chondrogenesis or whether the modulation of chondrogenesis results in the altered expression of N-cadherin remains to be clarified. However, our data clearly indicate that PKC and Erk-1 signaling is closely coupled with the regulation of N-cadherin expression during chondrogenesis.In this study, we presented evidence that the expression of fibronectin and its receptor integrin α5β1 is regulated by PKC through Erk-1 signaling pathway, especially at the later stage of micromass culture,i.e. in cells cultured for 4–5 days (Fig. 8). Thus, the increased expression of integrin α5β1 and fibronectin at the later stage of micromass culture upon inhibition or depletion of PKC is closely correlated with the inhibition of chondrogenesis. On the other hand, the decreased expression of these molecules upon Erk-1 inhibition is correlated with an enhancement of chondrogenesis. The above results are consistent with the facts that an interaction of cells with fibronectin via integrin α5β1 is necessary for cell condensation to occur and that a reduction of fibronectin and its integrin α5β1 receptor after cell aggregation is necessary for the progression of cartilage differentiation (10Tavella S. Bellese G. Castagnola P. Martin I. Piccini D. Doliana R. Colombatti A. Cancedda R. Tacchetti C. J. Cell Sci. 1997; 110: 2261-2270Crossref PubMed Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar, 38Frenz D.A. Jaikaria N.S. Newman S.A. Dev. Biol. 1989; 136: 97-103Crossref PubMed Scopus (127) Google Scholar). Indeed, it has been known that enhanced expression of fibronectin exerts negative effects on chondrogenesis (39Gerstenfeld L.C. Finer M.H. Boedtker H. Mol. Cell. Biol. 1985; 5: 1425-1433Crossref PubMed Scopus (39) Google Scholar, 40Zanetti N.C. Solursh M. Dev. Biol. 1986; 113: 110-118Crossref PubMed Scopus (32) Google Scholar, 41Gregg B.C. Rowe A. Brickell P.M. Wolpert L. Development. 1989; 105: 769-777PubMed Google Scholar). We demonstrated in this study that PKC activity that inactivates Erk-1 is required to reduce the expression of integrin α5β1 and fibronectin and thereby to initiate the progression of chondrogenic differentiation. Because the reduction of fibronectin and integrin α5β1 occurs after the cell aggregation (29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar), our results suggest also that PKC and Erk-1 are not directly related to cell condensation. It is not yet known how the reduction of integrin α5β1 and fibronectin expression causes progression of chondrogenesis and how Erk-1 regulates expression of cell adhesion molecules and ECM components during chondrogenesis. We postulate that Erk-1 modulates synthesis and/or activation of transcription factors that regulate the expression of cell adhesion molecules and ECM components. Further work is needed to define downstream events that lead to the modulation of cell adhesion molecule expression. Prechondrogenic mesenchymes differentiate to chondrocytes when they become closely packed in the limb buds of chick embryo or duringin vitro micromass culture of mesenchymes (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 3Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (9958) Google Scholar). Chondrogenesis in vitro requires transient proliferation of cells to increase the number of chondrogenic competent cells, and the increased number of cells undergoes extensive cell-cell interaction such as precartilage condensation, which is one of the earliest morphogenetic events (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 3Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Crossref PubMed Scopus (9958) Google Scholar). Precartilage condensation is regulated by several cell adhesion molecules such as N-cadherin (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar, 5Tsonis P.A. Rio-Tsonis K.D. Millan J.L. Wheelock M.J. Exp. Cell Res. 1994; 213: 433-437Crossref PubMed Scopus (52) Google Scholar, 6Oberlender S.O. Tuan R.S. Development. 1994; 120: 177-187Crossref PubMed Google Scholar) and extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate. 1The abbreviations used are: ECM, extracellular matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate.molecules including fibronectin, type I collagen, laminin, and tenascin (7Mackie E.J. Thesleft I. Chiquet-Ehrismann R. J. Cell Biol. 1987; 105: 2569-2579Crossref PubMed Scopus (291) Google Scholar, 8Maleski M.P. Knudson C.B. Exp. Cell Res. 1996; 225: 55-66Crossref PubMed Scopus (86) Google Scholar, 9Downie S.A. Newman S.A. Dev. Biol. 1994; 162: 195-208Crossref PubMed Scopus (71) Google Scholar, 10Tavella S. Bellese G. Castagnola P. Martin I. Piccini D. Doliana R. Colombatti A. Cancedda R. Tacchetti C. J. Cell Sci. 1997; 110: 2261-2270Crossref PubMed Google Scholar). Thereafter, a large amount of cartilage-specific ECM components such as sulfated proteoglycans and type II collagen are synthesized until individual chondrocytes are surrounded by the ECM (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar,2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar). Several groups of extracellular molecules have been found to regulate chondrogenesis during the stages of cell proliferation and precartilage condensation. For example, a fibroblast growth factor appears to enhance chondrogenesis by increasing the number of chondrogenic competent cells (11Savage M.P. Hart C.E. Riley B.B. Sasse J. Olwin B.B. Falion J.F. Dev. Dyn. 1993; 198: 159-170Crossref PubMed Scopus (148) Google Scholar), whereas transforming growth factor β family members enhance chondrogenic differentiation by promoting precartilage condensation (12Duprez D.M. Coltey M. Amthor H. Brikell P.M. Tickle C. Dev. Biol. 1996; 174: 448-452Crossref PubMed Scopus (115) Google Scholar, 13Roark E.F. Geer K. Dev. Dyn. 1994; 200: 103-116Crossref PubMed Scopus (121) Google Scholar). Although much progress has been made in finding a mechanism governing chondrogenesis at levels of cell proliferation and precartilage condensation, the signal transduction pathways involved in chondrogenesis are poorly understood. Increasing evidence indicates that protein kinase C (PKC) might play a key role in chondrogenic differentiation (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar, 15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar, 16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar), as shown by the fact that PKC activities in both cytosol and particulate membrane fractions of mesenchymes increased during chondrogenesis (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar). The activities of PKC seem to be required for chondrogenic differentiation of mesenchymes because inhibition or down-regulation of PKC blocks chondrogenesis (15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar) and also causes dedifferentiation of rabbit costal and articular chondrocytes (16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar). PKC is a multigene family composed of 11 known isoforms (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar, 19Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2353) Google Scholar, 20Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). Chick limb bud mesenchymes express α, ε, ζ, and λ/ι isoforms, and depletion of PKCα and ε is sufficient to inhibit chondrogenesis (15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar), suggesting that PKCα and/or ε induces chondrogenic differentiation of mesenchymes. Although the requirement of PKC in chondrogenesis has been clearly established, its molecular mechanism in the regulation of chondrogenesis is not evident. To elucidate a possible mechanism involved in PKC action, our effort in this study has been focused on whether the regulatory role of PKC in chondrogenesis is mediated by mitogen-activated protein (MAP) kinase and also on whether PKC and/or MAP kinase regulates chondrogenesis at stages of proliferation of chondrogenic competent cells and precartilage condensation. DISCUSSIONSeveral lines of evidence support the hypothesis that chondrogenic differentiation of mesenchymes is regulated by PKC (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar, 15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar, 16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar). PKC was initially thought to regulate chondrogenesis in a negative way because staurosporine, a potent inhibitor of PKC, enhanced chondrogenesis (30Kulyk W.M. Dev. Biol. 1991; 146: 38-48Crossref PubMed Scopus (38) Google Scholar). However, it was found later that the stimulatory effect of staurosporine was caused by effects other than its ability to inhibit PKC (31Kulyk W.M. Hoffman L.M. Exp. Cell Res. 1996; 223: 290-300Crossref PubMed Scopus (28) Google Scholar). PKC has been implicated in a variety of cellular processes including growth and differentiation of many types of cells (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar). Regulation of cellular processes by PKC has been suggested to be mediated by direct regulation of gene expression and/or regulation of activities of other signaling molecules such as MAP kinase (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar, 19Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2353) Google Scholar, 20Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). It is well known that direct stimulation of PKC with phorbol ester leads to an activation of MAP kinase (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar) which is a common intermediate in intracellular signaling cascades (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar).Studies on PKC regulation of MAP kinase indicated that stimulation of PKC activated MAP kinase signaling (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Unexpectedly, in the present study we found that the inhibition or down-regulation of PKC resulted in the activation of Erk-1 and that the activated Erk-1 was responsible for the inhibitory role of PKC in chondrogenesis. This conclusion was derived from the observations that phosphorylation of Erk-1 was related to the inhibition of chondrogenesis (Fig. 2) and that the inhibition of Erk-1 phosphorylation relieved the inhibition of chondrogenesis induced by PKC (Fig. 4). The signaling pathway leading to the activation of Erk-1 by the inhibition or down-regulation of PKC is not currently known. Because stimulation of PKC is known to activate Erk-1 and -2 by the stimulation of upstream signaling molecules such as Raf (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), it is not likely that activation of Erk-1 is induced by the stimulation of Raf as a consequence of PKC inhibition or depletion. Rather, it is quite possible that inhibition or depletion of PKC inactivates MAP kinase phosphatase, which enhances MAP kinase activation (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar).Subtypes of MAP kinase, Erk-1 and -2, are thought to play a key role in the signaling process of many types of cellular differentiation, acting as an inhibitor or stimulator for cellular differentiation, depending on the types of differentiation. For instance, activation of both Erk-1 and -2 is required for the differentiation of fibroblasts to adipocytes (34Sale E.M. Atkinson P.G. Sale G.J. EMBO J. 1995; 14: 674-684Crossref PubMed Scopus (245) Google Scholar), and maturation of immature T cells also requires MAP kinase activity because expression of catalytically inactive MAP kinase kinase inhibits T cell maturation (35Alberola-Ila J. Forbush K.A. Seger R. Krebs E.G. Perimutter R.M. Nature. 1995; 373: 620-623Crossref PubMed Scopus (369) Google Scholar). In contrast to the above, it has been suggested that inactivation of Erk-2 is required for C2C12 myoblasts to initiate myogenesis (36Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (303) Google Scholar). Inactivation of MAP kinase is also observed during enterocyte differentiation (37Mamajiwalla S.N. Burgess D.R. Oncogene. 1995; 11: 377-386PubMed Google Scholar). We demonstrated in this study that inactivation of Erk-1 is required for the initiation of chondrogenesis.Extracellular molecules known to regulate chondrogenesis exert their effects at the level of cell proliferation and/or precartilage condensation (11Savage M.P. Hart C.E. Riley B.B. Sasse J. Olwin B.B. Falion J.F. Dev. Dyn. 1993; 198: 159-170Crossref PubMed Scopus (148) Google Scholar, 12Duprez D.M. Coltey M. Amthor H. Brikell P.M. Tickle C. Dev. Biol. 1996; 174: 448-452Crossref PubMed Scopus (115) Google Scholar, 13Roark E.F. Geer K. Dev. Dyn. 1994; 200: 103-116Crossref PubMed Scopus (121) Google Scholar). Therefore, we investigated the role of PKC and Erk-1 in the regulation of proliferation of chondrogenic competent cells and the expression of cell adhesion molecules known to be involved in precartilage condensation. Because inhibition or down-regulation of PKC reduces cell proliferation (Fig. 6), PKC activity appeared to be required for the proliferation of chondrogenic competent cells. However, the inhibition of cell proliferation by PKC was eliminated not by inhibiting Erk-1 (Fig. 6), whereas chondrogenesis was recovered significantly (Fig. 4). In addition, the inhibition of Erk-1 in the absence of PKC modulation, a condition that enhanced chondrogenesis, did not affect proliferation of chondrogenic competent cells, and PD98059 enhanced chondrogenesis at low cell density without affecting cell proliferation (Fig. 7). Therefore, we conclude that the stimulation and recovery of chondrogenesis by Erk-1 inhibition were not the result of enhancement of cell proliferation.Precartilage condensation is a process that reduces intercellular spaces and formation of extensive cell-cell contacts between prechondrogenic mesenchymes (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar). The biochemical events leading to cell condensation are not yet understood clearly. However, both cell-cell and cell-ECM interactions have been considered to play a role in precartilage condensation. Therefore, the possibility of whether PKC and MAP kinase regulate chondrogenesis by modulating the expression of cell adhesion molecules and ECM components such as N-cadherin, integrin α5β1, and fibronectin was investigated.N-Cadherin has been known to be expressed in prechondrogenic mesenchymes and during cell condensation (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar, 5Tsonis P.A. Rio-Tsonis K.D. Millan J.L. Wheelock M.J. Exp. Cell Res. 1994; 213: 433-437Crossref PubMed Scopus (52) Google Scholar), and blockage of its function inhibited precartilage condensation (6Oberlender S.O. Tuan R.S. Development. 1994; 120: 177-187Crossref PubMed Google Scholar). However, it was not expressed in chondrocytes that were completely surrounded by cartilage-specific ECM molecules (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar). Our results (Fig. 8) also indicated that N-cadherin was expressed at the early period of micromass culture, and its expression decreased as chondrogenesis proceeded. Inhibition or down-regulation of PKC resulted in sustained expression of N-cadherin, whereas inhibition of Erk-1 enhanced the decrease of N-cadherin expression at the later period of micromass culture. In addition, the effect of PKC on N-cadherin expression was blocked by the inhibition of Erk-1, suggesting that PKC action was mediated by Erk-1. A question of whether the modulation of N-cadherin expression by PKC and Erk-1 is a determinant of chondrogenesis or whether the modulation of chondrogenesis results in the altered expression of N-cadherin remains to be clarified. However, our data clearly indicate that PKC and Erk-1 signaling is closely coupled with the regulation of N-cadherin expression during chondrogenesis.In this study, we presented evidence that the expression of fibronectin and its receptor integrin α5β1 is regulated by PKC through Erk-1 signaling pathway, especially at the later stage of micromass culture,i.e. in cells cultured for 4–5 days (Fig. 8). Thus, the increased expression of integrin α5β1 and fibronectin at the later stage of micromass culture upon inhibition or depletion of PKC is closely correlated with the inhibition of chondrogenesis. On the other hand, the decreased expression of these molecules upon Erk-1 inhibition is correlated with an enhancement of chondrogenesis. The above results are consistent with the facts that an interaction of cells with fibronectin via integrin α5β1 is necessary for cell condensation to occur and that a reduction of fibronectin and its integrin α5β1 receptor after cell aggregation is necessary for the progression of cartilage differentiation (10Tavella S. Bellese G. Castagnola P. Martin I. Piccini D. Doliana R. Colombatti A. Cancedda R. Tacchetti C. J. Cell Sci. 1997; 110: 2261-2270Crossref PubMed Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar, 38Frenz D.A. Jaikaria N.S. Newman S.A. Dev. Biol. 1989; 136: 97-103Crossref PubMed Scopus (127) Google Scholar). Indeed, it has been known that enhanced expression of fibronectin exerts negative effects on chondrogenesis (39Gerstenfeld L.C. Finer M.H. Boedtker H. Mol. Cell. Biol. 1985; 5: 1425-1433Crossref PubMed Scopus (39) Google Scholar, 40Zanetti N.C. Solursh M. Dev. Biol. 1986; 113: 110-118Crossref PubMed Scopus (32) Google Scholar, 41Gregg B.C. Rowe A. Brickell P.M. Wolpert L. Development. 1989; 105: 769-777PubMed Google Scholar). We demonstrated in this study that PKC activity that inactivates Erk-1 is required to reduce the expression of integrin α5β1 and fibronectin and thereby to initiate the progression of chondrogenic differentiation. Because the reduction of fibronectin and integrin α5β1 occurs after the cell aggregation (29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar), our results suggest also that PKC and Erk-1 are not directly related to cell condensation. It is not yet known how the reduction of integrin α5β1 and fibronectin expression causes progression of chondrogenesis and how Erk-1 regulates expression of cell adhesion molecules and ECM components during chondrogenesis. We postulate that Erk-1 modulates synthesis and/or activation of transcription factors that regulate the expression of cell adhesion molecules and ECM components. Further work is needed to define downstream events that lead to the modulation of cell adhesion molecule expression. Several lines of evidence support the hypothesis that chondrogenic differentiation of mesenchymes is regulated by PKC (14Sonn J.-K. Solursh M. Differentiation. 1993; 53: 155-162Crossref PubMed Scopus (24) Google Scholar, 15Choi B. Chun J.-S. Lee Y.-S. Sonn J.-K. Kang S-.S. Biochem. Biophys. Res. Commun. 1995; 216: 1034-1040Crossref PubMed Scopus (45) Google Scholar, 16Bouakka M. Legendre P. Jouis V. Langris M. Beliard R. Loyau G. Bocquet J. Biochem. Biophys. Res. Commun. 1988; 153: 690-698Crossref PubMed Scopus (10) Google Scholar, 17Takigawa M. Fukuo K. Takano T. Suzuki F. Cell Differ. 1983; 13: 283-291Crossref PubMed Scopus (21) Google Scholar). PKC was initially thought to regulate chondrogenesis in a negative way because staurosporine, a potent inhibitor of PKC, enhanced chondrogenesis (30Kulyk W.M. Dev. Biol. 1991; 146: 38-48Crossref PubMed Scopus (38) Google Scholar). However, it was found later that the stimulatory effect of staurosporine was caused by effects other than its ability to inhibit PKC (31Kulyk W.M. Hoffman L.M. Exp. Cell Res. 1996; 223: 290-300Crossref PubMed Scopus (28) Google Scholar). PKC has been implicated in a variety of cellular processes including growth and differentiation of many types of cells (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar). Regulation of cellular processes by PKC has been suggested to be mediated by direct regulation of gene expression and/or regulation of activities of other signaling molecules such as MAP kinase (18Goodnight J. Mischak H. Mushinski J.F. Adv. Cancer Res. 1994; 64: 159-209Crossref PubMed Google Scholar, 19Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2353) Google Scholar, 20Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (845) Google Scholar). It is well known that direct stimulation of PKC with phorbol ester leads to an activation of MAP kinase (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar) which is a common intermediate in intracellular signaling cascades (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar). Studies on PKC regulation of MAP kinase indicated that stimulation of PKC activated MAP kinase signaling (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Unexpectedly, in the present study we found that the inhibition or down-regulation of PKC resulted in the activation of Erk-1 and that the activated Erk-1 was responsible for the inhibitory role of PKC in chondrogenesis. This conclusion was derived from the observations that phosphorylation of Erk-1 was related to the inhibition of chondrogenesis (Fig. 2) and that the inhibition of Erk-1 phosphorylation relieved the inhibition of chondrogenesis induced by PKC (Fig. 4). The signaling pathway leading to the activation of Erk-1 by the inhibition or down-regulation of PKC is not currently known. Because stimulation of PKC is known to activate Erk-1 and -2 by the stimulation of upstream signaling molecules such as Raf (32Chao T.-S.O. Foster D.A. Rapp U.R. Rosner M.R. J. Biol. Chem. 1994; 269: 7337-7341Abstract Full Text PDF PubMed Google Scholar, 33Ueda Y. Hirai S. Osda S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), it is not likely that activation of Erk-1 is induced by the stimulation of Raf as a consequence of PKC inhibition or depletion. Rather, it is quite possible that inhibition or depletion of PKC inactivates MAP kinase phosphatase, which enhances MAP kinase activation (24Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar, 25Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 26Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar). Subtypes of MAP kinase, Erk-1 and -2, are thought to play a key role in the signaling process of many types of cellular differentiation, acting as an inhibitor or stimulator for cellular differentiation, depending on the types of differentiation. For instance, activation of both Erk-1 and -2 is required for the differentiation of fibroblasts to adipocytes (34Sale E.M. Atkinson P.G. Sale G.J. EMBO J. 1995; 14: 674-684Crossref PubMed Scopus (245) Google Scholar), and maturation of immature T cells also requires MAP kinase activity because expression of catalytically inactive MAP kinase kinase inhibits T cell maturation (35Alberola-Ila J. Forbush K.A. Seger R. Krebs E.G. Perimutter R.M. Nature. 1995; 373: 620-623Crossref PubMed Scopus (369) Google Scholar). In contrast to the above, it has been suggested that inactivation of Erk-2 is required for C2C12 myoblasts to initiate myogenesis (36Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (303) Google Scholar). Inactivation of MAP kinase is also observed during enterocyte differentiation (37Mamajiwalla S.N. Burgess D.R. Oncogene. 1995; 11: 377-386PubMed Google Scholar). We demonstrated in this study that inactivation of Erk-1 is required for the initiation of chondrogenesis. Extracellular molecules known to regulate chondrogenesis exert their effects at the level of cell proliferation and/or precartilage condensation (11Savage M.P. Hart C.E. Riley B.B. Sasse J. Olwin B.B. Falion J.F. Dev. Dyn. 1993; 198: 159-170Crossref PubMed Scopus (148) Google Scholar, 12Duprez D.M. Coltey M. Amthor H. Brikell P.M. Tickle C. Dev. Biol. 1996; 174: 448-452Crossref PubMed Scopus (115) Google Scholar, 13Roark E.F. Geer K. Dev. Dyn. 1994; 200: 103-116Crossref PubMed Scopus (121) Google Scholar). Therefore, we investigated the role of PKC and Erk-1 in the regulation of proliferation of chondrogenic competent cells and the expression of cell adhesion molecules known to be involved in precartilage condensation. Because inhibition or down-regulation of PKC reduces cell proliferation (Fig. 6), PKC activity appeared to be required for the proliferation of chondrogenic competent cells. However, the inhibition of cell proliferation by PKC was eliminated not by inhibiting Erk-1 (Fig. 6), whereas chondrogenesis was recovered significantly (Fig. 4). In addition, the inhibition of Erk-1 in the absence of PKC modulation, a condition that enhanced chondrogenesis, did not affect proliferation of chondrogenic competent cells, and PD98059 enhanced chondrogenesis at low cell density without affecting cell proliferation (Fig. 7). Therefore, we conclude that the stimulation and recovery of chondrogenesis by Erk-1 inhibition were not the result of enhancement of cell proliferation. Precartilage condensation is a process that reduces intercellular spaces and formation of extensive cell-cell contacts between prechondrogenic mesenchymes (1Solursh M. Curr. Opin. Cell Biol. 1989; 1: 989-998Crossref PubMed Scopus (37) Google Scholar, 2Reddi H.A. Curr. Opin. Gen. Dev. 1994; 4: 737-744Crossref PubMed Scopus (327) Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar). The biochemical events leading to cell condensation are not yet understood clearly. However, both cell-cell and cell-ECM interactions have been considered to play a role in precartilage condensation. Therefore, the possibility of whether PKC and MAP kinase regulate chondrogenesis by modulating the expression of cell adhesion molecules and ECM components such as N-cadherin, integrin α5β1, and fibronectin was investigated. N-Cadherin has been known to be expressed in prechondrogenic mesenchymes and during cell condensation (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar, 5Tsonis P.A. Rio-Tsonis K.D. Millan J.L. Wheelock M.J. Exp. Cell Res. 1994; 213: 433-437Crossref PubMed Scopus (52) Google Scholar), and blockage of its function inhibited precartilage condensation (6Oberlender S.O. Tuan R.S. Development. 1994; 120: 177-187Crossref PubMed Google Scholar). However, it was not expressed in chondrocytes that were completely surrounded by cartilage-specific ECM molecules (4Tavella S. Raffo P. Tacchetti C. Cancedda R. Castagnola P. Exp. Cell Res. 1994; 215: 354-362Crossref PubMed Scopus (160) Google Scholar). Our results (Fig. 8) also indicated that N-cadherin was expressed at the early period of micromass culture, and its expression decreased as chondrogenesis proceeded. Inhibition or down-regulation of PKC resulted in sustained expression of N-cadherin, whereas inhibition of Erk-1 enhanced the decrease of N-cadherin expression at the later period of micromass culture. In addition, the effect of PKC on N-cadherin expression was blocked by the inhibition of Erk-1, suggesting that PKC action was mediated by Erk-1. A question of whether the modulation of N-cadherin expression by PKC and Erk-1 is a determinant of chondrogenesis or whether the modulation of chondrogenesis results in the altered expression of N-cadherin remains to be clarified. However, our data clearly indicate that PKC and Erk-1 signaling is closely coupled with the regulation of N-cadherin expression during chondrogenesis. In this study, we presented evidence that the expression of fibronectin and its receptor integrin α5β1 is regulated by PKC through Erk-1 signaling pathway, especially at the later stage of micromass culture,i.e. in cells cultured for 4–5 days (Fig. 8). Thus, the increased expression of integrin α5β1 and fibronectin at the later stage of micromass culture upon inhibition or depletion of PKC is closely correlated with the inhibition of chondrogenesis. On the other hand, the decreased expression of these molecules upon Erk-1 inhibition is correlated with an enhancement of chondrogenesis. The above results are consistent with the facts that an interaction of cells with fibronectin via integrin α5β1 is necessary for cell condensation to occur and that a reduction of fibronectin and its integrin α5β1 receptor after cell aggregation is necessary for the progression of cartilage differentiation (10Tavella S. Bellese G. Castagnola P. Martin I. Piccini D. Doliana R. Colombatti A. Cancedda R. Tacchetti C. J. Cell Sci. 1997; 110: 2261-2270Crossref PubMed Google Scholar, 29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar, 38Frenz D.A. Jaikaria N.S. Newman S.A. Dev. Biol. 1989; 136: 97-103Crossref PubMed Scopus (127) Google Scholar). Indeed, it has been known that enhanced expression of fibronectin exerts negative effects on chondrogenesis (39Gerstenfeld L.C. Finer M.H. Boedtker H. Mol. Cell. Biol. 1985; 5: 1425-1433Crossref PubMed Scopus (39) Google Scholar, 40Zanetti N.C. Solursh M. Dev. Biol. 1986; 113: 110-118Crossref PubMed Scopus (32) Google Scholar, 41Gregg B.C. Rowe A. Brickell P.M. Wolpert L. Development. 1989; 105: 769-777PubMed Google Scholar). We demonstrated in this study that PKC activity that inactivates Erk-1 is required to reduce the expression of integrin α5β1 and fibronectin and thereby to initiate the progression of chondrogenic differentiation. Because the reduction of fibronectin and integrin α5β1 occurs after the cell aggregation (29Maini P.K. Solursh M. Int. Rev. Cytol. 1991; 129: 91-133Crossref PubMed Scopus (52) Google Scholar), our results suggest also that PKC and Erk-1 are not directly related to cell condensation. It is not yet known how the reduction of integrin α5β1 and fibronectin expression causes progression of chondrogenesis and how Erk-1 regulates expression of cell adhesion molecules and ECM components during chondrogenesis. We postulate that Erk-1 modulates synthesis and/or activation of transcription factors that regulate the expression of cell adhesion molecules and ECM components. Further work is needed to define downstream events that lead to the modulation of cell adhesion molecule expression. We are grateful to Drs. Woon Ki Paik for critical reading of the manuscript and Mahn Joon Ha (Ajou University) for helpful discussions." @default.
W2027254518 created "2016-06-24" @default.
W2027254518 creator A5015590017 @default.
W2027254518 creator A5021258379 @default.
W2027254518 creator A5024848021 @default.
W2027254518 creator A5039435063 @default.
W2027254518 creator A5052488200 @default.
W2027254518 creator A5055064361 @default.
W2027254518 creator A5057974303 @default.
W2027254518 date "1998-07-01" @default.
W2027254518 modified "2023-09-30" @default.
W2027254518 title "Protein Kinase C Regulates Chondrogenesis of Mesenchymes via Mitogen-activated Protein Kinase Signaling" @default.
W2027254518 cites W1528752221 @default.
W2027254518 cites W1576671418 @default.
W2027254518 cites W1641731911 @default.
W2027254518 cites W1662546471 @default.
W2027254518 cites W1804065695 @default.
W2027254518 cites W1865792554 @default.
W2027254518 cites W1972018349 @default.
W2027254518 cites W1975358464 @default.
W2027254518 cites W1979004076 @default.
W2027254518 cites W1986176372 @default.
W2027254518 cites W1993950414 @default.
W2027254518 cites W1994589542 @default.
W2027254518 cites W1995205975 @default.
W2027254518 cites W1997117663 @default.
W2027254518 cites W2001517274 @default.
W2027254518 cites W2004489402 @default.
W2027254518 cites W2006237807 @default.
W2027254518 cites W2011015637 @default.
W2027254518 cites W2031170198 @default.
W2027254518 cites W2032380793 @default.
W2027254518 cites W2035404931 @default.
W2027254518 cites W2035936653 @default.
W2027254518 cites W2049285163 @default.
W2027254518 cites W2062557303 @default.
W2027254518 cites W2064669451 @default.
W2027254518 cites W2068502127 @default.
W2027254518 cites W2072374837 @default.
W2027254518 cites W2075104267 @default.
W2027254518 cites W2076926358 @default.
W2027254518 cites W2080034804 @default.
W2027254518 cites W2087178104 @default.
W2027254518 cites W2087309480 @default.
W2027254518 cites W2104437462 @default.
W2027254518 cites W2121848695 @default.
W2027254518 cites W2133526415 @default.
W2027254518 cites W2140034535 @default.
W2027254518 cites W2163421864 @default.
W2027254518 cites W28018459 @default.
W2027254518 cites W4292820908 @default.
W2027254518 doi "https://doi.org/10.1074/jbc.273.30.19213" @default.
W2027254518 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9668109" @default.
W2027254518 hasPublicationYear "1998" @default.
W2027254518 type Work @default.
W2027254518 sameAs 2027254518 @default.
W2027254518 citedByCount "88" @default.
W2027254518 countsByYear W20272545182012 @default.
W2027254518 countsByYear W20272545182013 @default.
W2027254518 countsByYear W20272545182014 @default.
W2027254518 countsByYear W20272545182016 @default.
W2027254518 countsByYear W20272545182018 @default.
W2027254518 countsByYear W20272545182019 @default.
W2027254518 countsByYear W20272545182020 @default.
W2027254518 crossrefType "journal-article" @default.
W2027254518 hasAuthorship W2027254518A5015590017 @default.
W2027254518 hasAuthorship W2027254518A5021258379 @default.
W2027254518 hasAuthorship W2027254518A5024848021 @default.
W2027254518 hasAuthorship W2027254518A5039435063 @default.
W2027254518 hasAuthorship W2027254518A5052488200 @default.
W2027254518 hasAuthorship W2027254518A5055064361 @default.
W2027254518 hasAuthorship W2027254518A5057974303 @default.
W2027254518 hasConcept C124160383 @default.
W2027254518 hasConcept C132149769 @default.
W2027254518 hasConcept C137361374 @default.
W2027254518 hasConcept C159479382 @default.
W2027254518 hasConcept C184235292 @default.
W2027254518 hasConcept C185592680 @default.
W2027254518 hasConcept C55493867 @default.
W2027254518 hasConcept C59143045 @default.
W2027254518 hasConcept C82495950 @default.
W2027254518 hasConcept C86803240 @default.
W2027254518 hasConcept C90934575 @default.
W2027254518 hasConcept C95444343 @default.
W2027254518 hasConcept C97029542 @default.
W2027254518 hasConcept C99405784 @default.
W2027254518 hasConceptScore W2027254518C124160383 @default.
W2027254518 hasConceptScore W2027254518C132149769 @default.
W2027254518 hasConceptScore W2027254518C137361374 @default.
W2027254518 hasConceptScore W2027254518C159479382 @default.
W2027254518 hasConceptScore W2027254518C184235292 @default.
W2027254518 hasConceptScore W2027254518C185592680 @default.
W2027254518 hasConceptScore W2027254518C55493867 @default.
W2027254518 hasConceptScore W2027254518C59143045 @default.
W2027254518 hasConceptScore W2027254518C82495950 @default.
W2027254518 hasConceptScore W2027254518C86803240 @default.
W2027254518 hasConceptScore W2027254518C90934575 @default.
W2027254518 hasConceptScore W2027254518C95444343 @default.