Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta.

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using Gö6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.

Matrix metalloproteinases (MMPs), 1 a family of endopeptidases, have the ability to degrade extracellular matrix pro-teins, and therefore, play a fundamental role in tissue remodeling (1)(2)(3)(4)(5)(6). MMPs are implicated in the remodeling processes of the heart during chronic heart failure and following myocardial infarction (6 -10). MMP-2 (gelatinase A) and MMP-9 (gelatinase B), also called type IV collagenases, contain fibronectin-like domains for collagen binding (11) and are known to cleave matrix substrates including gelatin and collagen Type I, IV, V, VII, and X (5). Cardiac fibroblasts in culture constitutively express MMP-2, not MMP-9. Inflammatory cytokines, such as interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣, increase expression of MMP-2 and -9 (12,13). Although both MMP-2 and -9 are activated in the heart during myocardial remodeling, the temporal changes in the expression of these enzymes are different (14). MMP-9 activity increases 1 day after MI, whereas MMP-2 activity starts to increase within 4 days after MI. Furthermore, MMP-9 abundance increases in both ischemic and non-ischemic dilated cardiomyopathy, whereas the abundance of MMP-2 increases only in non-ischemic dilated cardiomyopathy (9). These observations suggest differential regulation of MMP-2 and -9 in the heart.
Inflammatory cytokines such as IL-1␤ and tumor necrosis factor-␣ are increased during chronic heart failure (15) and following myocardial infarction (16,17). We have shown that IL-1␤ activates ERK1/2 and JNKs, not p38 kinase in adult rat cardiac fibroblasts (18). We have also provided evidence that IL-1␤ activates PKC, specifically PKC-, in cardiac fibroblasts and that activation of PKC plays a critical role in the regulation of MMP-2 and -9 (12). In addition to the activation of MAPKs and PKC, IL-1␤ has been shown to activate NF-B in several different cell types (19 -21). The present study was undertaken to investigate the signaling pathways, including MAPKs (ERK1/2 and JNKs), PKC (␣, ␤1,, , and ⑀), and NF-B, involved in the expression and activity of MMP-2 and -9 in response to IL-1␤ in adult rat cardiac fibroblasts. The data presented here suggest that different isoforms of PKC differentially activate ERK1/2, JNKs, and NF-B and that activation of ERK1/2, JNKs, and NF-B differentially modulates the expression and activity of MMP-2 and -9.
Rat Cardiac Fibroblast Culture and Treatment-Adult rat cardiac fibroblasts were isolated from the supernatant of adult rat cardiac myocytes as described (13). The cells were grown to confluence and serum-starved for 48 h before use. Cells from the first and second passages were used for all the experiments. The cells were treated with IL-1␤ (4 ng/ml) for 48 h to measure MMP activity or for 15 min to measure activation of MAPKs, IKK␣/␤, and IB-␣. To study the role of MAPKs, PKC isoforms, or NF-B, cells were pretreated with PD98059 (10 M), SP600125 (10 M), chelerythrine (5 M), Gö6976 (5 or 10 nM), PKC-⑀-TIP (5 M), PKC--PS inhibitor peptide (50 g/ml), PKC--PS (25 M), or SN50 (100 g) for 30 min followed by treatment with IL-1␤. Western Blot Analyses-Activation of MAPKs (ERK1/2 and JNKs), IKK␣/␤, and IB-␣ was studied using Western blot analyses as described (18). Cells lysates were prepared using lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, and 0.5% Nonidet P-40), and protein contents were measured using the Bradford assay (Bio-Rad). Equal amounts of protein (30 g) were subjected to SDS-PAGE, and proteins were transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were then probed with phospho-specific ERK1/2, JNKs, c-Jun, IKK␣/␤, or IB-␣ antibodies. The membranes were then incubated with appropriate secondary antibody and analyzed as described (18). The protein loading of each sample was verified by staining the membrane with 0.1% Ponceau S solution. Protein levels for MMP-2 and -9 were measured in the conditioned media using monoclonal anti-MMP-2 and anti-MMP-9 antibodies.
In-gel Zymography-MMP activity in conditioned media was measured as described (13). Briefly, the media were collected, centrifuged for 5 min at 500 ϫ g to remove cells and debris, and lyophilized to dryness. The pellet was resuspended in water, and protein content was determined by Bradford assay (Bio-Rad). Samples (500 ng of protein) were loaded under non-reducing conditions onto 4% stacking, 10% separating SDS-polyacrylamide gel polymerized with 1 mg/1 ml gelatin (type A from porcine skin, Sigma) as a substrate and electrophoresed at 15-20 mA. Following electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 min with gentle shaking followed by a 30-min wash in distilled water. The gels were incubated overnight at 37°C in substrate buffer (50 mM Tris-HCl, pH 8.0, 5 mM CaCl 2 , and 0.02% NaN 3 ), stained in Coomassie Blue (R-250), and then destained using 7% acetic acid and 40% methanol solution. Clear and digested regions representing MMP activity were quantified using a Kodak gel documentation system (Eastman Kodak Co.), and molecular weights were estimated using prestained molecular weight markers.
Statistical Analyses-The data are expressed as mean Ϯ S.E. Statistical analysis was performed using the Student's t test or a one-way analysis of variance followed by a post hoc Tukey's test. Probability (p) values of Ͻ0.05 were considered to be significant.
Previously, we have shown that PD98059 and SP600125 specifically inhibit IL-1␤-stimulated increases in ERK1/2 and JNKs activity, respectively. SP600125 from 2-20 M concentration had no effect on IL-1␤-stimulated activation of ERK1/2 (18). To explore the possibility of whether a combination of PD98059 and SP600125 could be effective in the inhibition of MMP-2 activity and protein expression, cells were pretreated FIG. 1. Inhibition of ERK1/2 inhibits MMP-9 activity. Confluent cultures of cardiac fibroblasts were pretreated with PD (10 M) for 30 min followed by stimulation with IL-1␤ (4 ng/ml) for 48 h. MMP activity and protein levels in conditioned media were measured using in-gel zymography and Western blot analysis, respectively. A is a representative zymogram demonstrating the changes in MMP activities. CTL, control. B and C represent MMP-9 and MMP-2 activities, respectively, expressed as -fold increase versus control (n ϭ 4); *, p Ͻ 0.001 versus control; #, p Ͻ 0.01 versus IL-1␤. D, Western blot analysis of conditioned media using monoclonal anti-MMP-2 and anti-MMP-9 antibodies. The experiments were repeated three times with similar results.
with PD98059 ϩ SP600125 (PD ϩ SP) followed by treatment with IL-1␤ for 48 h. In-gel zymography and Western blot analyses indicated that inhibition of both ERK1/2 and JNKs together significantly inhibits IL-1␤-stimulated MMP-9 activity and protein levels but has no significant effect on MMP-2 (Fig.  3, A and B).
Inhibition of PKC Inhibits IL-1␤-stimulated Activation of ERK1/2 and JNKs-Recently, we have shown that IL-1␤-stimulated activation of PKC plays an important role in the expression and activity of MMP-2 and -9 (12). Activated PKC may act upstream in the activation of ERK1/2 and JNKs (24). To explore this possibility, cells were pretreated with chelerythrine (5 M) for 30 min followed by stimulation with IL-1␤ for 15 min. Analysis of cell lysates using phospho-specific antibodies indicated that inhibition of PKC almost completely inhibits IL-1␤stimulated activation of ERK1/2 (Fig. 4A) and JNKs (Fig. 4B, as reflected by the phosphorylation of c-Jun).
Chelerythrine is a specific (but not isoform-selective) PKC inhibitor and represents a unique class of PKC inhibitors that competitively interfere with the phosphate acceptor site and non-competitively inhibits the ATP-binding site (25). To identify PKC isoforms involved in the activation of ERK1/2 and JNKs, we used isoform-specific inhibitors of PKC. Gö6976, a selective inhibitor for PKC-␣ and -␤1 (IC 50 ϭ 2.3 and 6.2 nM, respectively) at 5 and 10 nM, inhibited IL-1␤-stimulated in-creases in c-Jun phosphorylation but had no significant effect on ERK1/2 phosphorylation (Fig. 4, C and D). PKC-⑀ translocation inhibitor peptide (PKC-⑀-TIP) had no significant effect on IL-1␤-stimulated activation of ERK1/2 and JNKs (Fig. 4, C and D). PKC pseudosubstrates (PS) are specific inhibitory peptides and are known to suppress PKC activity by interacting with the substrate-binding pocket in the catalytic domain (26). Pretreatment with PKC--PS completely inhibited IL-1␤-stimulated activation of ERK1/2 and JNKs (as reflected by c-Jun phosphorylation), whereas PKC--PS exhibited a partial effect (Fig. 4, C and D).
PKC Isoforms Differentially Modulate Expression and Activity of MMP-2 and -9 -To determine the role of PKC isoforms on MMP-2 and -9 expression and activity, cells were pretreated with Gö6976 (5 and 10 Fig. 5A). Western blot analysis of conditioned media demon-strated that Gö6976 also reduces MMP-9, not MMP-2, protein levels (Fig. 5B). Pretreatment with PKC--PS and PKC--PS almost completely inhibited IL-1␤-stimulated increases in MMP-9 and -2 protein levels, although PKC--PS seemed more effective than PKC--PS since it also inhibits the basal protein levels of MMP-2.

PKC-and -Act Upstream of NF-B, and Inhibition of NF-B Inhibits IL-1␤-stimulated Expression and Activity of
MMP-2 and-9 -Inflammatory cytokines are known to activate NF-B in different cell types (19 -21). To determine whether IL-1␤ activates NF-B and the role of PKC in the activation of NF-B, we studied phosphorylation of IKK␣/␤ and IB-␣. Western blot analysis demonstrated that IL-1␤ increases phosphorylation of both IKK␣/␤ and IB-␣ within 15 min of treatment, suggesting activation of NF-B (Fig. 6, A-D). Pretreatment with Gö6976 and PKC-⑀-TIP failed to inhibit IL-1␤-stimulated increases in IKK␣/␤ and IB-␣ phosphorylation. PKC--PS completely inhibited IL-1␤-stimulated increases in IKK␣/␤ and IB-␣ phosphorylation, whereas PKC--PS only exhibited a partial effect (Fig. 6, A and B). To test whether IL-1␤-induced activation of NF-B is mediated through MAPKs, cells were pretreated with PD98059 and SP600125. Pretreatment of cells with PD98059 and SP600125 alone or in combination had no effect on IL-1␤-stimulated increases in IKK␣/␤ and IB-␣ phosphorylation (Fig. 6, C and D).

DISCUSSION
IL-1␤ activates ERK1/2 and JNKs, not p38 kinase, in adult rat cardiac fibroblasts (18). Recently, we found that IL-1␤ activates PKC and that activation of PKC plays a critical role in the regulation of MMP-2 and -9 expression and activity (12). The major new findings of this study are that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-␣/␤1 act upstream of JNKs, not ERK1/2; 3) PKC-and -, not PKC-⑀, act upstream of ERK1/2, JNKs, and NF-B; and 4) activation of NF-B stimulates expression and activity of MMP-2 and -9.
MMPs are proposed to play a critical role in myocardial remodeling process following MI (10). IL-1␤, increased in the heart following MI, increases expression and activity of MMP-2 and -9 in adult rat cardiac fibroblasts (12). Here, we show that inhibition of ERK1/2 and JNKs alone or in combination significantly, but partially, inhibits IL-1␤-stimulated increases in MMP-9, not MMP-2, protein levels and activity. These data suggest the involvement of MAPK-independent pathway(s) in the regulation of both MMP-2 and -9 expression and activity. These findings are consistent with the observations that inducible MMPs (MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, and MMP-13) contain one or more AP-1-binding site(s) in their promoter, whereas the promoter region of constitutive MMPs (MMP-2 and MMP-11) does not contain an AP-1-binding site (27,28). The transcription factor AP-1 is formed by dimerization of different members of Fos and Jun protein family members. The expression of Fos and Jun is mainly regulated via the activation of ERK1/2 and JNKs, respectively. It is interesting to note that activation of ERK1/2 pathway is shown to play a critical role in the regulation of MMP-2 activity in transformed cells (29).
The possibility of involvement of MAPK-independent pathway(s) and that transcription factors, other than AP-1, are involved in the regulation of both MMP-2 and -9 is supported by our recently published data that inhibition of PKC using chelerythrine completely inhibits IL-1␤-stimulated increases in MMP-2 and -9 expression and activities (12). Human MMP-9 promoter has a binding site for transcription factor polyoma enhancer activator-3 (PEA-3), which is regulated by PKC (28,30). The human MMP-2 promoter contains AP-2 and SP-1 transcription factor-binding sites. In hepatoma cells, activation of PKC and protein kinase A activated AP-2 and MMP-2 gene expression (28,31). PKC activation is suggested to be necessary for the induction of MMP gene expression in response to proinflammatory cytokines, such as IL-1␤ and tumor necrosis factor-␣ in non-cardiac fibroblasts and tumor cell lines (32)(33)(34)(35). PKC comprises a large family of serine-threonine kinases and plays an important role in the regulation of cardiovascular functions. The documented 12 members of the PKC family are classified according to their structure and substrate requirements (24,36,37). In this study, using PKC isoform-selective inhibitors, we show that PKC-␣/␤1 play a critical role in the regulation of MMP-9 expression and activity, whereas activation of PKC-and PKC-plays a critical role in the regulation of expression and activity of MMP-2 and -9. The data presented here suggest that different isoforms of PKC differentially regulate the expression and activity of MMPs in cardiac fibroblasts in response to IL-1␤.
Various isoforms of PKC are known to modulate various signaling pathways (24). In cardiac myocytes, endothelin-1 stimulated activation of ERK1/2 is selectively mediated via PKC-⑀, whereas PKC-␦ preferentially activated JNKs and p38 kinase (38,39). The data presented here suggest that PKC-␣ and -␤1 act upstream of JNKs, whereas PKC-and -act upstream of both ERK1/2 and JNKs. Activation of neither ERK1/2 nor JNKs requires PKC-⑀. The differential effects of PKC isoforms on the regulation of MAPK activity in different cell types may reflect differences in activation of different isoforms of PKC and their downstream coupling. Of note, IL-1␤ has been shown to activate ERK1/2, JNKs, and p38 kinase via PKC-independent pathways in intestinal myofibroblasts (40).
Another interesting finding of this study is that PKC-and -, not PKC-␣, -␤1, or -⑀, act upstream in the activation of NF-B signaling pathway and that inhibition of NF-B inhibits IL-1␤-stimulated increases in MMP (MMP-2 and -9) expression and activity. Here, activation of NF-B was studied using increased phosphorylation of IKK␣/␤ and IB-␣ kinase as an index. Increased phosphorylation and activation of IKK␣/␤ increases phosphorylation of IB-␣. Upon phosphorylation, the IB-␣ protein is rapidly degraded by the proteasome, thereby allowing NF-B to enter into the nucleus (41). Inhibition of ERK1/2 and JNKs failed to inhibit IL-1␤-stimulated increases in phosphorylation of IKK␣/␤ and IB-␣, suggesting activation of NF-B independent of MAPK pathways. The data presented here suggest both PKC-and -are involved in the activation of NF-B. However, the effect of PKC-seemed more prominent than PKC-. These data are consistent with the findings of Anrather et al. (42) in which PKC-has been shown to be essential for the transcriptional activity of NF-B in endothelial cells.
NF-B has been suggested to play a role in the regulation of MMP expression (43,44). The proximal stimulatory region of the MMP-9 promoter has a functional NF-B site (28). NF-B activity is shown to be required for the inducibility of MMP-9 production in smooth muscle cells in response to cytokines (45,46). Inhibition of NF-B activation using SN50 inhibits MMP-9 expression and activity induced by IL-1␤ in adult cardiac fibroblasts. However, our data that NF-B activity plays an essential role in the regulation of IL-1␤-stimulated increases in MMP-2 in cardiac fibroblasts are interesting because no consensus NF-B-binding site has been identified in MMP-2 promoter (28). Activation of latent MMP-2 is regulated by membrane-type 1 MMP (MT1-MMP) in conjunction with tissue inhibitors of MMPs (47). MT1-MMP has an NF-B-binding site in its promoter (44). Therefore, it is possible that IL-1␤-stimulated activation of NF-B increases MMP-2 activity indirectly via the increased expression of MT1-MMP. It is also possible that NF-B directly modulates MMP-2 expression and activity via its interaction with other transcription factors that regulate MMP-2 expression. The later possibility is supported by our data that inhibition of NF-B also inhibits protein levels as well as activity of MMP-2.
The data presented here indicate differential regulation of MMP-2 and -9 expression and activity in adult rat cardiac fibroblasts. PKC-␣/␤1 activates JNKs, leading to increased protein levels and activity of MMP-9, whereas PKC-and PKCactivate JNKs, ERK1/2, and NF-B, resulting in increased protein levels and activity of both MMPs (Fig. 8). Thus, IL-1␤stimulated increases in MMP-9 expression and activity involve PKC-␣/␤1, PKC-, PKC-, ERK1/2, JNKs, and NF-B signaling molecules, whereas IL-1␤-stimulated increases in MMP-2 expression and activity are regulated via PKC-, PKC-, and NF-B pathways. Although these observations are made in vitro, activation of PKC, NF-B, and AP-1 has been observed in the non-infarcted myocardium of rats after MI (48,49). NF-B and AP-1 are also activated in congestive heart failure due to ischemic and dilated cardiomyopathy (49). We have observed increased phosphorylation of PKC-in mice after MI (12), whereas others have shown increased expression of PKC-␣ and PKC-␦, not PKC-⑀, in rat after MI (48). Thus, our findings raise the possibility that activation of these signaling pathways could contribute to pathologic remodeling of the heart by differentially regulating the expression and activity of MMP-2 and -9. A better understanding of the regulation of individual MMP is essential for the development of pharmacological strategies for the prevention of excessive matrix degradation and remodeling during chronic heart failure and after MI.