TGF-{beta}1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k

Carmen G. Lechuga,1,2 Zamira H. Hernández-Nazara,1,3 José-Alfredo Domínguez Rosales,1,3 Elena R. Morris,4 Ana Rosa Rincón,5 Ana María Rivas-Estilla,1 Andrés Esteban-Gamboa,2 and Marcos Rojkind1,3,6

1Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461; 2Department of Biochemistry, Faculty of Medicine, Universidad Autónoma de Madrid, and Department of Experimental Endocrinology, Hospital Universitario Puerta de Hierro, Madrid, Spain 28029; 3Experimental Pathology and 4Chemistry Sections, Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, DC 20307; 5Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029; and 6Departments of Biochemistry and Molecular Biology and of Pathology, The George Washington University Medical Center, Washington, D.C. 20037

Submitted 17 June 2003 ; accepted in final form 26 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Transforming growth factor-{beta}1 (TGF-{beta}1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-{beta}1 induces a transient elevation of {alpha}1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the {alpha}1(I) collagen mRNA. In the present study, we report that TGF-{beta}1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of {alpha}1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-{beta}1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70S6k. Altogether, our data suggest that regulation of MMP-13 by TGF-{beta}1 is a complex process involving transcriptional and posttranscriptional mechanisms.

fibrosis; fibrogenesis; collagenase; collagen degradation


LIVER FIBROSIS INDUCED IN animals is a reversible process, and resolution can be obtained within 3 to 6 mo on discontinuation of the noxious agent used to induce liver damage. However, liver fibrosis can become irreversible if one prolongs the administration of the injurious agents (38, 46). In contrast with these animal studies, liver cirrhosis in humans is considered to be the endstage of all chronic liver diseases resulting in generalized damage to the tissue. Accordingly, for many years, liver fibrosis was considered to be an irreversible process. Information gathered over the past 15 yr has shown that in some instances, fibrosis in humans can be ameliorated (3, 28, 32, 39, 40). Initial attempts to treat liver fibrosis were directed at key steps required for proline incorporation into collagen hydroxyproline (44). Additional studies focused on posttranslational modifications of collagen, such as the hydroxylation of proline residues and the formation of lysine-derived cross-links (44). More recently, attempts have been made to prevent the activation of hepatic stellate cells (HSC), to interfere with TGF-{beta} signaling, to decrease inflammation, and to prevent oxidative stress (2). However, little work has been performed to enhance the mechanisms involved in collagen degradation. Thus a better understanding of the molecular mechanisms involved in the expression, processing, and regulation of matrix metalloproteinases (MMPs) could result in novel therapies for this disease.

Overall, resolution of liver fibrosis is a complex process requiring discontinuation of the injurious agent and restoration of the proper balance of factors that will result in decreased deposition with enhanced degradation of type I collagen. Of the many MMPs described thus far, only the so-called, interstitial MMPs, such as types 1, 8, and 13 (4, 22, 29, 45, 49, 56), the gelatinase MMP2 (1) and the membrane type I collagenase MT1-MMP (MMP-14) (37) have the potential of degrading type I collagen and therefore in playing a role in matrix remodeling. Other MMPs are not true collagenases, they are gelatinases with a much wider specificity that can cleave collagens containing interruptions in their helical domains, such as type IV and other noncollagenous components of the extracellular matrix (45, 49, 56). Whereas MMP-8 is expressed by neutrophils and is the one with highest selectivity for type I collagen (20, 21), MMP-1 and MMP-13 are produced by many mesenchymal cells (29). In the liver, MMP-1 and MMP-13 are expressed by Kupffer cells and hepatic stellate cells (HSC) (22, 29). These enzymes have greater selectivity for type III and type II compared with type I collagen, respectively (13, 30). However, type II collagen is not present in the liver, and the main collagen in scar tissue is type I. In murine livers, levels of expression of MMP-1 are extremely low and only detectable by RT-PCR (Domínguez-Rosales JA and Rojkind M, unpublished observations). MMP-2 is expressed mainly in HSC/myofibroblasts (5, 29, 51). When this enzyme is free of inhibitors, it digests interstitial collagens with the same efficiency as MMP-1. Moreover, it produces the three-fourths- to one-fourth-length fragments characteristic of the cleavage produced by interstitial collagenases (1). MMP-14 is expressed in all liver cells (29), but its activity against native interstitial collagens is only one-eighth that of MMP-1 (37). Nonetheless, its activity is greatly enhanced in the presence of MMP-2 (37). Overall, these data suggest, although many cells in the liver can produce MMPs, the actual role of each cell type and/or the MMPs produced by them in collagen degradation, and resolution of liver fibrosis remains to be fully elucidated.

TGF-{beta}1, the main fibrogenic cytokine whose role in patients with cirrhosis and in animal-induced liver fibrosis has been clearly established (7, 9), also modulates the expression of several MMPs. Albeit the information is controversial (43, 47, 53, 57), this cytokine may play a key role in extracellular matrix remodeling. Therefore, we have performed experiments to better understand the role of MMP-13 in liver collagen remodeling and the molecular mechanisms involved in its regulation in HSC. We have already reported that HSC are heterogeneous regarding the expression of MMP-2 and MMP-13 mRNAs and that the expression of the latter can be enhanced by coculturing HSC with hepatocytes and by injuring the cultured hepatocytes with galactosamine (48). In the present study, we investigated the effect of TGF-{beta}1 on the temporal expression of {alpha}1(I) collagen and MMP-13 mRNAs in mouse HSC (mHSC). We showed that {alpha}1(I) collagen and MMP-13 mRNAs are reciprocally modulated by TGF-{beta}1. As expected, the decrease in steady-state levels of MMP-13 mRNA occurred at the time when {alpha}1(I) collagen gene was upregulated by the cytokine. However, the subsequent downfall of collagen gene expression, which followed TGF-{beta}1 administration at later time points, resulted in significant upregulation of MMP-13 mRNA. This TGF-{beta}1-mediated effect was posttranscriptionally regulated and required de novo protein synthesis. We further showed that p38MAPK, phosphatidylinositol 3-kinase (PI3-kinase), AKT, and p70S6k kinases are required for TGF-{beta}1-mediated regulation of steady-state levels of MMP-13 mRNA.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Chemicals. SB-203580, U-0126, LY-294002, wortmannin, and rapamycin were purchased from Calbiochem-Novabiochem (San Diego, CA) Transforming growth factor-{beta}1 (TGF-{beta}1) was purchased from Boehringer-Mannheim-Roche (Indianapolis, IN). Actinomycin D, L-{alpha}-phosphatidylinositol, and cycloheximide were purchased from Sigma (St. Louis, MO). The random priming labeling kit was purchased from Amersham International (Arlington Heights, IL). The mouse monoclonal antibody (Clone LIPCO IID1), which recognizes latent and active forms of MMP-13, was purchased from NeoMarkers (Fremont, CA). The following antibodies were purchased from Cell Signaling-New England BioLabs (Beverly, MA): polyclonal (Ser473) phospho-AKT (cat. no. 9217), total AKT (cat. no. 9272), polyclonal antibody to total p70S6k (cat. no. 9202), (Thr389) phospho-p70S6k (cat. no. 9430), total p42/44 (cat. no. 9102), phosphorylated p42/44 (cat. no. 9101), and the kits to determine p38MAPK and p42/44 MAPK activities (cat. nos. 9820 and 9800). The polyclonal antibody against {beta}-tubulin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). A goat anti-type I collagen antibody (cat. no. 1310–01) was obtained from Southern Biotechnology (Birmingham AL).

Cell cultures. All the experiments were performed by using mHSC in their activated phenotype (passages 5–7) isolated from adult B6D2F2 mice as previously described (24). Cells were cultured in MEM (Cellgro; Mediatech, Herndon, VA) supplemented with 10% FBS (HyClone, Logan, UT), nonessential amino acids, kanamycin, and penicillin/streptomycin (Life Technologies, Grand Island, NY). mHSC were maintained in FBS-containing medium for 36 h followed by 12 h of starvation in serum-free medium that contained 0.2% BSA fraction V (Sigma). All incubations were performed in this medium, and the cells were maintained at 37°C in a 5% CO2 incubator. mHSC were incubated with TGF-{beta}1 (8 ng/ml) for the times indicated in each of the figures. For the experiments using protein kinase inhibitors, actinomycin D (10 µg/ml) or cycloheximide (0.3 mM) were added 30 min before the cytokine. Concentrations of MEK 1/2, p38MAPK, PI3-kinase, and p70S6k inhibitors were used as follows: U-0126 (10 µM), SB-203580 (10 µM), LY-294002 (50 µM), wortmannin (200 nM), and rapamycin (50 nM). At these concentrations of inhibitors, kinase activities were inhibited, whereas cell viability was >90%. In some experiments, culture media from three cultures were collected separately, proteins were precipitated with cold acetone, and the presence of MMP-13 and type I collagen was determined by Western blot analysis.

Protein kinase assays. PI3-Kinase signal pathway was analyzed measuring the phosphorylation state of AKT as well as the activity of PI3-kinase. Determination of total and phosphorylated AKT was determined by Western blot analysis using the antibodies described above. PI3-kinase activity was determined after immunoprecipitation of the enzyme with an antibody against p85 regulatory subunit (cat. no. 06–195; Upstate Signaling) and measuring the incorporation of 32PO4 into phosphatidylinositol (8, 50). p38MAPK and p42/44 MAPK activities were determined by immunoprecipitation of the enzyme and measuring the phosphorylation of recombinant ATF-2 and ELK-1, respectively.

RNA extraction and Northern blot analysis. Total RNA was extracted as described (10). For Northern blot analysis, 15 µg of total RNA per lane was electrophoresed on 1% formaldehyde-agarose gels and transferred to Gene Screen membranes (New England Nuclear Life Science Products, Boston, MA). Hybridization was performed as described (15) using [32P]cDNA probes for {alpha}1(I) procollagen, MMP-13, S14 riboprotein, or S18 rRNA. Relative intensity of the signals was determined either by densitrometric analysis of the X-ray films, or by phosphorimaging analysis of the blots. Values are means ± SE of ≥3 experiments, and were corrected for loading differences using S14 riboprotein or rRNA S18 as probes.

Semiquantitative RT-PCR assays. Total RNA extraction was performed as described above; reverse transcription and cDNA amplification were performed as previously described (48) by using a PCR amplification instrument (model 9700; Perkin Elmer, Norwalk, CT). The following primers were used to amplify MMP-13 and S14 riboprotein, respectively: sense 5'-GCCCTATCCCTTGATGCCATT-3', anti-sense 5'-GTGAACCGCAGCGCTCAGTC-3'; and sense 5'-TGGAGACGACGATCAGAAAT-3', anti-sense 5'-TCCTCAATCCGCCCAATCTT-3'. All the reactions contained the two sets of primers. The best condition for proper amplification of both cDNAs was determined experimentally and was found to be 35 cycles using 0.2 µM/0.1 µM concentrations of MMP-13 and S14 primers, respectively (48). Signal intensities were determined by densitometric analysis of ethidium bromide-stained gels and the MMP-13/S14 signal ratio expressed as arbitrary units. Values are means ± SE of ≥3 experiments and are reported as fold change compared with controls.

Run-on transcription assay. Confluent cultures of mHSC maintained as described above were used (see Cell cultures). TGF-{beta}1 was added at a final concentration of 8 ng/ml for 1, 6, and 24 h. Nuclei were isolated and used to determine rates of {alpha}1(I) collagen, MMP-13, and riboprotein S14 transcription as previously described (17).

Western blot analysis. Culture media of three samples were pooled and concentrated by acetone precipitation. Albumin present in the acetone precipitate was removed by affi-gel chromatography (Bio-Rad, Hercules, CA). Aliquots containing 20 µg protein were used for SDS-PAGE as described below. Cells remaining after removing the culture media and three successive washes with cold PBS were lysed (in mM: 20 Tris, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 1 Na3VO4, 1 PMSF, and 1% Triton X-100 plus 1 µg/ml each leupeptin, antipain, chymostatin, anad pepstatin), 20 µg of protein from three cell extracts applied per lane, proteins resolved by SDS-PAGE using 12% gels, transferred to Immobilon-P membranes (Milipore, Bedford, MA), and blocked overnight with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, as previously described (15). Membranes to detect MMP-13 were first incubated for ≥2 h with a 1:500 dilution of the first antibody, followed by several washes with Tris-buffered saline containing 0.1% Tween 20 and then incubated for one additional hour with horseradish peroxidase-conjugated goat anti-mouse (1:3,000; Bio-Rad). To detect total AKT, (Ser473) phospho-AKT, phospho-ATF-2 (Thr71), (Ser383) phospho-Elk-1, total p70S6k, and (Thr389) phospho-p70S6k, membranes were incubated overnight with 1:1,000 dilution of the corresponding primary antibodies, washed 5 times as described, and incubated with a peroxidase-tagged anti-rabbit immunoglobulin (Bio-Rad) (1:3,000). Proteins were visualized by enhanced chemiluminiscence (Renaissance; New England Nuclear Life Science Products). Signal intensities of immunoreactive proteins were determined by densitometry and are the means ± SE of three experiments. To normalize for loading differences, Ponceu S staining (Sigma) of the membranes was performed. In some instances, loading differences were normalized after reprobing the blots and determining the amount of immunoreactive {beta}-tubulin (1:250). The expression of type I collagen was determined by dot blot analysis using an antibody that recognizes the native protein (1/500).

Statistical analysis. Data are expressed as means ± SE of three experiments. Statistical differences between experimental groups were analyzed by Student’s t-test. Pearson correlations coefficients (r) were determined by using Sigma Plot 2002 for Windows version 8.0 (SPSS). The half-life of MMP-13 mRNA was estimated from double-exponential curves. P values ≤ 0.05 were significantly different.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
TGF-{beta}1 has a biphasic effect on MMP-13 mRNA expression. Previous studies (48) from our laboratory demonstrated that {alpha}1(I) collagen and MMP-13 mRNAs are reciprocally modulated in fibroblasts and HSC. Therefore, we investigated whether TGF-{beta}1, a cytokine known to induce {alpha}1(I) collagen mRNA in mHSC, had any effect on MMP-13 mRNA expression. To this end, mHSC were incubated with 8 ng/ml of TGF-{beta}1 as described in MATERIALS AND METHODS, and total RNA was extracted at 0, 6, 12, 24, 48, and 72 h postcytokine administration. As expected, TGF-{beta}1 induced the expression of {alpha}1(I) collagen mRNA in a time-dependent manner, reaching maximal values by 12 h (3.9 ± 1.3-fold; P < 0.05) and then decreasing steadily approaching control values by 72 h (0.63 ± 0.09-fold; P < 0.05) (Fig. 1). mHSC expressed basal levels of MMP-13 mRNA, although levels of this mRNA decreased with time in culture and were 0.53 ± 0.23-fold (P < 0.05) and 0.16 ± 0.06-fold (P < 0.005) by 24 and 48 h, respectively, compared with zero time. After treatment with TGF-{beta}1, the early increase in {alpha}1(I) collagen gene (col1a1) expression was accompanied by a rapid decrease in steady-state levels of MMP-13 mRNA that reached 28.5 ± 7.8% (P < 0.05) and 45 ± 0.19% (P < 0.05) of control values by 6 and 12 h, respectively. However, MMP-13 mRNA expression was upregulated thereafter, reaching by 48 h values that were 10.9 ± 4.3-fold (P < 0.001) above those found in untreated controls and remained elevated for ≤72 h after exposure to TGF-{beta}1 (Fig. 1).



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Fig. 1. Time-course of col1a1 and matrix metalloproteinase-13 (MMP-13) gene expression in mouse hepatic stellate cells (mHSC) treated with transforming growth factor-{beta}1 (TGF-{beta}1) as determined by Northern blot analysis. A: studies were performed with total RNA extracted from control untreated and TGF-{beta}1-treated cells at the time points indicated. Signal intensities were calculated in arbitrary units after densitometric analysis. Because levels of expression in controls vary with time in culture, for each experimental time point, an identical untreated control sample was used. Results were calculated as fold change compared with controls and are means ± SE of 3 experiments after correcting for loading differences using expression of S14 mRNA as an internal control. *P < 0.05 and **P < 0.01 vs. control untreated. B: representative Northern blot.

 
To determine whether changes in MMP-13 mRNA induced by TGF-{beta}1 in mHSC were accompanied by increased MMP-13 protein production, mHSC were treated with TGF-{beta}1 for 48 h, after which time, cells and medium were harvested separately (See MATERIALS AND METHODS). Western blot analysis performed with culture media and cell extracts showed the presence of two immunoreactive bands, possibly corresponding to pro-MMP-13 (~60 kDa) and MMP-13 (~48 kDa), respectively (Fig. 2, A and C). MMP-13 present in cell extracts corresponds to enzyme present inside the cells and/or that associated with the extracellular matrix produced by the cells (48). It is well known that without the use of inhibitors of collagen cross-links, the protein is deposited and remains as part of the cell layer (26). Moreover, because collagenases have a high affinity for collagen, they bind to this protein in vitro and in vivo (35).



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Fig. 2. Western blot analysis of MMP-13 (AC) and type I collagen associated with cells and cell layers in control and TGF-{beta}1-treated mHSC (DF). For these experiments, mHSC were treated with 8 ng/ml of TGF-{beta}1 for 48 h after which time the medium was removed as described in MATERIALS AND METHODS, and cells and associated cell layers were dissolved with lysis buffer and incubated with a 1:500 dilution of a monoclonal MMP-13 antibody followed by a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (see MATERIALS AND METHODS). Proteins were visualized by enhanced chemiluminescence and signal intensities of immunoreactive proteins, were determined by densitometry. A: representative Western blot analysis of MMP-13 obtained from cell-extracts. B: values are means ± SE of 3 experiments after correcting for loading differences using the membrane stained with Ponceu S. C: representative Western blot of MMP-13 from culture media of 3 samples collected 48 h after TGF-{beta}1 administration and concentrated by acetone precipitation. D: representative dot blot of type I collagen present in total cell and cell layer extract. The goat anti-type I collagen antibody was used at a 1:500 dilution. The primary antibody was detected by using a peroxidase-labeled, donkey anti-goat antibody (1:3,000). E: values are means ± SE of ≥3 experiments. F: representative dot blot of type I collagen present in culture media (see MATERIALS AND METHODS). *P < 0.05 and **P < 0.01 vs. untreated controls.

 
Densitometric analysis of the main band obtained from cells treated with TGF-{beta}1 showed a 2.4 ± 0.55-fold (P < 0.01) increase above control values (Fig. 2B). As shown in Fig. 2C, MMP-13 present in culture medium was also increased after incubation with TGF-{beta}1 for 48 h. No attempts were made to quantify total MMP-13 produced by mHSC, because signal obtained from culture media was generated with pooled samples concentrated by acetone precipitation, whereas that from cell extracts corresponds to individual samples. However, it is important to note that most of the enzyme present in the cultures was associated with the cells and/or the extracellular matrix produced by the cells (cell layer). In agreement with the aforementioned finding, collagen produced by HSC was also found associated with the cell layer. As illustrated in Fig. 2D, TGF-{beta}1 increased the expression of type I collagen by 24 h (3.7 ± 1.3-fold; P < 0.05) as determined by a dot blot assay using an antibody that reacts with native type I collagen. As also shown in Fig. 2E, no additional accumulation of collagen was observed at 48 h compared with 24 h. Similar to our findings with MMP-13, small amounts of type I collagen were present in the culture medium (Fig. 2F).

TGF-{beta}1 modulates MMP-13 mRNA expression by transcriptional, posttranscriptional, and de novo protein synthesis-dependent mechanisms. To determine the molecular mechanisms involved in TGF-{beta}1-mediated MMP-13 mRNA modulation, we performed several experiments. We first performed run-on transcription assays with nuclei of mHSC obtained at different time points after TGF-{beta}1 administration. Three assays revealed that MMP-13 transcription was elevated 2.3 ± 0.2 (P < 0.05)-, 2.0 ± 0.3 (P < 0.05)- and 1.3 ± 0.2 [P not significant (NS)]-fold in mHSC treated with TGF-{beta}1 for 1, 6, and 24 h, respectively (Fig. 3). Because gene transcription was increased at the time points in which MMP-13 mRNA were downregulated by TGF-{beta}1, it was important to investigate other possible mechanisms whereby this cytokine modulates the expression of MMP-13 mRNA. Therefore, we studied whether this cytokine had any effect on the half-life of the message. To this end, we determined the effect of actinomycin D, an inhibitor of transcription, on MMP-13 mRNA levels in control and TGF-{beta}1-treated mHSC. Levels of mRNA expression were corrected for loading using S18 rRNA, because actinomycin D treatment induced a rapid degradation of S14 riboprotein. As illustrated in Fig. 4A, the administration of actinomycin D resulted in a rapid degradation of MMP-13 mRNA. Addition of TGF-{beta}1 further enhanced the actinomycin effect. With values obtained from three experiments and using double exponential curves, we estimated the half-life for MMP-13 mRNA in the presence or absence of TGF-{beta}1. The initial decline in MMP-13 mRNA appeared to occur with a half-time of 5–10 min. However, this value should be taken with caution because of the lack of additional time points. Moreover, MMP mRNAs appear to be degraded when the cells are detached. We have already shown that MMP-2 mRNA, which is expressed at high levels in HSC cultured for 24 to 48 h is completely destroyed after detaching the cells (48). As shown in Fig. 4B, the half-life of MMP-13 mRNA in control untreated cells was 62.4 h (r2 = 0.93) and in TGF-{beta}1-treated HSC was 11 h (r2 = 0.97).



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Fig. 3. MMP-13 gene transcription showing run-on transcription assays performed with nuclei obtained from control untreated and TGF-{beta}1-treated mHSC 1, 6, and 24 h after the addition of the cytokine. Signal intensities of autoradiographies were determined by densitometric analysis. Values were corrected for loading differences using signals obtained with S14 as control. Values are expressed as fold increase compared with controls and are means ± SE of ≥3 experiments. *P < 0.05 vs. untreated controls.

 


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Fig. 4. Effect of actinomycin D on MMP-13 mRNA expression in control and TGF-{beta}1-treated mHSC. A: cultures incubated with or without TGF-{beta}1 were treated with 10 µg/ml actinomycin D for the times indicated in the figure, after which time total RNA was extracted for Northern blot analysis. B: half-life (t1/2) of MMP-13 mRNA was estimated from double exponential curves using mean ± SE obtained from 3 experiments.

 
To determine whether de novo protein synthesis was required for TGF-{beta}1-mediated upregulation of MMP-13 mRNA, mHSC were incubated for 24 h with a dose of cycloheximide that inhibits by 95% protein synthesis in mHSC. As shown in Fig. 5, both basal and TGF-{beta}1-dependent upregulation of MMP-13 mRNA was inhibited by cycloheximide. Cell viability was not significantly affected by treatment with cycloheximide (data not shown).



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Fig. 5. Effect of cycloheximide on MMP-13 mRNA expression in control untreated and TGF-{beta}1-treated mHSC. Cells were treated with cycloheximide (0.3 mM) in the absence or presence of TGF-{beta}1 for 24 h after which time total RNA was extracted for Northern blot analysis (see MATERIALS AND METHODS). A: values are means ± SE of ≥3 experiments. *P < 0.05 and **P < 0.01 vs. untreated controls; ***P < 0.001 vs. TGF-{beta}1-treated. B: representative Northern blot.

 
TGF-{beta}1 regulates expression of MMP-13 mRNA by a signaling pathway dependent on the p38MAPK p70S6k and PI3-kinase families of protein kinases. To analyze possible signal transduction pathways involved in TGF-{beta}1-dependent upregulation of MMP-13 mRNA, we first determined whether this cytokine had any effect on p38MAPK activity and whether inhibition of p38MAPK by SB-203580 had any effect on MMP-13 expression. To this end, mHSC treated with or without TGF-{beta}1 were incubated for 24 or 48 h in the presence or absence of inhibitors of the various kinases (vide infra), and the expression of MMP-13 was determined by either Northern blot analysis or RT-PCR as well as by Western blot analysis of the protein. As shown in Fig. 6A, TGF-{beta}1 induced a rapid increase in p38MAPK activity as determined by phosphorylation of recombinant ATF-2. Values increased to 412.6 ± 13.2% (P < 0.05) after 5 min and sharply decreased to below control values by 30 min (29.6 ± 13.6%, P < 0.05). The activity of p38MAPK was also determined after 24 h after the administration of TGF-{beta}1 and was similar to that in control cultures (data not shown). To test whether p38MAPK was involved in TGF-{beta}1-mediated upregulation of MMP-13 mRNA expression, cells treated with the cytokine for 48 h were incubated with the p38MAPK inhibitor SB-203580. As shown in Fig. 6B, although SB-203580 had no effect on basal expression of MMP-13 mRNA, it significantly inhibited TGF-{beta}1-dependent induction (66.5 ± 28.3%, P < 0.05) occurring 48 h postcytokine administration. However, the amount of MMP-13 protein present in the cell culture was not significantly altered by this inhibitor (2.9 ± 0.5 with TGF-{beta}1 vs. 3.8 ± 2.5 with SB-203580 + TGF-{beta}1, P not significant) (Fig. 6C).



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Fig. 6. A: p38 MAPK activity was assessed by immunoprecipitation of the active enzyme with a specific antibody and measuring in vitro phosphorylation of recombinant ATF-2 ({circ}P-ATF-2) (A) (see MATERIALS AND METHODS). Role of p38MAPK on TGF-{beta}-dependent expression of MMP-13 mRNA (B) and protein (C). Cells were incubated with TGF-{beta}1 with or without the kinase inhibitor SB-203580 (10 µM) for the times indicated in the figure. This figure depicts MMP-13 mRNA (B) and protein levels (C) determined by Northern and Western blot analyses 48 h after cytokine administration. Proteins were visualized by enhanced chemiluminiscence and signal intensities determined by densitometric analysis. Values are means ± SE of ≥3 experiments. *P < 0.05 and **P < 0.01 vs. untreated controls; ***P < 0.05 vs. TGF-{beta}1 treated.

 
TGF-{beta}1 also induced a transient elevation in p42/44 MAPK activity, as determined by phosphorylation of ELK-1, that reached its maximum by 5 min (162.6 ± 32.5%, P < 0.05). Values returned to normal levels by 10 min and decreased to 27.6 ± 38.6% (P < 0.05) of controls by 30 min (Fig. 7A). Similar to our findings with p38MAPK, activation of p42/44 MAPK, as determined by its degree of phosphorylation, was similar to that in control cultures at 24 h after the administration of TGF-{beta}1 (Fig. 7B). To investigate whether these changes in protein kinase activity were involved in TGF-{beta}1-induced MMP-13 upregulation, we used the specific MEK 1/2 inhibitor U-0126. As illustrated in Fig. 7B, U-0126 inhibited by 67 ± 9 and 63 ± 7% (P < 0.01) phosphorylation of p42/44 MAPK in control as well as in TGF-{beta}1-treated cells, respectively; however, it had no effect on basal (0.93 ± 0.24 vs. 1.0 ± 0, P = NS) or TGF-{beta}1-induced expression of MMP-13 mRNA (Fig. 8A) (4.1 ± 0.3 vs. 4.2 ± 0.1, P = NS). In contrast with these findings, U-0126 inhibited by 37.8 ± 0.5% (P < 0.05) TGF-{beta}1-induced MMP-13 protein levels (Fig. 8B).



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Fig. 7. Effect of TGF-{beta}1 on p42/44 MAPK activity (A) and on the phosphorylation of p42/44 MAPK (B). A: cells were incubated with TGF-{beta}1 for the times indicated in the figure, and p42/44 MAPK activity was assessed by immunoprecipitation of the active enzyme with a specific antibody and in vitro phosphorylation of recombinant ELK-1 ({circ}P-ELK-1). B: phosphorylation state of p42/44 MAPK was determined by Western blot analysis performed 24 h after cytokine administration. The effect of the MEK 1/2 inhibitor (10 µM U-0126) on p42/44 MAPK phosphorylation was assessed by adding the inhibitor 30 min before incubation with TGF-{beta}1. Values reported are means ± SE of ≥3 experiments. *P < 0.05 and **P < 0.01 vs. untreated controls; ***P < 0.01 vs. TGF-{beta}1 treated.

 


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Fig. 8. Effect of MEK 1/2 inhibitor (U-0126) on MMP-13 mRNA (A) and protein (B) expression in control and TGF-{beta}1-treated HSC. Semiquantitative RT-PCR amplification of MMP-13 and S14 mRNAs expressed by control and TGF-{beta}1-treated mHSC. Amplification was performed by using total RNA extracted from mHSC 48 h after incubation with TGF-{beta}1 in the presence or absence of MEK 1/2 inhibitor U-0126. U-0126 was added to cultured cells 30 min before cytokine administration. The reverse and forward primers used for MMP-13 and S14 amplification are described in MATERIALS AND METHODS. The primer concentration and number of cycles required to obtain linear amplification of the transcripts was determined experimentally. MMP-13 mRNA values represent means ± SE of ≥3 experiments after correcting for levels of expression of S14 transcript. B: HSC were incubated with TGF-{beta}1 for 48 h, and MMP-13 levels were determined by Western blot analysis as described in Western blot analysis. The same blot was stripped and incubated with an anti-tubulin antibody. Proteins were visualized by enhanced chemiluminiscence, and signal intensities were determined by densitometric analysis. MMP-13 values were corrected for levels of tubulin expression and are means ± SE of ≥3 experiments. *P < 0.001 vs. untreated controls; **P < 0.05 vs. TGF-{beta}1-treated.

 
In additional experiments, we investigated the role of the PI3-kinase pathway and p70S6k on TGF-{beta}1-mediated upregulation of MMP-13 mRNA. We first measured total and phosphorylated AKT in extracts of control and TGF-{beta}1-treated mHSC. As shown in Fig. 9A, TGF-{beta}1 decreased levels of phosphorylated AKT (P-AKT) by 10 min post-TGF-{beta}1 administration (52 ± 17%, P < 0.01). Surprisingly, when levels of P-AKT were determined by 24 h, values increased 341 ± 38.8% (P < 0.01) above controls (Fig. 9B). These TGF-{beta}1-dependent events were prevented by wortmannin (Fig. 9B) and by LY-294002 (Fig. 9C). Because AKT can be phosphorylated by PI3-kinase, we measured this enzyme activity in control and TGF-{beta}1-treated cells cultured in the presence or absence of wortmannin. As illustrated in Fig. 10A, PI3-kinase activity was increased by 40 ± 14% (P < 0.05) 24 h after TGF-{beta}1 administration, and this increase in activity was prevented by wortmannin. However, wortmannin had no effect on TGF-{beta}1-dependent upregulation of MMP-13 mRNA (Fig. 10B). Nonetheless, wortmannin inhibited MMP-13 protein expression by 50% but had no effect in its processing, because most of the protein was of 48 kDa [1.37 ± 0.34 vs. 2.69 ± 0.55 (P < 0.05)] (Fig. 11). In contrast with wortmannin, the competitive inhibitor of PI3-kinase (LY-294002) completely prevented AKT phosphorylation (Fig. 9C) and abrogated basal and TGF-{beta}1-mediated expression of MMP-13 mRNA (0.04 ± 0.04-fold, P < 0.001 and 0.35 ± 0.17-fold, P < 0.05 compared with controls, respectively) (Fig. 12). On the other hand, LY-294002 enhanced the accumulation of MMP-13 protein in controls (1.81 ± 0.1) but prevented its upregulation by TGF-{beta}1 (1.37 ± 0.34). In addition, LY-294002 inhibited MMP-13 protein processing by 90 ± 5, (P < 0.001) and 84 ± 16% (P < 0.001), respectively. As shown in Fig. 11, the immunoreactive protein extracted from cells and cell layers was larger in molecular weight (~60,000).



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Fig. 9. Cellular levels of total and phosphorylated AKT ({circ}P-AKT) (A and B) in mHSC incubated with or without TGF-{beta}1 in the presence or absence of 200 nM wortmannin (Wort; B) or 50 µM LY-294002 (C). Cells were incubated with TGF-{beta}1 for the times indicated in A or for 24 h as in B and C, and levels of total AKT and {circ}P-AKT were determined by Western blot analysis. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors were added 30 min before incubation with the cytokine. Proteins were visualized by enhanced chemiluminiscence, and signal intensities were determined by densitometric analysis. Values are means ± SE of ≥3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated controls; ****P < 0.001 vs. TGF-{beta}1 treated.

 


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Fig. 10. PI3-kinase activity (A) and MMP-13 mRNA levels (B) in mHSC were incubated with or without TGF-{beta}1 for 24 h (A) or 48 h (B) in the presence or absence of wortmannin. A: PI3-kinase activity was determined after immunoprecipitation of the enzyme and measuring incorporation of 32PO4 into phosphatidylinositol. B: MMP-13 mRNA expression was determined by Northern blot analysis using the expression of S14 mRNA to correct for loading differences. Levels of MMP-13 mRNA expression were determined as described (Fig. 1). Values of PI3-kinase activity and MMP-13 mRNA levels are means ± SE of ≥3 experiments. *P < 0.05 and ***P < 0.01 vs. untreated controls; **P < 0.05 vs. TGF-{beta}1-treated.

 


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Fig. 11. Comparative analysis of the effects of 200 nM wortmannin and 50 µM LY-294002 on the expression of MMP-13 protein by mHSC treated with TGF-{beta}1 for 48 h. The PI3-kinase inhibitors were added 30 min before incubation with the cytokine. Proteins were visualized by enhanced chemiluminiscence, and signal intensities were determined by densitometric analysis. The same blot was stripped and incubated with an anti-tubulin antibody. Results are means ± SE of ≥3 experiments. *P < 0.05 vs. untreated controls; **P < 0.05 vs. TGF-{beta}1 treated.

 


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Fig. 12. Semiquantitative RT-PCR amplification of MMP-13 and S14 mRNAs expressed by control and TGF-{beta}1-treated mHSC. Amplification was performed by using total RNA extracted from mHSC 48 h after incubation with TGF-{beta}1 in the presence or absence of LY-294002. LY-294002 was added 30 min before TGF-{beta}1 administration. The reverse and forward primers used for MMP-13 and S14 amplification are described in MATERIALS AND METHODS. The primer concentration and number of cycles required to obtain linear amplification of the transcripts was determined experimentally. MMP-13 mRNA values represent means ± SE of ≥3 experiments after correcting for levels of expression of S14 transcript. *P vs. TGF-{beta}1-treated cells; ***P < 0.001 vs. untreated controls.

 
Levels of phosphorylated p70S6k were transiently elevated by 5 min after TGF-{beta}1 administration and decreased to values close to 50% of controls by 30 min (0.55 ± 0.19; P < 0.05) (Fig. 13A). However, in accordance with our findings, namely the increased levels of phosphorylated AKT induced by TGF-{beta}1, phosphorylated p70S6k values were also significantly increased by the cytokine by 24 h (214.2 ± 72.8%, P < 0.05) (see Fig. 13B). Rapamycin, an inhibitor of the p70S6k pathway decreased basal levels of phosphorylated p70S6k and abrogated the TGF-{beta}1-dependent effect (2.96 ± 3.5 and 2.14 ± 3.4%, P < 0.001 compared with control). (Fig. 13B). In agreement with these findings, rapamycin abrogated TGF-{beta}1-dependent upregulation of MMP-13 mRNA (1.1 ± 0.22 vs. 2.83 ± 1.1%; P < 0.05), and prevented cytokine-mediated upregulation of MMP-13 protein associated with the cell layer (0.99 ± 0.1 vs. 1.8 ± 0.3%, P < 0.05) (see Fig. 13, C and D).



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Fig. 13. Effect of TGF-{beta}1 and of rapamycin on cellular concentration of p70S6k and phosphorylated p70S6k ({circ}P-p70S6k; A and B), and on the expression of MMP-13 mRNA (24 h) (C) and protein (48 h) (D) in control untreated and TGF-{beta}1-treated mHSC. Cells were incubated with or without TGF-{beta}1 in the presence or absence of 50 nM rapamycin for the times indicated in A, 24 h in B and C, or 48 h in D. The p70S6k inhibitor was added 30 min before incubation with the cytokine. Levels of total p70S6k, phosphorylated p70S6k (p-p70S6k) and MMP-13 protein were determined by Western blot analysis. Proteins were visualized by enhanced chemiluminiscence, and signal intensities were determined by densitometric analysis. D: levels of MMP-13 protein were corrected for loading differences using tubulin levels as a control. C: MMP-13 mRNA expression was determined by Northern blot analysis using the expression of S14 mRNA to correct for loading differences. Values are means ± SE of ≥3 experiments. *P < 0.05 vs. untreated controls; **P < 0.05 and ***P < 0.001 vs. TGF-{beta}1-treated.

 

    DISCUSSION
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Liver fibrosis results from an imbalance between the amount of extracellular matrix components produced (namely type I and type III collagens) and deposited into the extracellular space and that degraded by MMPs. Whereas collagen deposition depends primarily on the amount of protein produced and that degraded intracellularly, removal of deposited collagen depends on the expression of pro-MMPs, their activation, and the presence of their tissue inhibitors (TIMPs) (45). There are additional factors, however, that may hamper collagen degradation. These include among others, the presence of inter- and intramolecular cross-links and the thickness of the collagen bundles. Newly synthesized collagen is degraded more rapidly, and thick collagen bundles containing cross-linked collagen are degraded more slowly. Indeed, studies performed in vivo in rats treated for 7 wk with CCl4 have revealed that although the amount of collagen measured by hydroxyproline content remains elevated for 30 days after discontinuation of CCl4 treatment, the half-life of type I and type III collagens was reduced by 50% (45). These unexpected results suggested that mainly recently produced collagen was being removed efficiently, whereas old, cross-linked collagen was being digested at a slower pace. These and other experiments raise some questions regarding the role of MMPs in collagen removal, the cells that produce them, and the role of TIMPs in preventing collagen degradation. Regardless of these findings, different cell types of the liver express one or more of the MMPs, and these could play a role in remodeling of the loose connective tissue matrix of the liver and/or in resolving the fibrotic process when the injurious stimulus has ceased.

Studies performed in rats with CCl4-induced liver cirrhosis have shown that during the time-course of the induction of liver fibrosis, the expression of MMP-13 mRNA that is elevated at early time points decreases, and it is very low by 12 wk of treatment. However, during the recovery phase, steady-state levels of MMP-13 mRNA increase in some but not all HSC (55). It was shown that ~50% of the cells expressing MMP-13 transcripts stained positive with antibodies to {alpha}-smooth muscle actin, suggesting that both activated and nonactivated HSC express MMP-13. In other studies (25), resolution of CCl4-induced hepatic fibrosis was accompanied by an increase in collagenolytic activity that occurred at the same time as the myofibroblasts within the fibrous septa were undergoing apoptosis. These findings further indicate that nonactivated HSC may play a role in expressing MMP-13 during the recovery phase in this animal model. However, the expression of MMP-13 mRNA, which was 20% above controls in the cirrhotic animals, did not change during resolution. Taking together these and other findings (48), we speculate that regardless of whether they are activated or not, HSC produce MMP-13 and that terminal differentiated myofibroblasts do not play a role in resolution of liver fibrosis.

Studies pertaining to the molecular mechanisms that control MMP-13 gene expression have shown that although the coding sequences for rat and human MMP-13 genes are very similar (86%), regulation of the protein differs in rat and humans. Expression of human MMP-13 in human cells is silenced by an alternative spliced product of the T cell-restricted intracellular antigen, which is not expressed in rats. This event is controlled by the 3'-untranslated region of this gene (57). Overall, these findings indicate the need of further studies to unravel the molecular mechanisms involved in MMP-13 gene expression and detect possible sites for pharmacological intervention.

Studies presented in the present study show the complexity of MMP-13 regulation in mouse HSC. Although the transcription of the MMP-13 gene in mouse HSC treated with TGF-{beta}1 is increased by 2.5-fold, nonetheless, the expression of its mRNA is decreased due to a fivefold drop in half-life. TGF-{beta}1 is one of the main fibrogenic cytokines whose expression has been associated with increased collagen deposition in the liver (7, 9). This cytokine orchestrates multiple events, including the removal of transcriptional repressor complexes (33, 34) and the formation of transcriptional activator complexes that upregulate the expression of multiple genes, including those encoding type I collagen (6, 16). TGF-{beta}1 is expressed by HSC, and these cells respond to this cytokine with increased production of type I collagen by a mechanism involving the formation of H2O2 and the activation of p38MAPK (15, 54). TGF-{beta}1 alters the balance of collagen synthesis and degradation by enhancing expression of the type I collagen genes and inhibiting collagen degradation via the induction of TIMP-1 (6). TGF-{beta}1 also plays a role in MMP expression. However, results obtained with other cell types are controversial because of conflicting reports indicating that this cytokine can either induce or inhibit various MMPs (43, 47, 53). HSC produce matrix-degrading enzymes and, as indicated above, also produce various collagens types. Therefore, it is difficult to ascertain whether the same cell produces simultaneously enzymes (MMPs) and substrate (collagen), whether the expression of the former precludes the production of the latter, whether different populations of cells produce one or the other, and/or whether distinct routes of secretion of collagen and MMPs allow the same cell to produce and secrete both. As a first step toward addressing some of these issues, we took advantage of rat HSC lines and clones isolated in our laboratory, which have an activated phenotype and differ significantly in their capacity to express mRNAs encoding various extracellular matrix components, and MMPs involved in their degradation (18, 19, 48). We demonstrated that cells expressing high steady-state levels of {alpha}1(I) collagen mRNA do not express MMP-2 and -13. Thus we postulated that {alpha}1(I) collagen and MMP-13 mRNAs are reciprocally modulated in HSC (48). In the present study, we present additional evidence to support the reciprocal modulation of {alpha}1(I) collagen and MMP-13 mRNAs. We demonstrated that TGF-{beta}1-elicited {alpha}1(I) collagen mRNA upregulation is accompanied by downregulation of MMP-13 at early time points in postcytokine administration, whereas at late time points, {alpha}1(I) collagen mRNA levels decrease and those of MMP-13 increase. Our findings also indicate that MMP-13 modulation by TGF-{beta}1 is complex and recognizes opposing posttranscriptional and transcriptional mechanisms. TGF-{beta}1 induces the transcription of MMP-13 by 1 h and sustains it for several hours. By 24-h post-TGF-{beta}1 administration, transcription of MMP-13 is still 30% above controls. This occurs at a time in which TGF-{beta}1 is known to induce {alpha}1(I) collagen gene transcription in HSC (15). Thus our findings suggest that both col1a1 and MMP-13 are being transcribed at the same time. However, from these findings, we cannot determine whether the two mRNAs are transcribed simultaneously within the same cell. Because of the increased transcription of MMP-13 induced by TGF-{beta}1, we expected to find increased levels of its corresponding mRNA shortly after cytokine administration. Surprisingly, MMP-13 mRNA declined quite rapidly within the first 6 h. This decline in mRNA was the result of a rapid degradation of the message. As shown in Fig. 4, TGF-{beta}1 decreased the half-life of MMP-13 mRNA from 64 to 11 h. On the other hand, upregulation of MMP-13 mRNA by TGF-{beta}1 occurring at late time points is dependent on de novo protein synthesis as demonstrated by the inhibition in basal expression and abrogation of the cytokine-mediated induction of MMP-13 mRNA by cycloheximide. MMP-13 produced by HSC is mostly associated with the cell layer and in control untreated cells, there is a 41.5-to-58.5 ratio of proMMP-13 and MMP-13 as determined by densitometric analysis of the bands corresponding to 60 and 48 kDa. In this regard, it is noteworthy to mention that TGF-{beta}1 not only induced the expression of MMP-13 but appeared to enhance its processing, because most of the protein in the cytokine-treated cells had a molecular mass of 48 kDa. Thus TGF-{beta}1 may be inducing the expression of members of the plasminogen activation cascade (14, 52), key elements involved in processing of MMPs. Altogether, these findings suggest that TGF-{beta}1-mediated regulation of MMP-13 expression is quite complex. MMP-13 mRNA is transcriptionally regulated by a de novo protein synthesis-dependent mechanism, but mRNA levels reflect the balance between gene transcription and mRNA degradation. This TGF-{beta}1-mediated effect on MMP-13 mRNA degradation appeared to be selective, because the {alpha}1(I) collagen mRNA increased, whereas the levels of MMP-13 decreased and vice versa.

Previous studies (54) pertaining to the role of TGF-{beta}1 and TNF-{alpha} on collagen gene expression by HSC revealed that p38MAPK is a key kinase involved in its regulation. Whereas TGF-{beta}1 induced {alpha}1(I) collagen gene expression and increased the phosphorylation of p38MAPK, TNF-{alpha} and the specific inhibitor of p38MAPK, SB-203580, decreased p38MAPK phosphorylation and col1a1 gene expression (54). In the present study, we demonstrated that p38MAPK is also involved in TGF-{beta}1-mediated upregulation of MMP-13 mRNA. We showed that SB-203580 had no effect on basal expression of MMP-13 mRNA but abrogated the TGF-{beta}1-mediated response. Thus on the basis of these findings, it appears that p38MAPK is involved in type I collagen and MMP-13 mRNA modulation. These results are similar to findings of others in which the TGF-{beta}1-dependent expression of MMP-13 mRNA in various cell types is p38MAPK dependent (27, 31, 41, 42). Altogether, these findings suggest that p38MAPK is important in the regulation of col1a1 and MMP-13 mRNA expression. However, it does not seem to be a key player in explaining the mechanisms involved in the reciprocal modulation of the two genes. In contrast with these findings, p42/44 MAPK is not involved in TGF-{beta}1-dependent upregulation of MMP-13 mRNA. The inhibition of MEK 1/2 by U-0126 did not prevent MMP-13 mRNA upregulation by the cytokine. Nonetheless, p42/44 MAPK may be involved in posttranscriptional regulation, because protein levels were decreased in the presence of the inhibitor. In the present study, we also showed that inhibition of PI3-kinase and AKT phosphorylation by LY-294002 prevented TGF-{beta}1-mediated expression of MMP-13 protein and mRNA. In addition, it blocked its processing from procollagenase to collagenase. Although the mechanism whereby LY-294002 exerts this effect is unknown, it could be associated with changes in the expression of the proteolytic cascade (or the inhibitors) that modulate the activation of MMPs. In contrast with these results, although wortmannin blocked TGF-{beta}1-induced AKT phosphorylation as well as PI3-kinase activity, it had no effect on MMP-13 mRNA expression, but it prevented protein production without affecting processing. Wortmannin has been widely used as a specific inhibitor of the PI3-kinase signaling pathway; however, some of its effects are concentration-dependent. It has been demonstrated that concentrations of this inhibitor >100 nM inhibit other kinases such as some isoforms of PI4-kinase (36), phospholipase A2 (12), or p42/44 MAPK (8, 11, 23). On the other hand, LY-294002 is not free of side reactions. Inhibition of AKT activation by LY-294002 has been shown to increase Raf and ERK (p42/44 MAPK) activities in HEK-293 and MCF-7 cells (58) and in culture, rat hepatocytes (8). Therefore, additional experiments using expressing dominant-negative forms of these kinases may need to be performed to help in establishing the actual role of the PI3-kinase pathway in MMP-13 expression. In the present study, we also demonstrated that inhibition of p70S6k phosphorylation by rapamycin abrogated TGF-{beta}1-mediated expression of MMP-13 mRNA. This effect was specific, because the expression of {alpha}1(I) collagen mRNA was not affected by rapamycin (data not shown). The mechanism whereby p70S6K is involved in TGF-{beta}1-mediated upregulation of MMP-13 mRNA remains to be investigated.

In summary, our findings demonstrate the complexity of the regulatory mechanisms involved in MMP-13 expression. We showed that mRNA levels do not exactly match protein levels and that inhibitors of protein kinase pathways can affect either mRNA levels, protein levels, or both. Whereas inhibition of p38MAPK by SB-203580 abrogated TGF-{beta}1 induction of MMP-13 mRNA, protein levels appeared to be unaffected. On the other hand, MEK 1/2 inhibitors had no effect on mRNA expression but decreased protein levels. Finally, inhibition of PI3-kinase pathway by LY-294002 resulted in complete inhibition of MMP-13 mRNA expression, a decrease in MMP-13 protein levels, and a complete inhibition in processing of the immunoreactive latent protein of 60 kDa. In conclusion, our findings indicate that TGF-{beta}1 modulates the expression of MMP-13 by regulating the transcription of the gene, the half-life of its mRNA, and the translation of the protein and its activation.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 DISCLAIMER
 REFERENCES
 
This work was supported, in part, with National Institutes of Health Grants RO1-AA-09231 and RO1-AA-10541 (to M. Rojkind). C. G. Lechuga was supported, in part, by a grant from the Ministerio de Educación y Cultura de España.


    DISCLAIMER
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 ABSTRACT
 MATERIALS AND METHODS
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The opinions or assertions contained herein are the private views of the Authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.


    ACKNOWLEDGMENTS
 
The helpful discussions with Dr. Allan Wolkoff (Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY) pertaining to the calculation and interpretation of MMP-13 mRNA half-life is greatly acknowledged.

Present addresses: M. Rojkind, Department of Biochemistry and Molecular Biology and of Pathology, The George Washington University Medical Center, Washington, D.C. 20037; A. M. Rivas-Estilla, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Nuevo Leon, Monterrey, NL, 64460 México; and Z. H. Hernández-Nazara and J. A. D. Rosales, Departamento de Fisiología, Universidad de Guadalajara, Servicio de Biología Molecular en Medicina, Hospital Civil de Belén, Jalisco, 44280 México; and C. G. Lechuga, Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, 28029 España.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Rojkind, Research Professor of Biochemistry and Molecular Biology and of Pathology, The George Washington Univ. Medical Center, Ross Hall 522A, 2300 I St., NW, Washington, D.C. 20037 (E-mail: bcmmmr{at}gwumc.edu)

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.


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 DISCUSSION
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 REFERENCES
 

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