p38 Kinase-dependent and -independent Inhibition of Protein Kinase C ζ and -α Regulates Nitric Oxide-induced Apoptosis and Dedifferentiation of Articular Chondrocytes*

In articular chondrocytes, nitric oxide (NO) production triggers dedifferentiation and apoptotic cell death that is regulated by the converse functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase (ERK) and p38 kinase. Since protein kinase C (PKC) transduces signals that influence differentiation, survival, and apoptosis of various cell types, we investigated the roles and underlying molecular mechanisms of action of PKC isoforms in NO-induced dedifferentiation and apoptosis of articular chondrocytes. We report here that among the expressed isoforms, activities of PKCα and -ζ were reduced during NO-induced dedifferentiation and apoptosis. Inhibition of PKCα activity was independent of NO-induced activation of ERK or p38 kinase and occurred due to blockage of expression. On the other hand, PKCζ activity was inhibited as a result of NO-induced p38 kinase activation and was observed prior to proteolytic cleavage by a caspase-mediated process to generate enzymatically inactive fragments. Inhibition of PKCα or -ζ activities potentiated NO-induced apoptosis, whereas ectopic expression of these isoforms significantly reduced the number of apoptotic cells and blocked dedifferentiation. Ectopic expression of PKCα or -ζ did not affect p38 kinase or ERK but inhibited the p53 accumulation and caspase-3 activation that are required for NO-induced apoptosis of chondrocytes. Therefore, our results collectively indicate that p38 kinase-independent and -dependent inhibition of PKCα and -ζ, respectively, regulates NO-induced apoptosis and dedifferentiation of articular chondrocytes.

In articular chondrocytes, nitric oxide (NO) production triggers dedifferentiation and apoptotic cell death that is regulated by the converse functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase (ERK) and p38 kinase. Since protein kinase C (PKC) transduces signals that influence differentiation, survival, and apoptosis of various cell types, we investigated the roles and underlying molecular mechanisms of action of PKC isoforms in NO-induced dedifferentiation and apoptosis of articular chondrocytes. We report here that among the expressed isoforms, activities of PKC␣ and -were reduced during NO-induced dedifferentiation and apoptosis. Inhibition of PKC␣ activity was independent of NO-induced activation of ERK or p38 kinase and occurred due to blockage of expression. On the other hand, PKC activity was inhibited as a result of NO-induced p38 kinase activation and was observed prior to proteolytic cleavage by a caspase-mediated process to generate enzymatically inactive fragments. Inhibition of PKC␣ or -activities potentiated NO-induced apoptosis, whereas ectopic expression of these isoforms significantly reduced the number of apoptotic cells and blocked dedifferentiation. Ectopic expression of PKC␣ or -did not affect p38 kinase or ERK but inhibited the p53 accumulation and caspase-3 activation that are required for NO-induced apoptosis of chondrocytes. Therefore, our results collectively indicate that p38 kinase-independent and -dependent inhibition of PKC␣ and -, respectively, regulates NO-induced apoptosis and dedifferentiation of articular chondrocytes.
Proinflammatory cytokines, such as interleukin-1␤ and tumor necrosis factor-␣, play significant roles in structural and biochemical alterations in chondrocytes and cartilage during arthritic disease (1)(2)(3)(4). One of the leading mechanisms by which cytokines elicit their effects on cartilage involves the stimulation of nitric oxide (NO) 1 production via inducible NO synthase (4,5). Although NO-induced cartilage destruction is caused in various ways, increased apoptotic cell death (6 -8) and loss of differentiated phenotype of articular chondrocytes (9,10) appear to be important contributors.
Direct production of NO by treatment with the NO donor, sodium nitroprusside (SNP), during primary culture of rabbit articular chondrocytes causes dedifferentiation and apoptotic cell death (11). The effects of NO on differentiation and apoptosis of chondrocytes are regulated by the converse functions of two mitogen-activated protein kinase subtypes, specifically, extracellular signal-regulated kinase (ERK) and p38 kinase, in association with the elevation of p53 protein levels, caspase-3 activation, and differentiation status. SNP treatment stimulated the activation of both ERK-1/-2 and p38 kinase. Activated ERK-1/-2 induces dedifferentiation and inhibits NO-induced apoptosis, whereas p38 mitogen-activated protein kinase plays a role in the maintenance of the differentiated status and induction of chondrocyte apoptosis. In addition to NO-induced apoptosis and dedifferentiation, ERK-1/-2 regulates chondrogenesis and the maintenance of differentiated phenotypes of articular chondrocytes. For example, down-regulation of ERK-1/-2 activity during chondrogenesis of mesenchymal cells enhanced differentiation (12,13). In contrast, ERK-1/-2 activity in differentiated articular chondrocytes was dramatically increased during dedifferentiation caused by a serial monolayer culture. Moreover, inhibition of ERK-1/-2 activity subsequently blocked dedifferentiation (14). ERK-1/-2 activity in chondrogenesis is regulated by protein kinase C (PKC) ␣, whereas ERK-1/-2 and PKC␣ independently regulate dedifferentiation due to serial subcultures. PKC␣ expression was down-regulated during subculturing of chondrocytes, which in turn resulted in cellular dedifferentiation.
PKC comprises a family of serine/threonine kinases with 11 isoforms (15). Individual PKC isoforms exhibit a high degree of homology in the catalytic region but vary with regard to tissue distribution and activation requirements. In addition to regulating cell differentiation, PKC proteins play an important role in the modulation of apoptosis by acting as either pro-or antiapoptotic signals. Whether PKC induces or protects against apoptosis varies with the cell type, extracellular stimuli, and the specific isoforms that are activated or inhibited. Among the PKC isoforms, PKC␦ is consistently reported as proapoptotic (16,17). However, other isoforms (such as PKC␣) function as both antiapoptotic (18 -22) and proapoptotic signals (23), depending on the experimental system. The specific role of PKC␣ in apoptosis is dependent on the modulation of both activity (22,23) and expression levels of the protein (19 -21). Some PKC isoforms, including ␦ (16, 24), ⑀, (25), (26), and (27)(28)(29)(30), additionally undergo proteolytic cleavage by caspases during apoptosis. This cleavage leads to the production of an enzymatically active catalytic domain in PKC ␦ (16,24), whereas that of the isoform generates an inactive catalytic domain (28,29). Consistent with this observation, a number of studies show that inhibition of PKC activity required for the apoptosis of various cell types precedes its cleavage in various experimental conditions (28,29,31,32).
Because a role of PKC in the regulation of NO-induced apoptosis has not been clearly elucidated yet, we addressed in this study the potential involvement of PKC isoforms in the regulation of NO-induced dedifferentiation and apoptosis of rabbit articular chondrocytes by using SNP as a NO donor. We additionally investigate the specific mechanisms of regulation of NO-induced apoptosis by these PKC isoforms. We report here that p38 kinase-independent and -dependent inhibition of PKC␣ and -, respectively, is required for both NO-induced dedifferentiation and accumulation of proapoptotic p53 that in turn induces apoptosis of chondrocytes.
Determination of Caspase-3 Activity-Caspase-3 activity was determined by measuring the absorbance at 405 nm following cleavage of the synthetic substrate, Ac-Asp-Glu-Val-Asp-chromophore p-nitroaniline (Ac-DEVD-pNA). Briefly, chondrocytes were lysed on ice for 10 min in cell lysis buffer provided in the CLONTECH A ApoAlert TM CPP32 colorimetric assay kit. Lysates were reacted with 50 M Ac-DEVD-pNA in reaction buffer (0.1 M Hepes, 20% glycerol, 10 mM dithiothreitol, and protease inhibitors including 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, pH 7.4). Mixtures were maintained at 37°C for 1 h and subsequently analyzed in an enzyme-linked immunosorbent assay reader. Enzyme activity was calculated from a standard curve prepared using p-nitroaniline. Levels of relative p-nitroaniline were normalized against the protein concentration of each extract.
Quantitation of Apoptosis-Apoptotic cell death was quantified by a flow cytometric assay based on the number of cells containing fragmented DNA, as previously described (11). Briefly, cells were harvested by centrifugation and fixed in 80% ethanol precooled to Ϫ20°C. Cells were resuspended in phosphate-buffered saline containing 50 g/ml propidium iodide, 0.1% Nonidet P-40, and 100 g/ml RNase A (Sigma) and incubated for 1 h. The number of cells containing fragmented DNA was quantified using 1 ϫ 10 4 cells on a FACSort flow cytometer.
Transfection-Chondrocytes were transfected with empty vector or expression vector containing wild-type PKC␣, PKC, or the catalytic domain of PKC (38), a dominant negative form of ERK-2 or p38 kinase (39), or wild-type or dominant negative p53 (p53 273 ). Transfection of the expression vector was performed as described previously (11). The expression vector was introduced to cells using LipofectAMINE PLUS (Invitrogen) using the procedure recommended by the manufacturer. The transfected cells were cultured in complete medium for 24 h and used for further assay as indicated in each experiment.
Northern Blot Analysis-Total RNA isolated by a single-step guanidinium thiocyanate-phenol chloroform method using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) was denatured and fractionated on formaldehyde/agarose gels (15 g/lane). RNA was then transferred to Schleicher & Schuell Nytran N nylon membranes. Prehybridization and hybridization were performed in 250 mM Na 2 HPO 4 (pH 7.2), 7% SDS, 1 mM EDTA, 250 mM NaCl, 5% dextran sulfate, 50% formamide, and 100 g/ml denatured single strand DNA for 3 and 12 h, respectively. PKC␣ transcript was probed with partial cDNA generated by reverse tran- scriptase PCR. The forward PCR primer for PKC␣ was 5Ј-TCAAC-CCCGAGTGGAACGAGACA-3Ј, and the reverse primer was 5Ј-GGAAATAGTGTTGGTCGTCTTTT-3Ј. High specific activity randomprimed probes were prepared from the PCR product (324 bp) using the T7 QuickPrime kit (Amersham Biosciences) as specified by the supplier. Filters were washed three times with 0.2ϫ SSC/0.1% SDS and exposed to Kodak X-OMAT film with intensifying screens at Ϫ80°C.
Western Blot Analysis-Whole cell lysates were prepared as described previously (11). Briefly, proteins from chondrocytes were extracted with buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with inhibitors for proteases and phosphatases. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The nitrocellulose sheet was blocked with 3% non-fat dry milk in Tris-buffered saline, and proteins were detected using the following antibodies: rabbit anti-p53 polyclonal antibody and active caspase-3 (New England Biolabs, Beverly, MA), PKC and ERK-1/-2 (BD Transduction Laboratories), p38 kinase (Santa Cruz Biotechnology Inc.), and phosphorylation-specific antibody for ERK (Cell Signaling Technology, Beverly, MA). Blots were developed using a peroxidase-conjugated secondary antibody and an ECL system.

NO Generation Leads to Reduced PKC␣ and -Protein Levels in Articular
Chondrocytes-Western blotting analyses revealed that rabbit joint articular chondrocytes express ␣, ⑀, , and / isoforms of PKC (14). We initially determined the effects of SNP (1 mM) on the expression pattern of PKC isoforms in chondrocytes. Among the expressed PKC isoforms, levels of ␣ and decreased in a time-and dose-dependent manner, whereas ⑀ and / expression patterns remain unchanged at all the time points and SNP concentrations measured (Fig. 1, A  and B). The reduction in PKC␣ protein levels appears to be due to a dose-dependent decrease in transcription upon SNP treatment (Fig. 1C). In contrast, the decrease in PKC protein levels was accompanied by a concomitant increase in an ϳ46-kDa fragment (Fig. 1D). Since the epitope of the anti-PKC antibody employed was localized at the C-terminal region, the detected protein is similar to the reported PKC fragment generated by the action of caspase in other cell types during apoptosis (28 -30).
Reduction of PKC␣ and -Expression Is Required for NOinduced Dedifferentiation of Articular Chondrocytes-A recent study by our group demonstrated that SNP treatment of articular chondrocytes causes both dedifferentiation and apoptotic cell death (11). Therefore, we first examined whether the observed decrease in PKC␣ expression and degradation of PKC affects phenotypic loss of differentiated chondrocytes by inducing ectopic expression of wild-type PKC␣ and -. Consistent with our previous data (11), SNP treatment led to a reduction in sulfated proteoglycan accumulation and expression of type II collagen, as determined by Alcian blue staining and Western blot analysis, respectively (Fig. 2), thus signifying dedifferentiation of chondrocytes. Ectopic expression of PKC␣ or -significantly blocked dedifferentiation of SNP-treated cells, as determined by the rescue of type II collagen expression (Fig.  2B) and accumulation of sulfated proteoglycan ( Fig. 2A). Therefore, the above results indicate that reduction of PKC␣ andparticipates in the regulation of SNP-induced dedifferentiation of chondrocytes.
Reduction of PKC␣ and -Expression Is Required for NOinduced Apoptosis-Treatment of chondrocytes with SNP (1 mM) for 24 h causes apoptotic cell death in a dose-and timedependent manner, which is detectable as early as 6 h (11). Therefore, the possible involvement of PKC␣ and -in SNPinduced apoptosis was determined by inhibiting the activity or ectopic expression of these PKC isoforms. Treatment of chon- drocytes with indicated concentrations of PKC inhibitors (GF109203X (40) or Go6976 (41)) enhanced SNP-induced apoptotic death of chondrocytes. Stimulation of apoptosis was evident at 1 M, and more cells underwent apoptosis at higher concentrations (Fig. 3A). In contrast, ectopic expression of PKC␣ or -significantly blocked SNP-induced apoptotic death of chondrocytes (Fig. 3, B and C). Quantitation of apoptotic cells using a FACSort flow cytometer with propidium iodide staining disclosed that the percentage of apoptotic cells (23 Ϯ 2%) was reduced to 7 Ϯ 2% and 9 Ϯ 2% upon ectopic expression of PKC␣ and -, respectively (Fig. 3C), indicating that both isoforms are required for SNP-induced apoptosis.

Inhibition of Expression and Activity PKC␣ Is Independent of SNP-induced Activation of ERK and p38
Kinase-A decrease in PKC␣ activity was noted during SNP-induced apoptosis (Fig.  4A, upper panel), consistent with reduced mRNA and protein levels (Fig. 4A, lower panel). To elucidate the signaling pathways leading to decreased PKC␣ expression and activity, chondrocytes were treated (prior to the addition of SNP) with inhibitors for signaling molecules involved in apoptosis and dedifferentiation such as ERK-1/-2, p38 kinase, and caspase-3. As shown in Fig. 4B, inhibition of ERK-1/-2 with PD98059, p38 kinase with SB203580, or caspase-3 with z-DEVD (data not shown) did not affect SNP-induced reduction of PKC␣ expression or activity. Expression of dominant negative forms of ERK-2 or p38 kinase also had no effects on the expression and activity of PKC␣ (Fig. 4B). However, inhibition of ERK potentiated SNP-induced apoptosis, whereas inhibition of p38 kinase or caspase-3 blocked apoptotic cell death (Fig. 4C), which is consistent with our previous results (11). The above results thus indicate that inhibition of PKC␣ activity is a result of decreased expression and is independent of known signaling molecules involved in apoptosis, including ERK-1/-2, p38 kinase, and caspase-3.
Activation of p38 Kinase Inhibits PKC Activity Prior to Its Cleavage-In the next series of experiments, the functional relationship between degradation and enzymatic activity of PKC was determined. In cells treated with SNP for various time periods, PKC activity decreased in a time-dependent manner, consistent with the pattern of degradation (Fig. 5A). This decrease in PKC protein level was blocked upon treatment of cells with z-DEVD (Fig. 5B), a relatively specific inhibitor of caspase-3 (33), suggesting that the protein is cleaved by a process involving caspase-3. This result was further supported by the observation that inhibition of p38 kinase with SB203580 (conditions that inhibit SNP-induced caspase-3 activation) blocked PKC cleavage, whereas that of ERK-1/-2 with PD98059 (potentiating caspase-3 activation) enhanced PKC cleavage (Fig. 5C) (11).
Cleavage of PKC by caspases produces either an active or inactive catalytic domain, depending on the experimental system (28 -30). Since the antibody used for PKC recognizes the C-terminal region, the generated fragment that contains the catalytic domain is enzymatically inactive. The decrease of PKC activity is evident 12 h after SNP treatment and preceded its cleavage by caspase-3 (Fig. 5B). This was further supported by the observation that blockage of caspase-3 with z-DEVD significantly decreased cleavage of PKC but did not rescue PKC activity (Fig. 5B). Inhibition of PKC activity was completely abrogated when SNP-induced p38 kinase activation was blocked with SB203580. However, blocking ERK activity with PD98059 did not affect SNP-induced inhibition of PKC activity (Fig. 5C), indicating that PKC activity is regulated by p38 kinase. The functional significance of PKC activity in SNP-induced apoptosis was determined by inhibition or activation of the protein. Inhibition of PKC activity by treatment of cells with a specific inhibitory peptide (36) (Fig. 6A, upper  panel) potentiated SNP-induced apoptosis (Fig. 6B), whereas increase in PKC activity by ectopic expression of the constitu-FIG. 5. Inhibition of PKC activity precedes cleavage and is required for SNP-induced apoptosis. Chondrocytes were treated with 1 mM SNP for the indicated time periods (A). Intact PKC was detected by Western blot analysis. PKC activity was determined by immune complex kinase assay. Cells were left untreated (Ϫ) or treated (ϩ) with 20 M z-DEVD for 30 min and exposed to 1 mM SNP for the indicated time periods (B). Intact PKC size and activity were determined by Western blotting and immune complex kinase assay, respectively. Cells were left untreated (Control) or treated with 1 mM SNP for 24 h that was pretreated for 30 min with 20 M PD98059 (PD) or 20 M SB203580 (SB) followed by determination of intact PKC size and activity (C). Relative amounts of intact PKC protein and activity were quantified from four independent experiments (lower panels). tively active catalytic domain of this protein (Fig. 6A, lower panel) reduced apoptotic cell death (Fig. 6C). Therefore, the above results collectively indicate that inhibition of PKC activity, which precedes its cleavage, is required for SNP-induced apoptosis of chondrocytes.
Inhibition of PKC␣ and -Is Required for Accumulation of p53 and Activation of Caspase-3-Our recent study demonstrated that SNP treatment of articular chondrocytes causes apoptotic cell death by the converse functions of ERK and p38 kinase in association with accumulation of p53 and activation of caspase-3 (11). To elucidate the mechanisms of the antiapoptotic function of PKC␣ and -, we examined the effects of ectopic expression of the PKC isoforms on the activation of signaling pathway involved in SNP-induced apoptosis. As shown in Fig. 7A, ectopic expression of PKC␣ and -did not affect activation of either p38 kinase or ERK-1/-2. However, increased PKC␣ and -expression blocked accumulation of p53 and activation of caspase-3 (Fig. 7, A and B).
Because the above results indicate that inhibition of both PKC␣ and -is necessary for the accumulation of p53, we next determined whether the increase in p53 protein level is directly involved in NO-induced apoptosis. Consistent with our previous observation (11), the increased apoptosis by the inhibition of ERK-1/-2 (Fig. 4C) accompanied increased accumulation of p53 (Fig. 8A), whereas the decreased apoptosis by the inhibition of p38 kinase (Fig. 4C) accompanied decreased accumulation of p53 (Fig. 8A). In addition, ectopic expression of wild-type p53 results in a significant increase in caspase-3 activity and apoptosis (Fig. 8, B and C), whereas forced expression of the dominant negative mutant form of p53 (p53 273 ) blocked SNPinduced caspase-3 activation and apoptosis of chondrocytes (Fig. 8, B and C), indicating an essential role for p53 in chondrocyte apoptosis. Taken together, the above results indicated that inhibition of PKC␣ and -is required for the accumulation of p53 that is essential for NO-induced apoptosis of articular chondrocytes. DISCUSSION We demonstrate in this study that a decrease in PKC␣ andactivities is required for both SNP-induced dedifferentiation and apoptosis of rabbit knee joint articular chondrocytes. The PKC protein family comprises 11 isoforms that are well conserved across species. Although individual isoforms display a high degree of homology in the catalytic regions, they vary with regard to tissue distribution and cofactor requirement for activation, suggesting specific functions (15). The findings that PKC␣ is down-regulated during SNP-induced dedifferentiation and that ectopic expression blocks this process are consistent with the expected role of this isoform in the regulation of chondrocyte differentiation. For example, PKC␣ expression increases during chondrogenesis of mesenchymal cells induced by micromass culture. Conversely, inhibition or down-regulation of this protein abrogates chondrogenesis (12,13). On the other hand, PKC␣ expression is decreased during serial subculturing of articular chondrocytes, and this down-regulation of PKC␣ appears sufficient to induce dedifferentiation (14). Therefore, PKC␣ functions as a critical signaling molecule during differentiation and in the maintenance of the differentiated phenotype of chondrocyte. In addition to PKC␣, we also demonstrated in this study that expression and activity of PKC plays an important role in the maintenance of the differentiated phenotype of articular chondrocytes. Although dedifferentiation of chondrocytes caused by serial subculture does not accompany any modulation of PKC expression and activity (14), this study clearly demonstrated that inhibition of PKC activity is necessary for SNP-induced dedifferentiation chondrocytes.
Our data also demonstrate for the first time that activities of PKC␣ and -activity are inhibited via different mechanisms and that inhibition of both PKC␣ and -activity is required in NO-induced apoptosis of chondrocytes. Although we cannot rule out the possibility of decreased mRNA stability, reduction in PKC␣ activity appears to be a result of decreased mRNA FIG. 7. Ectopic expression of PKC␣ or -does not inhibit ERK and p38 kinase but inhibits the region downstream of p38 kinase. Chondrocytes were transfected with empty vector or wild-type PKC␣ or -. Transfected cells were cultured in complete medium for 24 h and treated with 1 mM SNP for an additional 24 h. Phosphorylation of ERK-1/-2, p53 accumulation, and activation of caspase-3 were detected by Western blot analysis. p38 kinase activity was determined with a kinase assay using ATF-2 as a substrate (A). Caspase-3 activity was determined as described under "Experimental Procedures" (B). Data represent results of a typical experiment (A) and average values with standard deviation (B) (n ϭ 4).

FIG. 8. Proapoptotic function of p53 in SNP-treated articular chondrocytes.
Chondrocytes were transfected with empty vector or a dominant negative form of ERK-2 (⌬ERK) or p38 kinase (⌬p38), cultured in complete medium for 24 h, and treated with 1 mM SNP for an additional 24 h (A). Levels of p53 protein were determined by Western blot analysis. Cells were transfected with empty vector or vector containing wild-type p53 cDNA (p53) or a dominant negative form of p53 (p53m) (B and C). Following incubation of cells in complete medium for 24 h, the cells were untreated or treated with 1 mM SNP for 24 h. Levels of p53 and active caspase-3 were determined by Western blot analysis (B). Apoptotic cells and caspase-3 activity was determined as described under "Experimental Procedures" (C). The data represent a typical result or average values with standard deviation (n ϭ 4). transcription levels in SNP-treated cells (Fig. 4). In addition, no fragmentation of PKC␣ was detected during SNP-induced apoptosis (data not shown). In contrast to PKC␣, PKC activity was inhibited in a p38 kinase-dependent manner. The results are consistent with the reported antiapoptotic functions of PKC␣ and -in various cell types induced by different extracellular stimuli. For instance, a decrease in expression and/or activity of PKC␣ is associated with induction of apoptosis (18 -22), although the isoform also functions as a proapoptotic signal depending on the experimental system (23). The inhibition of PKC activity is also required for apoptosis that is caused by various extracellular stimuli (28,29,31,32). Consistent with earlier reports (28,29), our data show that inhibition of PKC activity occurs prior to cleavage via caspase-3 to produce an enzymatically inactive catalytic domain.
Although the mechanisms underlying regulation of PKC␣ expression remain to be elucidated, it is clear that expression is not regulated by ERK-1/-2 and p38 kinase that oppositely regulate apoptosis and dedifferentiation. The promoter region of mouse PKC␣ contains a TATA-like box (but no CAAT box), a silencer element, and retinoic acid response element (42). We are currently investigating the signaling pathway and regulatory mechanism of PKC␣ transcription in rabbit articular chondrocytes. In contrast to PKC␣, inhibition of p38 kinase activity completely abrogates SNP-induced inhibition of PKC activity, confirming that PKC activity is inhibited via p38 kinase activation (Fig. 5). We did not characterize the molecular mechanism of PKC inhibition in this study. However, a variety of mechanisms for the regulation of PKC activity have been reported. Since phosphorylation of PKC regulates processing of the protein that is required for activation (43), modulation of protein phosphatases controls PKC activity. For instance, PKC␣ activity is blocked by dephosphorylation via activation of protein phosphatases upon exposure of cells to ceramide (44) or vitamin E analog (24). The modulation of inhibitory proteins for PKC isoforms is another suggested mechanism for PKC inhibition. For example, Par-4 interacts with the regulatory domain of PKC through its leucine zipper domain and inhibits kinase activity (36). It is generally believed that the inhibition of PKC activity is the principal mechanism by which Par-4 induces apoptosis (28,45), although the protein may also modulate Bcl-2 expression and the transcription function of WT-1 (45,46).
The ability of PKC isoforms to protect cells from apoptosis may originate from regulation of cell survival signals, such as Akt (20), or transcription factors, such as NFB (27,47), Ap-1 (48), and Sp-1 (49), which control the expression of genes that mediate cell survival and apoptosis. Among the transcription factors, NFB protects cells from apoptosis in most cases. However, the factor also displays proapoptotic function, depending on the cell type and extracellular stimuli (50). NFB (which exists in a latent form in the cytoplasm bound to an inhibitory protein IB) is activated via phosphorylation and subsequent degradation of IB, following which the released protein translocates to the nucleus to activate target genes such as antiapoptotic Bcl-2 (51) and proapoptotic p53 (52). We recently found that SNP treatment activates NFB that acts as a proapoptotic signal in chondrocytes. 2 p38 kinase appears to activate NFB via the phosphorylation of IB by IB kinase and subsequent degradation of IB, as revealed by data showing that ectopic expression of dominant negative IB kinase blocks NFB activation. Our data also indicated that p38 kinase-dependent stimulation of NFB functions as a proapoptotic signal by stimulating p53 transcription. We observed in this study that ectopic expression of PKC␣ and -blocks IB degradation and NFB activation (data not shown), indicating that inhibition of PKC␣ and -in SNP-treated chondrocytes is required for NFB activation. Therefore, we speculate that NFB is activated through both p38 kinase-dependent inhibition of PKC and p38 kinase-independent inhibition of PKC␣ to increase levels of proapoptotic p53 and subsequent activation of caspases.