Protein Kinase C Regulates Chondrogenesis of Mesenchymes via Mitogen-activated Protein Kinase Signaling*

Sung-Hee ChangDagger , Chun-Do OhDagger , Myung-Soon YangDagger , Shin-Sung KangDagger , Young-Sup Lee§, Jong-Kyung Sonn, and Jang-Soo ChunDagger parallel

From the Department of Dagger  Biology and § Biochemistry, College of Natural Sciences and  Teacher's College, Kyungpook National University, Taegu 702-701, Korea

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 5beta 1 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Prechondrogenic mesenchymes differentiate to chondrocytes when they become closely packed in the limb buds of chick embryo or during in vitro micromass culture of mesenchymes (1-3). 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 (1-3). Precartilage condensation is regulated by several cell adhesion molecules such as N-cadherin (4-6) and extracellular matrix (ECM)1 molecules including fibronectin, type I collagen, laminin, and tenascin (7-10). 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 (1, 2). 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 (11), whereas transforming growth factor beta  family members enhance chondrogenic differentiation by promoting precartilage condensation (12, 13).

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 (14-17), as shown by the fact that PKC activities in both cytosol and particulate membrane fractions of mesenchymes increased during chondrogenesis (14). The activities of PKC seem to be required for chondrogenic differentiation of mesenchymes because inhibition or down-regulation of PKC blocks chondrogenesis (15) and also causes dedifferentiation of rabbit costal and articular chondrocytes (16, 17). PKC is a multigene family composed of 11 known isoforms (18-20). Chick limb bud mesenchymes express alpha , epsilon , zeta , and lambda /iota isoforms, and depletion of PKCalpha and epsilon  is sufficient to inhibit chondrogenesis (15), suggesting that PKCalpha and/or epsilon  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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Micromass Culture of Mesenchymes-- Mesenchymes were derived from the distal tips of Hamburger-Hamilton stage 23/24 embryo limb buds (3) of fertilized white Leghorn chicken eggs as described previously (15). The cells were suspended at a density of 2.0 × 107 cells/ml in Ham's F-12 medium containing 10% fetal calf serum, unless otherwise indicated. Chondrogenesis was induced by adding the cells in 15-µl drops to 35-mm or 60-mm culture dishes. The cells were incubated for 60 min at 37 °C to allow attachment and then cultured in Ham's F-12 medium containing 10% fetal calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin either in the absence or presence of various reagents as described in each experiment. Chondrogenic differentiation of mesenchymes was determined immunocytochemically by examining the expression of type II collagen as described below. Alternatively, cells cultured for various time periods were stained with Alcian blue to localize the sulfated cartilage matrix. Alcian blue bound to sulfated glucosaminoglycans was extracted with 4 M guanidine-HCl and quantitated by measuring the absorbance at 600 nm (15).

Immunofluorescence Microscopy and Immunocytochemistry-- Mesenchymes cultured for the indicated time periods were fixed with 3% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature, and the fixed cells were washed and incubated for 45 min with anti-collagen type II monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) containing 5% fetal calf serum. The cells were then washed with phosphate-buffered saline and incubated with rhodamine-conjugated goat anti-mouse IgG antibody for another 45 min. The specimen samples were covered with glycerol and examined with a Nikon Optiphot fluorescence microscope. Alternatively, the cells incubated with anti-collagen type II monoclonal antibody were washed with phosphate-buffered saline and decorated with biotinylated anti-mouse IgG for 30 min. The cells were incubated with Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) for 30 min, and type II collagen was visualized by developing with DAB substrate solution kit (Vector Laboratories, Inc.) by the procedure recommended by the manufacturer.

Cell Fractionation-- Separation of cytosolic and particulate membrane fractions was performed as described previously (21, 22). Briefly, cells micromass cultured for the indicated time periods were scraped in buffer A (20 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose, 2 mM EGTA, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). The cells were sonicated for 6 s twice and centrifuged at 100,000 × g for 1 h. The supernatant was designated as the cytosolic fraction. The pellet was extracted with buffer B (20 mM Tris-HCl, pH 7.5, containing 1% SDS, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, and protease inhibitors as described above). After centrifugation at 15,000 × g for 15 min, the supernatant was saved as the particulate membrane fraction.

Western Blot Analysis-- Total cell lysate was prepared from cells micromass cultured for various time periods by extracting proteins with a lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% deoxycholate). Proteins were separated by 8% polyacrylamide gel electrophoresis containing 0.1% SDS and transferred to nitrocellulose membrane. The nitrocelluose sheet was blocked with 3% nonfat dry milk in Tris-buffered saline. PKC isoforms were detected with isoform-specific anti-PKC monoclonal antibodies for beta , delta , epsilon , theta , lambda /iota , and µ (Transduction Laboratories, Lexington, KY) or with polyclonal antibodies for alpha , gamma , and zeta  (Santa Cruz Biotechnology, Santa Cruz, CA) and eta  (Biomol, Plymouth Meeting, PA). Expression of cell adhesion molecules was determined by using antibodies purchased from following sources: rabbit anti-chick N-cadherin polyclonal antibody from Sigma Chemical Co. (St. Louis, MO), rabbit anti-human fibronectin polyclonal antibody from Upstate Biotechnology Inc. (Lake Placid, NY), mouse anti-chick integrin alpha 5 monoclonal antibody from Developmental Studies Hybridoma Bank, and rabbit anti-human integrin beta 1 polyclonal antibody from Chemicon (Temecula, CA). The blots were developed using a peroxidase-conjugated secondary antibody, and proteins were visualized by the ECL system (21, 22).

MAP Kinase Assay-- Mesenchymes were micromass cultured in the absence or presence of various reagents for the indicated time periods. Proteins were extracted with a buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM 4-nitrophenyl phosphate). Expression of MAP kinase subtypes Erk-1 and -2 was determined by Western blotting using antibodies obtained from Santa Cruz Biotechnology. Activation of MAP kinase was examined by determining its phosphorylation state using antibody specific to phosphorylated Erk-1 and -2 (New England Biolabs, Beverly, MA).

Cell Proliferation-- Mesenchymes were micromass cultured (three spots/35-mm dish) in the absence or presence of various reagents for the indicated time periods. Individual cells were suspended with 0.1% each of trypsin and collagenase, and the number of viable cells was counted in triplicate using a hemocytometer.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

PKC Regulates Chondrogenesis by Modulation of MAP Kinase-- Micromass-cultured mesenchymes derived from chick limb buds underwent spontaneous differentiation into chondrocytes, demonstrated by the expression of type II collagen, a cartilage-specific molecule (Fig. 1A). Immunofluorescence microscopy revealed that the expressed type II collagen was localized in cartilage nodules (Fig. 1B). The expression of type II collagen was reduced when the cells were cultured in the presence of Go6976 to inhibit PKC (23) or phorbol 12-myristate 13-acetate (PMA) to down-regulate PKC (Fig. 1A). Similar results were obtained when the micromass-cultured cells were stained with Alcian blue to localize the sulfated cartilage matrix (data not shown). Quantitation of chondrogenesis by measuring an absorbance of Alcian blue extract indicated that inhibition or down-regulation of PKC reduced chondrogenesis to 29 and 32% of the control, respectively (Fig. 1C). The above results indicate that PKC activity is required for chondrogenic differentiation of mesenchymes in in vitro micromass culture.


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Fig. 1.   PKC is required for the chondrogenic differentiation of mesenchymes in vitro. Panel A, type II collagen expression. Mesenchymes were micromass cultured for 1 (a) or 4 (b) days in the presence of vehicle alone or for 4 days in the presence of 10 nM PMA (c) or 0.5 µM Go6976 (d). Expression of type II collagen was determined by immunocytochemistry. Panel B, localization of type II collagen in cartilage nodules. Mesenchymes were cultured for 4 days, and distribution of type II collagen was examined by immunofluorescence microscopy (a). Phase-contrast microscopy of the micromass culture is shown in (b). Panel C, quantitation of chondrogenesis. The cells cultured for 4 days in the presence of vehicle as a control, 0.5 µM Go6976, or 10 nM PMA, were stained with Alcian blue to localize sulfated proteoglycan. Chondrogenesis was quantitated by measuring an absorbance of extractable Alcian blue at 600 nm (n = 4).

Because Erk-1 and -2 subtypes of MAP kinase are downstream targets of PKC (24-26), it was felt of importance to examine whether regulation of chondrogenesis by PKC was mediated by MAP kinase signaling. Erk-1 was expressed at constant level during chondrogenesis (Fig. 2A); however, its phosphorylation was high at early time periods of micromass culture, i.e. culture day 1, and reduced as chondrogenesis proceeded (Fig. 2B). Erk-2 was not easily detectable with the antibody employed, probably because of its low level of expression. When a large amount of protein was used, the expression and phosphorylation patterns of Erk-2 were similar to those of Erk-1 (data not shown). In subsequent experiments, therefore, we examined only Erk-1. Phosphorylation of Erk-1 was almost completely blocked when mesenchymes were cultured in the presence of PD98059, a specific inhibitor of MAP kinase kinase (27). In contrast, Erk-1 phosphorylation was enhanced significantly when mesenchymes were cultured in the presence of PMA or Go6976 (Fig. 2B), reagents that inhibit chondrogenesis (Fig. 1). As shown in Fig. 2A, treatment of mesenchymes with PD98059, Go6976, or PMA during micromass culture of mesenchymes did not affect the expression level of Erk-1.


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Fig. 2.   Inhibition or down-regulation of PKC enhances Erk-1 phosphorylation. Mesenchymes were micromass cultured for the indicated time periods in the presence of vehicle alone as a control, 10 µM PD98059, 10 nM PMA, or 0.5 µM Go6976. Expression (A) and phosphorylation (B) of Erk-1 were determined by Western blotting. The data represent a typical experiment conducted more than five times (A and B) with similar results.

Enhancement of Erk-1 phosphorylation by inhibition or down-regulation of PKC suggested that phosphorylation and/or activation of Erk-1 could inhibit chondrogenesis. To explore this possibility, the effect of inhibition of Erk-1 with PD98059 on chondrogenic mesenchyme differentiation was determined. As shown in Fig. 3A, treatment of the cells with PD98059 enhanced type II collagen expression. Also, Alcian blue staining indicated that PD98059 treatment enhanced chondrogenesis up to 2-fold (Fig. 3B) at concentrations in the range of 1-20 µM. Thus, it is concluded that inactivation of Erk-1 resulted in an enhanced chondrogenesis, whereas Erk-1 activation by inhibition or depletion of PKC was inhibitory for chondrogenesis.


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Fig. 3.   Inhibition of Erk-1 enhances chondrogenesis. Panel A, mesenchymes were cultured for 4 days in the presence of vehicle alone (a) or 10 µM PD98059 (b), and the expression of type II collagen was examined by immunocytochemistry. Panel B, mesenchymes were cultured for the indicated time periods in the presence of vehicle alone as a control, 10 µM PD98059, 0.5 µM Go6976, or 10 nM PMA. Chondrogenesis was quantitated by staining sulfated proteoglycan with Alcian blue and reading the absorbance of bound Alcian blue extract at 600 nm. Panel C, mesenchymes were micromass cultured for 4 days in the presence of various concentrations of PD98059, and chondrogenesis was quantitated. The data in A represent results of a typical experiment; the data in B and C represent the average of six independent experiments with standard deviation.

We next explored the possibility of whether the inhibition of chondrogenesis by down-regulation or inhibition of PKC was caused by the activation of Erk-1. It was found that the inhibition of Erk-1 with PD98059 blocked PMA- or Go6976-induced reduction of type II collagen expression (Fig. 4A) or Alcian blue staining (Fig. 4B), suggesting that Erk-1 activation was responsible for the inhibition of chondrogenesis. PD98059 treatment, however, did not recover chondrogenesis completely when the cells were treated with high concentrations of PMA or Go6976, i.e. 10 nM and 0.5 µM, respectively (Fig. 4B). As expected, more Erk-1 was phosphorylated at high concentrations of PKC modulators, and consequently PD98059 was less effective in blocking Erk-1 phosphorylation (Fig. 4C). The ability of PD98059 to recover chondrogenesis was proportional to the degree of inhibition of Erk-1 phosphorylation. Taken together, our results suggest that regulation of chondrogenesis by PKC is mediated through Erk-1.


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Fig. 4.   Inhibition of Erk-1 relieves blocking of chondrogenesis induced by the inhibition or down-regulation of PKC. Panel A, cells were cultured in the presence of 5 nM PMA (a), 5 nM PMA and 10 µM PD98059 (b), 0.25 µM Go6976 (c), 0.25 µM Go6976 and 10 µM PD98059 (d) for 4 days. Chondrogenic differentiation was determined by staining type II collagen. The data represent results of a typical experiment conducted four times. Panel B, mesenchymes were micromass cultured in the presence of 10 µM PD98059 and various concentrations of PMA or Go6976 for 4 days, and chondrogenesis was quantitated by reading an absorbance of Alcian blue extract. The data represent the relative value of absorbance of Alcian blue extract against control culture (i.e. cells cultured in the absence of PMA, Go6976, and PD98059) and average values of four independent experiments with standard deviation. Panel C, mesenchymes were micromass cultured in the presence of 10 µM PD98059 and various concentrations of PMA or Go6976 for the indicated time periods. Phosphorylation of Erk-1 was determined by Western blotting. The data represent the results of a typical experiment conducted four times with similar results.

Stimulatory Effect of Erk-1 Inhibition on Chondrogenesis Is Not the Result of Modulation of PKC-- In addition to the inhibition of Erk-1, a possibility of PD98059 modulation of expression and activity of PKC isoforms in chondrogenesis was examined. We have shown previously (15) that mesenchymes derived from chick limb buds contained multiple PKC isoforms such as alpha , gamma , epsilon , zeta , and lambda /iota . However, anti-PKCgamma antibody (Transduction Laboratories Inc.) used in previous study cross-reacted with PKCalpha (according to the manufacturer). When PKCgamma -specific antibodies obtained from Santa Cruz Biotechnology or Life Technologies, Inc. were used in this study, no immunoreactive band was detected in mesenchymes cultured up to 5 days (data not shown), indicating that PKCgamma was expressed at an insignificant level in mesenchymes during chondrogenesis. In addition, PKCeta and µ, which were not examined in the previous study (15), were also expressed at insignificant levels, with the corresponding antibodies failing to detect these proteins (data not shown). We therefore examined the effect of Erk-1 inhibition on the expression and subcellular distribution of PKC isoforms alpha , epsilon , zeta , and lambda /iota . Although treatment of mesenchymes with PD98059 enhanced chondrogenesis up to 2-fold (Fig. 3), the amounts and expression patterns of PKC isoforms were essentially the same among the cells cultured in the presence or absence of PD98059 (Fig. 5A). In addition, translocation of cytosolic PKC isoforms to the particulate membrane fractions was not affected by the inhibition of Erk-1 (Fig. 5B). Thus, it is highly unlikely that the inhibition of Erk-1 enhances chondrogenesis by modulating PKC. Taken together, these results strongly suggest that Erk-1 is a downstream target of PKC and that the action of PKC on chondrogenesis is exerted through the modulation of Erk-1.


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Fig. 5.   Inhibition of Erk-1 does not affect expression and subcellular distribution of PKC isoforms. Panel A, mesenchymes were cultured up to 4 days in the absence or presence of 10 µM PD98059, and the expression of PKC isoforms was detected by Western blotting. Panel B, cells cultured in the absence or presence of 10 µM PD98059 for 4 days were fractionated into cytosolic (c) and particulate membrane (m) fractions. The distribution of PKC isoforms was detected by Western blotting. The data represent a typical experiment conducted four times (A and B) with comparable results.

Effects of PKC and MAP Kinase on the Proliferation of Chondrogenic Competent Cells-- Because chondrogenesis of mesenchymes is cell density-dependent and requires transient proliferation of chondrogenic competent cells (1, 2), a question arises as to whether PKC and Erk-1 are involved in chondrogenesis at the stage of cell proliferation. As shown in Figs. 6, A and B, compared with the control culture, treatment of cells with 10 nM PMA or 0.5 µM Go6976 for 4 days reduced cell proliferation by 43 and 51%, respectively; however, neither affected cell viability throughout the culture periods, as determined by trypan blue exclusion experiments: more than 97% of the cells was viable in all treatments (data not shown). Thus, the reduction in cell number by PKC modulation was caused by reduced cell proliferation. In contrast to the inhibitory effect of PKC modulation on cell proliferation, the inhibition of Erk-1 with PD98059, a condition that enhanced chondrogenesis (Fig. 3), did not affect proliferation of the cells (Fig. 6A). This indicates that the stimulatory effect of Erk-1 inhibition on chondrogenesis is not caused by enhanced cell proliferation. Furthermore, the inhibition of PMA- or Go6976-induced cell proliferation was not relieved by the inhibition of Erk-1 with PD98059 (Fig. 6B), although chondrogenesis was recovered by the inhibitor (Fig. 4).


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Fig. 6.   PKC but not Erk-1 regulates cell proliferation. Panel A, the number of cells was counted after micromass culture of mesenchymes for the indicated time periods in the presence of vehicle alone as a control, 10 nM PMA, 0.5 µM Go6976, or 10 µM PD98059. The data represent the percent increase in cell number against day 1 culture. Panel B, cells were cultured for 4 days in the presence of the indicated concentrations of PMA or Go6976 and in the presence or absence of 10 µM PD98059, and the number of cells was counted. The data represent the relative number of cells against control culture, i.e. cells cultured in the absence of PMA, Go6976, and PD98059. The data represent the average values of five independent experiments with standard deviation (A and B).

To confirm further that the stimulatory effect of PD98059 was independent of cell proliferation, we explored a question of whether PD98059 enhanced chondrogenesis at a low density of cells. When micromass culture was performed at reduced cell density of 2 × 107 cells/ml to 1, 0.5, or 0.25 × 107 cells/ml, chondrogenesis was reduced to 41, 13, and 8% of the control, as shown in Fig. 7, A and B. This confirms the fact that chondrogenesis in culture is cell density-dependent. PD98059 treatment of the cells at a density of 0.25 × 107 cells/ml did not induce chondrogenesis; a slight but significant increase at 0.5 × 107 cells/ml, however, enhanced chondrogenesis dramatically at a density of 1 × 107 cells/ml (1.8-fold increase against control). This suggested that there was a certain threshold density of cells to undergo differentiation even in the presence of PD98059. In addition to the modulation of chondrogenesis, micromass culture of mesenchymes at a reduced cell density (1 or 0.5 × 107 cells/ml) resulted in enhancement of Erk-1 phosphorylation and less increase of PKCalpha and epsilon  expression compared with those in cells cultured at a density of 2 × 107 cells/ml (data not shown). It also appeared that the stimulatory effect of PD98059 on chondrogenesis at low cell density (i.e. 1 × 107 cells/ml) was independent of cell proliferation because PD98059 did not affect cell numbers at a density of 1 × 107 cells/ml as well as 2 × 107 cells/ml (Fig. 7C). These results clearly indicated that the inhibition of Erk-1 with PD98059 stimulated chondrogenesis without mediation of cell proliferation. The results also led us to conclude that the recovery of chondrogenesis by PD98059 in the cells treated with PMA or Go6976 was independent of proliferation of chondrogenic competent cells.


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Fig. 7.   Inhibition of Erk-1 enhances chondrogenesis at low cell density. Panel A, expression of type II collagen was determined by immunocytochemistry in mesenchymes micromass cultured at density of 2 × 107 cells/ml (a) or 1 × 107 cells/ml in the absence (b) or presence (c) of 10 µM PD98059 for 4 days. Panel B, mesenchymes were micromass cultured at the indicated density of cells in the absence (-) or presence (+) of 10 µM PD98059 for 4 days. Chondrogenesis was quantitated by reading an absorbance of Alcian blue extract. (panel C) The number of cells was counted in cells cultured at the indicated density for 4 days in the absence or presence of 10 µM PD98059. The data in panel A represent the results of a typical experiment; the data in panels B and C represent the average of four independent experiments with standard deviation.

Effects of PKC and MAP Kinase on the Expression of Cell Adhesion Molecules-- The role of PKC and Erk-1 in the expression of cell adhesion molecules and ECM components was investigated in an attempt to understand their roles in precartilage condensation. Expression of N-cadherin, which mediates cell-cell interaction (28), was high in 1-day culture and was reduced as chondrogenesis proceeded (Fig. 8). However, down-regulation of PKC with PMA or inhibition with Go6976 caused sustained expression of N-cadherin throughout the culture period. In contrast, the inhibition of Erk-1 accelerated the decrease of N-cadherin expression (Fig. 8). This indicated that activities of PKC and Erk-1 had opposing effects on N-cadherin expression.


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Fig. 8.   Regulation of cell adhesion molecule expression by PKC and Erk-1 during chondrogenesis. Mesenchymes were cultured in the presence of vehicle alone as a control, 10 nM PMA, 0.5 µM Go6976, and/or 10 µM PD98059 for the indicated time periods. The expression of N-cadherin, integrins alpha 5 and beta 1, and fibronectin was determined by Western blotting. The data represent a typical experiment conducted more than six times with similar results.

Because the interaction of cells with ECM in addition to cell-cell interaction had been known to regulate chondrogenesis at the stage of precartilage condensation (1, 2), a possibility of PKC and Erk-1 regulation of the expressions of integrin and its ligand during chondrogenesis was examined, particularly integrin alpha 5beta 1 and its ligand fibronectin. Expressions of integrins alpha 5 and beta 1 increased transiently during chondrogenesis; their expressions were detected in the cells cultured for 1 day, increased as chondrogenesis proceeded, and subsequently decreased at the later stage of chondrogenic differentiation, i.e. the 5th day of culture (Fig. 8). Down-regulation or inhibition of PKC caused a continuous increase of integrins alpha 5 and beta 1 expression throughout the culture period. In contrast, the inhibition of Erk-1 accelerated the decrease of integrins alpha 5 and beta 1 expression in the 4th and 5th day cultures. Similar to the above, the expression of fibronectin also increased transiently during chondrogenesis (Fig. 8), increased continuously throughout the culture period when PKC was down-regulated or inhibited, and finally Erk-1 inhibition promoted the decrease of fibronectin expression at the later period of micromass culture (Fig. 8). Therefore, the activity of PKC appeared to be required for the decrease of expression of integrins alpha 5 and beta 1 and fibronectin at the later period of micromass culture, whereas Erk-1 activation resulting from PKC inhibition or depletion increased the expression of these molecules. Because the reduction of integrin alpha 5beta 1 and its ligand fibronectin expression after cell condensation is necessary for chondrogenesis to progress (10, 29), regulation of the expression of these molecules by PKC and Erk-1 appears to be well correlated with the regulation of chondrogenesis.

Finally, we investigated the question of whether the modulation of expression of cell adhesion molecules by PKC was mediated by MAP kinase signaling. As shown in Fig. 8, the sustained expression of N-cadherin and increased expression of integrin alpha 5, beta 1, and fibronectin by the inhibition or down-regulation of PKC were abolished when Erk-1 activation was blocked with PD98059. The ability of PD98059 to block PKC-mediated modulation of these expressions was dependent on the concentration of PMA or Go6976 (data not shown). Thus, the degree of recovery of chondrogenesis by treating cells with PD98059 appeared to be proportional to the degree of PD98059 to block PKC. Taken together, these results indicate that the regulatory activity of PKC in the expression of cell adhesion molecules and ECM component is mediated by Erk-1 signaling.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Several lines of evidence support the hypothesis that chondrogenic differentiation of mesenchymes is regulated by PKC (14-17). PKC was initially thought to regulate chondrogenesis in a negative way because staurosporine, a potent inhibitor of PKC, enhanced chondrogenesis (30). However, it was found later that the stimulatory effect of staurosporine was caused by effects other than its ability to inhibit PKC (31). PKC has been implicated in a variety of cellular processes including growth and differentiation of many types of cells (18). 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 (18-20). It is well known that direct stimulation of PKC with phorbol ester leads to an activation of MAP kinase (32, 33) which is a common intermediate in intracellular signaling cascades (24-26).

Studies on PKC regulation of MAP kinase indicated that stimulation of PKC activated MAP kinase signaling (32, 33). 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 (32, 33), 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 (24-26).

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 (34), and maturation of immature T cells also requires MAP kinase activity because expression of catalytically inactive MAP kinase kinase inhibits T cell maturation (35). In contrast to the above, it has been suggested that inactivation of Erk-2 is required for C2C12 myoblasts to initiate myogenesis (36). Inactivation of MAP kinase is also observed during enterocyte differentiation (37). 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 (11-13). 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 (1, 2, 29). 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 alpha 5beta 1, and fibronectin was investigated.

N-Cadherin has been known to be expressed in prechondrogenic mesenchymes and during cell condensation (4, 5), and blockage of its function inhibited precartilage condensation (6). However, it was not expressed in chondrocytes that were completely surrounded by cartilage-specific ECM molecules (4). 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 alpha 5beta 1 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 alpha 5beta 1 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 alpha 5beta 1 is necessary for cell condensation to occur and that a reduction of fibronectin and its integrin alpha 5beta 1 receptor after cell aggregation is necessary for the progression of cartilage differentiation (10, 29, 38). Indeed, it has been known that enhanced expression of fibronectin exerts negative effects on chondrogenesis (39-41). We demonstrated in this study that PKC activity that inactivates Erk-1 is required to reduce the expression of integrin alpha 5beta 1 and fibronectin and thereby to initiate the progression of chondrogenic differentiation. Because the reduction of fibronectin and integrin alpha 5beta 1 occurs after the cell aggregation (29), 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 alpha 5beta 1 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.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Woon Ki Paik for critical reading of the manuscript and Mahn Joon Ha (Ajou University) for helpful discussions.

    FOOTNOTES

* This work was supported by grants from the Korea Ministry of Education (BSRI-97-4402), Korea Science and Engineering Foundation (971-0503-014-2), and Hallym Academy of Science, Hallym University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Dept. of Biology, College of Natural Sciences, Kyungpook National University, Pookgu, Taegu 702-701, Korea. Tel.: 82-53-950-5353; Fax: 82-53-953-3066; E-mail: jschun{at}bh.kyungpook.ac.kr.

1 The abbreviations used are: ECM, extracellular matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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