![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
in Endothelin-Induced Type I Collagen Expression in Cardiac Myofibroblasts Isolated from the Site of Myocardial Infarction
Department of Physiology, the Brody School of Medicine, East Carolina University, Greenville, North Carolina
Received April 23, 2004; accepted July 6, 2004.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
(PKC) and mitogen-activated protein kinase/extracellular signal-regulated kinase-1/2 (MAPK/ERK1/2) play a role in ET-induced type I collagen expression using specific pharmacological inhibitors. The present study also reveals the expression of various isoforms of PKC including PKC
, PKC
I, PKCII, PKC
, PKC, PKC
, PKC
, and PKC
in cardiac myoFb. Our results from mRNA and protein studies demonstrate that calphostin-C, a PKC inhibitor, decreased the ET-induced type I collagen expression suggesting a role for the PKC pathway. Further treatment with rottlerin, a PKC isoform-specific inhibitor, demonstrated attenuation of 80 to 90% of type I collagen expression induced by ET. However, Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo [3,4-c]carbazole]], an inhibitor of Ca2+-dependent PKC isoforms (PKC and PKCI), showed little to no effect on ET-stimulated type I collagen expression. Furthermore, the MAPK inhibitor PD98059 (2'-amino-3'-methoxyflavone) attenuated ET-dependent activation of p44/42 MAPK (pERK1/2) and also down-regulated type I collagen expression. Similarly, rottlerin inhibited the activation of p44/42 MAPK (pERK) implicating the involvement of PKC and MAPK/ERK1/2 in ET-induced type I collagen expression. Our protein/DNA array and reverse transcription-polymerase chain reaction results from ET-treated samples showed a significant increase in Sp1 expression. PD98059 and rottlerin decreased ET-induced Sp1 expression, suggesting a possible interaction of Sp1 with PKC and MAPK in ET-induced type I collagen expression in cardiac myoFb.
; Nguyen et al., 1998, 2001; Rossi et al., 1999; Thai et al., 1999; Bauersachs et al., 2000; Mulder et al., 2000; Fraccarollo et al., 2002; Pfeffer et al., 2000). The myofibroblasts (myoFb) found at the site of myocardial infarction (MI) are known to play a vital role in the accumulation of collagens during repair/remodeling (Campbell and Katwa, 1997; Katwa et al., 1997; Sun et al., 2000; Chintalgattu et al., 2003). Recent studies from our laboratory have shown ET-induced expression of type I collagen in cardiac myoFb (Katwa, 2003). The present study was designed to elucidate the involvement of PKC isoforms, MAPK, and transcriptional factors in ET-induced type I collagen expression in cardiac myoFb isolated from the site of MI.
The PKC family has at least 11 different isoforms of which the number and level of PKC isoform expression is tissue-specific, and their subcellular localization varies depending on the cell type (Mackay and Mochly-Rosen, 2001). Studies in other cell types, such as scleroderma fibroblasts and mesangial cells, have shown PKC involvement in TGF-1 mediated collagen biosynthesis (Jimenez et al., 2001; Runyan et al., 2003). Additionally, studies have suggested that stabilization of elastin mRNA in lung fibroblasts by TGF-1 requires Smads, PKC, and the MAP kinase p38 (Kucich et al., 2002). More recently, it has been shown that the PKC and ERK1/2 pathways are associated with ET-induced type I (IV) collagen expression in mesangial cells (Hua et al., 2001). Several reports have also suggested the involvement of various transcription factors including Sp1 in the collagen expression pathway (Chen et al., 1998; Ruiz et al., 2000; Czuwara-Ladykowska et al., 2001; Garcia-Ruiz et al., 2002). However, there are no reports on the role of PKC, MAPK, and Sp1 in the synthesis of collagen in cardiac myoFb.
Thus, in the present study, we investigated the possible role of PKC isoforms in ET-induced type I collagen expression in cardiac myoFb by pharmacological intervention using PKC isoform-specific inhibitors. Our data also revealed the involvement of PKC, MAPK, and Sp1 in ET-induced type I collagen expression in these cells.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
antibody and PKC-specific substrate were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-type I collagen and anti-mouse IgG-HRP were purchased from Sigma-Aldrich (St. Louis, MO). A protein DNA array kit and Sp1 reporter construct were purchased from Panomics (Redwood City, CA). Nuclear extraction kit was obtained from Active Motif (Carlsbad, CA). Other general laboratory grade chemicals were purchased from Fisher Scientific Co. (Pittsburgh, PA).
Cell Culture and Treatments. Myofibroblasts were isolated from rat MI using the previously described method (Chintalgattu et al., 2003). In this study, we used early passages (<12) of myoFb cultures to avoid possible morphological changes during sub-culturing. These cells were grown to 80% confluence in 10% DMEM in 100-mm plates. Prior to treatment, medium was replaced with serum-deprived medium (0.4% DMEM) for 12 h. Cells were pretreated in DMEM for 1 h with calphostin-C (10-7 M), rottlerin (1 µM), PD98059 (100 µM), and Go6976 (10 nM) followed by ET (10-9 M). Protein and RNA were isolated from treated cells after 3 and 24 h of incubation, respectively.
Protein Kinase Assay. The cells were grown in serum-deprived (0.4% fetal bovine serum) DMEM for 12 h and then treated with ET or ET + rottlerin in serum-free medium for various time periods. The total protein was isolated from cells using radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate; 0.1% SDS) containing 1x protease inhibitor cocktail from Sigma-Aldrich. Immunoprecipitation was carried out using 100 µg of protein with 4 µg of PKC antibody and incubated 3 h at 4°C and then 50 µl of protein G-Sepharose overnight at 4°C. Kinase assay was carried out using a PKC kinase assay kit from Upstate (Waltham, MA) with 10 µl of immunocomplexes. After 10 min of incubation, samples were spotted on P81 phosphocellulose paper and washed with 0.75% phosphoric acid. The amount of incorporated radioactivity (32P-ATP) into substrate was measured by scintillation counting. The kinase assay was also repeated with crude protein (50 µg) and PKC-specific substrate from Santa Cruz Biotechnology, Inc. using the above kinase assay kit.
Luciferase Assay and Transient Transfection. Cells were grown in 6-well plates until 70% confluent and then serum-deprived medium was added for 12 h. In accordance with the manufacturer's instructions (Panomics), 2 µg of Sp1 reporter vector or 2 µg of pSilencer Sp1 siRNA along with 0.5 µg of pSV--galactosidase vector as a control for transfection efficiency were transfected into myoFb. After 18 h of transfection in Opti-MEM I (Invitrogen, Carlsbad, CA) with LipofectAMINE 2000 (Invitrogen), cells were treated with ET or ET + rottlerin for 24 h and measured for luciferase and -galactosidase activity according to the manufacturer's instructions (Promega, Madison, WI).
Western Blot and ELISA. After 24-h treatment, protein was isolated using lysis buffer (1x protease inhibitor cocktail, 10 mM Tris-HCl, pH 6.8; 150 mM NaCl; 1% Nonidet P-40). Protein samples were electrophoresed using a 4 to 12% SDS-polyacrylamide gel under reducing conditions as described previously (Chintalgattu et al., 2003). The proteins were transferred to polyvinylidene difluoride membranes and blocked with phosphate-buffered saline containing 5% nonfat milk powder for 1 h at room temperature. Membranes were incubated with the appropriate primary antisera. Type I collagen, MAPK, and pMAPK antiserum was used at 1:1000 dilutions. All PKC antibodies were used at 1:100 dilutions. HRP conjugated goat anti-rabbit or goat anti-mouse IgG was used as the secondary antisera at 1:5000 dilutions. Protein bands were visualized using a chemiluminescence detection system (ECL; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). Extracellular type I collagen was analyzed using ELISA as described previously (Katwa, 2003).
Protein/DNA Array. Nuclear extracts were prepared using Active Motif nuclear extraction kit. The protein/DNA array was carried out according to the manufacturer's instructions (Panomics). Briefly, 5 µg of nuclear extract was eletrophoresed on a 2% agarose gel at 120 V. The protein DNA complex was isolated from the gel after 15 min. The isolated protein DNA complex was incubated with a protein/DNA array membrane overnight at 42°C. The membrane was washed with buffer and developed with streptavidin-HRP conjugate. Expression levels of various transcription factors were analyzed using densitometric analysis.
RT-PCR. Total RNA was isolated from 80% confluent cells using the Trizol method. Reverse transcription was performed on 2 µg of total RNA with 0.5 µg/µl oligo(dT)12-18 primer and MMLV reverse transcriptase. PCR amplification of specific type I collagen, Sp1, and 2-microglobulin (2-MG) gene sequences was performed with the cDNA in a 25-µl reaction using a high fidelity PCR kit (Life Technologies). The following gene-specific oligonucleotide primers were used in the reaction: type I collagen: forward 5'-GCGAAGGCAACAGTCGATTC-3', reverse 5'-CCCAAGTTCCGGTGTGACTC-3'; Sp1: forward 5'-GATCATCAGGGACTAGTTCTCAAG-3', reverse 5'-CACAGTGACTGTGCCAGCAGGAAT-3'; 2-MG: forward 5'-AGTGACGTGTTTAACTCTGCAAGC-3', reverse 5'-CTCCCCAAATTCAAGTGTACTCTCG-3'. The above genes were amplified for 32 cycles at 94°C for 15 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min followed by a final extension at 72°C using GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, CA). PCR products were fractionated on a 1.5% agarose gel containing ethidium bromide and visualized under UV light.
Quantitative Real-Time RT-PCR (qRT-PCR). Analysis of type I collagen and GAPDH mRNA expression by qRT-PCR was performed utilizing a Cepheid Smart Cycler real-time PCR (Cepheid, Sunnyvale, CA). Type I collagen and GAPDH gene-specific primers and TaqMan probes were synthesized (Synthegen, LLC, Houston, TX) and are as follows: GAPDH primers: forward 5'-CGTGGAGTCTACTGTGAATCTTC-3', reverse 5'-GGCATGGACTGTGGTCATGAG-3'; probe HEX: 5'-TCATCCATGACAACTTTGGCATC-3'. Type I collagen probe TET: 5'-ACAGCACGCTTGTGGATGGCTGC-3'. The above described type I collagen RT-PCR primers were used in qRT-PCR. These primers were designed to span exon-intron junctions to prevent amplification of genomic DNA. TaqMan probes were labeled at the 5'-end with the reporter dye molecule TET and HEX (tetrachloro-6-carboxyfluorescein; absorbance 522 nm, emission 538 nm and hexachlorofluorescein; absorbance 536 nm, emission 552 nm) and at the 3'-end with the quencher dye molecule TAMRA (6 carboxytetramethylrhodamine; emission 582 nm). Thermoscript one-step quantitative RT-PCR platinum Taq kit (Life Technologies) was used to analyze the type I collagen, and GAPDH in qRT-PCR, and 25 µl of reactions were prepared according to the manufacturer's recommendation. Equal amounts of total RNA (100 ng) was used in all qRT-PCR reactions. The temperature profile included an initial 30 min at 50°C (for cDNA synthesis) then denaturation at 95°C for 2 min to deactivate the reverse transcriptase enzyme. This was followed by 30 cycles of denaturation at 95°C for 15 s, 60 s at 68°C annealing, and elongation with optics on for fluorescence monitoring. Standard curve extrapolation of gene copy number was performed for type I collagen and GAPDH genes.
Flow Cytometric Analysis. The confluent myoFb (1 x 10-6 cells/plate) cultures were subjected to 24 h of incubation in serum-free DMEM without (untreated control) and with various PKC inhibitors [rottlerin (1-6 µM), PD98059 (100 µM), and Go6976 (10 nm)]. Cells were trypsinized and centrifuged at 600 rpm for 10 min at 4°C. The cell pellets were washed with ice-cold phosphate-buffered saline, and propidium iodide (Sigma-Aldrich) was added to a final concentration of 10 µg/ml and incubated for 5 min at room temperature. The cells were finally analyzed in a FACScan flow cytometer (BD Biosciences, San Jose, CA) equipped with an argon ion laser tuned at 488 nm (modFit LT 3.0 software).
Statistical Analysis. Results are reported as mean ± S.E.M. for a minimum of three to six determinations each of which was performed in duplicate or triplicate. Statistical analysis was performed using one-factor analysis of variance with p < 0.05 being considered significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Kinase Activity in Cardiac Myofibroblasts. The number and levels of PKC isoform expression vary in different tissues. Our Western blot experiments showed the expression of various PKC isoforms such as PKC, PKCI, PKCII, PKC, PKC, PKC, PKC, and PKC in cardiac myoFb (Fig. 1A). These isoforms may be involved in various cellular mechanisms including the collagen synthesis pathway. There was a significant increase in PKC expression in ET-treated myoFb (Fig. 1B) clearly suggesting the participation of PKC in ET-mediated signaling mechanisms.
|
Isolated protein from myoFb treated with ET alone or along with rottlerin for 24 h was subjected to immunoprecipitation with PKC-specific antibody and then immunocomplexes were used for in vitro kinase assay. Results from Fig. 1C clearly demonstrate that ET induced a 2-fold increase in PKC activity and attenuation of ET-induced PKC activity by rottlerin; this suggests a role for PKC in ET-induced collagen expression. Although the endpoint for most of our experiments was 24 h, we observed a time-dependent increase in PKC activity with ET-treated protein samples (data not shown). We found similar results when we measured the kinase activity with PKC-specific substrate with crude protein from ET and rottlerin treatments. We found that rottlerin (1 µM) was able to inhibit the ET-induced collagen expression (Fig. 2), therefore, we used 1 µM rottlerin in all experiments in this study.
|
The Role of PKC and MAPK in ET-Induced Type I Collagen Expression by Western Blot and ELISA. In this study, we used Western blot and ELISA to analyze intracellular and extracellular expression of type I collagen, respectively. To understand the interaction of the PKC and MAPK/ERK1/2 in ET-stimulated expression of type I collagen, myoFb were treated with calphostin-C (which inhibits most of the PKC isoforms) and PD98059 (a MAPK inhibitor) along with ET. The results showed a decreased expression of type I collagen suggesting a role for the PKC and MAPK in ET-induced expression of type I collagen. Protein as well as medium samples from Go6976 (an inhibitor of Ca2+-dependent PKC and PKCI isoform) (Martiny-Baron et al., 1993) and ET-treated myoFb were analyzed using Western blot and ELISA. Results from ET + Go6976 treatment show little to no effect on type I collagen protein expression (Fig. 3, A and B). These findings clearly demonstrate the minimal role, if any, of the Ca2+-dependent PKC and PKCI isoforms on ET-stimulated type I collagen expression.
|
Myofibroblasts treated with rottlerin (1 µM), a specific inhibitor of PKC (Gschwendt et al., 1994), attenuated 80 to 90% of ET-induced type I collagen protein expression (Fig. 3A). Media samples from ET + rottlerin-treated myoFb showed similar decreases in extracellular type I collagen expression levels (Fig. 3B). These results confirm the involvement of the PKC in ET-induced type I collagen expression.
The Role of PKC and MAPK in ET-Induced Type I Collagen Gene Expression Using RT-PCR and Real-Time RT-PCR. As shown in Fig. 4A, calphostin-C and PD98059 reduced ET-induced type I collagen RNA expression in myoFb, indicating the involvement of PKC and MAPK/ERK1/2 in ET-stimulated type I collagen expression. Myofibroblasts treated with Go5976 did not show significant effects on ET-induced type I collagen expression, suggesting Ca2+-dependent PKC isoforms may not be involved in ET-induced type I collagen expression. Myofibroblasts treated with rottlerin and ET showed a 90% reduction of type I collagen RNA expression, indicating a predominant role for PKC in ET-stimulated type I collagen expression.
|
The RT-PCR data were further confirmed by qRT-PCR (Fig. 4B). The real-time PCR data also demonstrated a similar decrease in relative copy number of type I collagen mRNA in ET + rottlerin-treated samples (Fig. 4B). The data shown above from RNA as well as protein studies (Fig. 3, A and B; Fig. 4, A and B) clearly demonstrate the interaction between PKC (specifically PKC) and the MAPK pathways in ET-induced type I collagen expression.
Effect of the MAPK Inhibitor PD98059 on Endothelin-Mediated ERK Phosphorylation. To determine whether ET stimulates ERK1/2 activity, ERK1/2 phosphorylation was measured after 24 h of treatment with ET (Fig. 5). Our Western blot experiments revealed the up-regulation of pERK1/2 in ET-treated samples indicating possible interactions of ET with the MAP kinase cascade resulting in ERK1/2 activation. Thus having shown that ET induced ERK activation, we wanted to determine whether ERK phosphorylation was required for type I collagen protein production. If ERK phosphorylation is necessary for type I collagen expression, blocking the activation of ERK should attenuate the effects of ET in the up-regulation of type I collagen expression. We found that the MAPK inhibitor, PD98059, with the inhibition of ERK1/2 was able to suppress type I collagen protein expression at a concentration of 100 µM (Fig. 3, A and B; Fig. 4, A and B). Furthermore, PD98059 also abrogated the effects of ET on ERK1/2 activation at the same concentration (Fig. 5). These results further suggest a role for MAPK/ERK1/2 in ET-mediated type I collagen expression.
|
The PKC inhibitor rottlerin was used to further correlate the interaction between PKC, MAPK, and type I collagen protein expression. We found that rottlerin (1 µM) was able to inhibit the activation of pMAPK/pERK following ET treatment (Fig. 5). Taken together, our results (Fig. 3, A and B; Fig. 4, A and B; Fig. 5) strongly suggest that PKC mediates ET induction of type I collagen through MAPK.
ET-Induced Expression of Various Transcription Factors by Protein/DNA Array. Array membranes were treated with nuclear extracts isolated from control (untreated) and ET-treated myoFb. ET treatment resulted in 10-fold increases in Sp1 expression, whereas Smad3/4, Stat1, Stat3, and Stat4 showed 2- to 5-fold increases. Stat5 and Stat6 as well as AP-1 and -2 showed a 1- to 2-fold increase in their expression levels (Fig. 6). These results clearly indicated a role for Sp1 in ET-dependent signaling mechanisms. Our RT-PCR results from myoFb treated with ET + PD98059 and ET + rottlerin showed a decrease in Sp1 expression, suggesting a role for PKC in ET-stimulated Sp1 RNA expression (Fig. 7). These results suggest a possible interaction of Sp1 with the PKC and MAPK in ET-induced type I collagen expression.
|
|
Role of Rottlerin on ET-Induced Sp1 in Vivo Binding Activity. To determine the influence of rottlerin on ET-induced Sp1-binding activity in vivo, cells were transfected with 2 µg of Sp1 reporter vector and pSilencer containing siRNA Sp1 (Banchio et al., 2003) or empty vectors (pSilencer or Sp1 reporter vector) along with pSV--galactosidase vector (control for transfection) for 18 h. Cells were then treated with ET or ET + rottlerin for 24 h. The ET treatment significantly increased Sp1 binding activity, whereas ET + rottlerin or ET + pSilencer Sp1 decreased the ET-induced Sp1 activity suggesting a possible role for Sp1 in ET-induced collagen expression (Fig. 8).
|
Role of PKC/MAPK Inhibitors on Myofibroblast Cell Death (Apoptosis). To analyze and rule out the nonspecific (toxicity) role of pharmacological agents (rottlerin, Go6976, and PD98059) on myoFb cell death (apoptosis), myoFb was treated with various pharmacological inhibitors of PKC and - (rottlerin, 1-6 µM; Go6976, 10 nM) MAPK (PD98059, 100 µM) for 24 h and analyzed for flow cytometry as described under Materials and Methods. Results clearly demonstrated that more than 94% myoFb are viable after treatment with various concentrations of pharmacological agents (rottlerin, Go6976, and PD98059), suggesting no influence of these inhibitors on cell death (apoptosis) under these experimental conditions (Fig. 9).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
isoform-specific blocker) and PD98059 (a MAPK pathway blocker) reduced the ET-induced collagen expression in myoFb, suggesting the association of PKC and MAPK in ET-stimulated collagen biosynthesis pathway. Rottlerin treatment resulted in decreasing the ET-induced Sp1-binding activity and Sp1 expression, indicating its possible involvement in ET-induced collagen expression cascade. Our results demonstrate the involvement of PKC, MAPK, and Sp1 in ET-induced collagen synthesis.
Clinical and experimental studies on the heart have identified a number of conditions such as pathological cardiac hypertrophy, heart failure, and ischemic preconditioning in which PKCs are activated (Dunnmon et al., 1990; D'Angelo et al., 1997; Miyamae et al., 1998; Qiu et al., 1998; Bowling et al., 1999). Despite these known associations and the importance of PKC isoforms as being essential and ubiquitous signaling molecules, their contribution to clinical situations like abnormal deposition of extracellular matrix and tissue fibrosis in the heart is yet to be fully established. One study on osteoblastic cells suggests the involvement of PKC and ERK pathways in TGF-1-induced type I collagen expression (Palcy and Goltzman, 1999). Several previous reports have implicated the role of Sp1 and Sp3 in collagen transcription in various models (Li et al., 1995; Chen et al., 1998; Ihn and Tamaki, 2000; Ruiz et al., 2000; Czuwara-Ladykowska et al., 2001; Garcia-Ruiz et al., 2002). In the present study, we show a role for PKC, ERK1/2, and Sp1 in ET-induced type I collagen expression in cardiac myoFb.
Results from our Western blot demonstrate the expression of PKC isoforms PKC, PKCI, PKCII, PKC, PKC, PKC, PKC, and PKC by these cells. Mackay and Mochly-Rosen (2001) demonstrated the tissue-specific and differential cellular localization of PKC isoforms. The isoforms PKC, PKC, PKC, PKC, PKC, and PKC are consistently expressed, whereas expression of PKCI and PKCII were inconsistently reported in neonatal and adult ventricular myocytes (Mackay and Mochly-Rosen, 2001). Previous studies have also demonstrated a role for PKC isoforms in various pathological conditions in the heart; however, the role of these isoforms in cardiac myoFb is unclear. Since ET has been shown to play a key role in abnormal accumulation of type I collagen in these cells, we attempted to understand the role of PKC isoforms in this process using specific pharmacological inhibitors. Our in vitro kinase assay for PKC demonstrated that rottlerin resulted in attenuation of ET-induced PKC activity as well as ET-induced up-regulation of PKC expression in myoFb, suggesting a role for PKC in ET-induced collagen expression in myoFb. Similarly, Clerk et al. (1994) reported the ET-induced expression of various isoforms including PKC in neonatal ventricular myocytes.
In our study, experiments with isoform-specific inhibitors such as rottlerin, a PKC inhibitor, showed an 80 to 90% down-regulation of ET-induced type I collagen expression, suggesting a predominant role for the PKC isoform in the ET-induced collagen expression. Findings in dermal fibroblasts suggested the involvement of PKC in TGF-1-mediated collagen expression (Jimenez et al., 2001). More recently, Runyan et al. (2003) demonstrated TGF-1-stimulated collagen expression via PKC in human mesangial cells. Hua et al. (2001) suggested multiple PKC isoforms to be associated with ET-1-stimulated I (IV) expression in mesangial cells. Gschwendt et al. (1994) suggested that rottlerin not only specifically inhibit the PKC but also was able to differentiate, to some extent, between the various PKC isoforms. PKC required the lowest rottlerin (IC50, 3-6 µM) concentration for its inhibition, whereas other PKC isoforms required 5- to 10-fold higher concentrations, suggesting that at lower concentrations rottlerin exclusively inhibited PKC. Moreover, in this study we used a 1 µM concentration of rottlerin further diminishing the possibility of other PKC isoforms inhibition along with PKC by rottlerin. In addition, treatment with Ca2+-dependent inhibitor Go6976 (PKC and PKCI inhibitor) along with ET showed little to no effect on ET-stimulated type I collagen expression. These data clearly outline the predominant role of PKC in ET-1-mediated collagen expression in cardiac myoFb.
In this study, results from RNA and protein experiments revealed decreased expression of collagen in myoFb treated with PD98059 (a specific inhibitor of MAPK) and ET, implicating the involvement of the MAPK/ERK1/2 pathway in ET-induced collagen gene expression in cardiac myoFb. Furthermore, rottlerin and ET-treated myoFb showed decreased expression of the pERK and type I collagen suggesting ET-induced collagen expression may be mediated by PKC and ERK pathway. Similarly, Rodriguez-Barbero et al. (2002) demonstrated the p38 MAPK-mediated TGF-1-stimulated collagen expression in L6E9 myoblasts, whereas Hua et al. (2001) reported ERK1/2 involvement in ET-induced collagen expression in mesangial cells.
In the present study, protein/DNA array experiments demonstrated the ET-induced expression of various transcriptional factors. On the basis of previous studies and our array data, the transcriptional factors relevant to collagen expression were analyzed. Our results revealed ET-stimulated expression of Sp1, Smad, and Stat1-6 in cardiac myoFb. Among them, Sp1 levels were significantly increased when compared with controls in these cells. Findings from previous studies have also demonstrated the Sp1/Sp3 association with the collagen biosynthetic pathway (Chen et al., 1998; Ruiz et al., 2000; Czuwara-Ladykowska et al., 2001; Garcia-Ruiz et al., 2002). Our RT-PCR data also demonstrate ET-induced expression of Sp1, whereas rottlerin and PD98059 attenuated the ET-induced expression of Sp1. Rottlerin and Sp1-siRNA reduced the ET-induced Sp1 in vivo binding activity. This suggests the involvement of Sp1 in ET-mediated type I collagen expression.
In conclusion, our data demonstrates the expression of various PKC isoforms and the involvement of PKC, ERK1/2, and Sp1 in ET-induced expression of type I collagen in cardiac myoFb. Our data strongly support the use of pharmacological intervention via PKC inhibitors to reduce the ET-induced collagen expression in myoFb, which may serve as a potential therapeutic agent in preventing the abnormal accumulation of collagen/fibrosis seen in myocardial infarction.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: ET-1, endothelin-1; myoFb, myofibroblasts; MI, myocardial infarction; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; TGF-1, transforming growth factor-1; ERK1/2, extracellular signal-regulated kinase-1/2; RT-PCR, reverse transcription-polymerase chain reaction; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo [3,4-c]carbazole; PD98059, 2'-amino-3'-methoxyflavone; HRP, horseradish peroxidase; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; 2-MG, 2-microglobulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Address correspondence to: Dr. Laxmansa C. Katwa, Department of Physiology, 6N-98, Brody Medical Sciences Bldg., East Carolina University Brody School of Medicine, 600 Moye Blvd., Greenville, NC 27834. E-mail: katwal{at}mail.ecu.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Banchio C, Schang LM, and Vance DE (2003) Activation of CTP: phosphocholine cytidylyltransferase alpha expression during the S phase of the cell cycle is mediated by the transcription factor Sp1. J Biol Chem 278: 32457-32464.
Bauersachs J, Braun C, Fraccarollo D, Widder J, Ertl G, Schilling L, Kirchengast M, and Rohmeiss P (2000) Improvement of renal dysfunction in rats with chronic heart failure after myocardial infarction by treatment with the endothelin A receptor antagonist, LU 135252. J Hypertens 18: 1507-15014.[CrossRef][Medline]
Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, Mintze K, Pickard T, Roden R, Bristow MR, et al. (1999) Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 99: 384-391.
Campbell SE and Katwa LC (1997) Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts. J Mol Cell Cardiol 29: 1947-1958.[CrossRef][Medline]
Chen SJ, Artlett CM, Jimenez SA, and Varga J (1998) Modulation of human alpha1 (I) procollagen gene activity by interaction with Sp1 and Sp3 transcription factors in vitro. Gene 215: 101-110.[CrossRef][Medline]
Chintalgattu V, Nair DM, and Katwa LC (2003) Cardiac myofibroblasts: a novel source of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR. J Mol Cell Cardiol 35: 277-286.[CrossRef][Medline]
Clerk A, Bogoyevitch MA, Anderson MB, and Sugden PH (1994) Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem 269: 32848-32857.
Czuwara-Ladykowska J, Shirasaki F, Jackers P, Watson DK, and Trojanowska M (2001) Fli-1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway. J Biol Chem 276: 20839-20848.
D'Angelo DD, Sakata Y, Lorenz JN, Boivin G, Walsh RA, Liggett SB, and Dorn GW II (1997) Transgenic Gq overexpression induces cardiac contractile function in mice. Proc Natl Acad Sci USA 94: 8121-8126.
Dunnmon PM, Iwaki K, Henderson SA, Sen A, and Chien KR (1990) Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells. J Mol Cell Cardiol 22: 901-910.[CrossRef][Medline]
Fraccarollo D, Galuppo P, Bauersachs J, and Ertl G (2002) Collagen accumulation after myocardial infarction: effects of ETA receptor blockade and implications for early remodeling. Cardiovasc Res 54: 559-567.
Garcia-Ruiz I, de la Torre P, Diaz T, Esteban E, Fernandez I, Munoz-Yague T, and Solis-Herruzo JA (2002) Sp1 and Sp3 transcription factors mediate malondialdehyde-induced collagen alpha 1(I) gene expression in cultured hepatic stellate cells. J Biol Chem 277: 30551-30558.
Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, and Marks F (1994) Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 93-98.[CrossRef][Medline]
Guarda E, Katwa LC, Myers PR, Tyagi SC, and Weber KT (1993) Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovasc Res 27: 2130-2134.
Hua H, Goldberg HJ, Fantus IG, and Whiteside CI (2001) High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha1 (IV) collagen expression in response to endothelin-1: role of specific protein kinase C isoenzymes. Diabetes 50: 2376-2383.
Ihn H and Tamaki K (2000) Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: association with increased expression of the type I collagen gene. Arthritis Rheum 43: 2240-2247.[CrossRef][Medline]
Jimenez SA, Gaidarova S, Saitta B, Sandorfi N, Herrich DJ, Rosenbloom JC, Kucich U, Abrams WR, and Rosenbloom J (2001) Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts. J Clin Investig 108: 1395-1403.[CrossRef][Medline]
Katwa LC (2003) Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo. Am J Physiol Heart Circ Physiol 285: H1132-H1139.
Katwa LC, Campbell SE, Tyagi SC, Lee SJ, Cicila GT, and Weber KT (1997) Cultured myofibroblasts generate angiotensin peptides de novo. J Mol Cell Cardiol 29: 1375-1386.[CrossRef][Medline]
Kucich U, Rosenbloom JC, Abrams WR, and Rosenbloom J (2002) Transforming growth factor-beta stabilizes elastin mRNA by a pathway requiring active Smads, protein kinase C-delta and p38. Am J Respir Cell Mol Biol 26: 183-188.
Li L, Artlett CM, Jimenez SA, Hall DJ, and Varga J (1995) Positive regulation of human alpha 1 (I) collagen promoter activity by transcription factor Sp1. Gene 164: 229-234.[CrossRef][Medline]
Mackay K and Mochly-Rosen D (2001) Localization, anchoring and functions of protein kinase C isoenzymes in the heart. J Mol Cell Cardiol 33: 1301-1307.[CrossRef][Medline]
Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marmé D, and Schächtele C (1993) Selective inhibition of protein kinase C isoenzymes by indolocarbazole Gö 6976. J Biol Chem 268: 9194-9197.
Miyamae M, Rodriguez MM, Camacho SA, Diamond I, Mochly-Rosen D, and Figueredo VM (1998) Activation of protein kinase C correlates with a cardioprotective effect of regular ethanol consumption. Proc Natl Acad Sci USA 95: 8262-8267.
Mulder P, Boujedaini H, Richard V, Derumeaux G, Henry JP, Renet S, Wessale J, Opgenorth T, and Thuillez C (2000) Selective endothelin-A versus combined endothelin-A/endothelin-B receptor blockade in rat chronic heart failure. Circulation 102: 491-503.
Nguyen QT, Cernacek P, Calderoni A, Stewart DJ, Picard P, Sirois P, White M, and Rouleau JL (1998) Endothelin A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure after infarction in the rat. Circulation 98: 2323-2330.
Nguyen QT, Cernacek P, Sirois MG, Calderone A, Lapointe N, Stewart DJ, and Rouleau JL (2001) Long-term effects of nonselective endothelin A and B receptor antagonism in postinfarction rat: importance of timing. Circulation 104: 2075-2081.
Palcy S and Goltzman D (1999) Protein kinase signalling pathways involved in the up-regulation of the rat alpha1 (I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells. Biochem J 343: 21-27.
Pfeffer JM, Finn PV, Zornoff LA, and Pfeffer MA (2000) Endothelin-A receptor antagonism during acute myocardial infarction in rats. Cardiovasc Drugs Ther 14: 579-587.[CrossRef][Medline]
Qiu Y, Ping P, Tang X-L, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner S, and Bolli R (1998) Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved. J Clin Investig 101: 2182-2198.[Medline]
Rodriguez-Barbero A, Obreo J, Yuste L, Montero JC, Rodriguez-Pena A, Pandiella A, Bernabeu C, and Lopez-Novoa JM (2002) Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts. FEBS Lett 513: 282-288.[CrossRef][Medline]
Rossi GP, Cesari M, and Pessina AC (1999) Endothelins and cardiac fibrosis. Hypertension 34: e1.
Ruiz IG, de la Torre P, Diaz T, Esteban E, Morillas JD, Munoz-Yague T, and Solis-Herruzo JA (2000) Sp family of transcription factors is involved in iron-induced collagen alpha1 (I) gene expression. DNA Cell Biol 19: 167-178.[CrossRef][Medline]
Runyan CE, Schnaper HW, and Poncelet AC (2003) Smad3 and PKC mediate TGF-beta1-induced collagen I expression in human mesangial cells. Am J Physiol Renal Physiol 285: F413-F422.
Sun Y, Zhang JQ, Zhang J, and Lamparter S (2000) Cardiac remodeling by fibrous tissue after infarction in rats. J Lab Clin Med 135: 316-323.[CrossRef][Medline]
Thai HM, Van HT, Gaballa MA, Goldman S, and Raya TE (1999) Effects of AT1 receptor blockade after myocardial infarct on myocardial fibrosis, stiffness and contractility. Am J Physiol 276: H873-H880.
This article has been cited by other articles:
|
|
 |
|
  J. Villar, M. I. Arenas, C. M. MacCarthy, M. J. Blanquez, O. M. Tirado, and V. Notario PCPH/ENTPD5 Expression Enhances the Invasiveness of Human Prostate Cancer Cells by a Protein Kinase C{delta} Dependent Mechanism Cancer Res., November 15, 2007; 67(22): 10859 - 10868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. He, A. Dibas, T. Yorio, and G. Prasanna Parallel Signaling Pathways in Endothelin-1-Induced Proliferation of U373MG Astrocytoma Cells Experimental Biology and Medicine, March 1, 2007; 232(3): 370 - 384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Steinberg and M. A. Sussman Cardiac Hypertrophy Served With Protein Kinase C{epsilon}: {delta} Isoform Substitution Available at Additional Cost Circ. Res., April 15, 2005; 96(7): 711 - 713. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
