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Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.

Okada A, Tomasetto C, Lutz Y, Bellocq JP, Rio MC, Basset P - J. Cell Biol. (1997)

Bottom Line: Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T.Shinagawa, E.Seiki. 1994.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, C.U. de Strasbourg, France.

ABSTRACT
Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.

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MT1-MMP gene expression in rat fibroblasts and proGelA activation in the presence of Con A. (A) Northern blot  analysis. 10 μg of total RNA obtained from RAT1 (lanes 1 and 2)  or FR3T3 (lanes 3 and 4) rat fibroblasts cultured in the absence  (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml)  were used in each lane. Blots were hybridized with 32P-labeled  rat MT1-MMP and GelA cDNA probes. Blots were reprobed  with the 36B4 cDNA used as a loading control (Masiakowski et al.,  1982). (B) Western blot analysis. Crude cell membrane extracts  corresponding to half a culture plate of subconfluent RAT1 (lanes  1 and 2) or FR3T3 (lanes 3 and 4) fibroblasts cultured in the  absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50  μg/ml) were analyzed. Proteins were detected using anti-rat  MT1-MMP mouse monoclonal antibody, 1MMP-1C1. (C) Gelatin zymography. 20 μl of medium conditioned by COS-1 cells  transiently transfected with pTL1GelA (lane 1), or pTL1GelA  and pTL1MT1-MMP (lane 2), and 20 μl of medium conditioned  by RAT1 (lanes 3 and 4) and FR3T3 (lanes 5 and 6) rat fibroblasts cultured in the absence (lanes 3 and 5) or presence (lanes 4  and 6) of Con A (50 μg/ml), were used.
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Figure 5: MT1-MMP gene expression in rat fibroblasts and proGelA activation in the presence of Con A. (A) Northern blot analysis. 10 μg of total RNA obtained from RAT1 (lanes 1 and 2) or FR3T3 (lanes 3 and 4) rat fibroblasts cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml) were used in each lane. Blots were hybridized with 32P-labeled rat MT1-MMP and GelA cDNA probes. Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982). (B) Western blot analysis. Crude cell membrane extracts corresponding to half a culture plate of subconfluent RAT1 (lanes 1 and 2) or FR3T3 (lanes 3 and 4) fibroblasts cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml) were analyzed. Proteins were detected using anti-rat MT1-MMP mouse monoclonal antibody, 1MMP-1C1. (C) Gelatin zymography. 20 μl of medium conditioned by COS-1 cells transiently transfected with pTL1GelA (lane 1), or pTL1GelA and pTL1MT1-MMP (lane 2), and 20 μl of medium conditioned by RAT1 (lanes 3 and 4) and FR3T3 (lanes 5 and 6) rat fibroblasts cultured in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of Con A (50 μg/ml), were used.

Mentions: During rat skin wound healing, MT1-MMP and GelA transcripts were specifically found in wound stromal cells (Fig. 3, B, C, I, and J). This finding prompted us to examine MT1-MMP and GelA gene expression in two different rat fibroblast cell lines, RAT1 and FR3T3. Both cell lines were found to express high levels of MT1-MMP and GelA transcripts (Fig. 5 A, lanes 1 and 3). However, the MT1-MMP protein could not be detected either in crude cell membrane extracts or in cytosolic fractions by Western blot analysis (Fig. 5 B, lanes 1 and 3, and data not shown). Consistently, media conditioned by these cells contained mainly pro-GelA, as shown by gelatin zymography (Fig. 5 C, lanes 3 and 5). Since pro-GelA activation has been demonstrated to be induced by Con A treatment in human fibroblasts (Overall and Sodek, 1990), we then examined whether treatment with con A of these rat fibroblasts was associated with pro-GelA activation. Although both cell lines stimulated by Con A showed only a weak increase in MT1-MMP and GelA transcript levels (Fig. 5 A, lanes 2 and 4), the MT1-MMP protein could now be easily detected by Western blot analysis of crude cell membrane fractions prepared from the Con A–treated cells (Fig. 5 B, lanes 2 and 4). Furthermore, as shown by gelatin zymography, the mature form of GelA was found to be the major GelA species represented in media conditioned by Con A–treated cells (Fig. 5 C, lanes 4 and 6).


Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.

Okada A, Tomasetto C, Lutz Y, Bellocq JP, Rio MC, Basset P - J. Cell Biol. (1997)

MT1-MMP gene expression in rat fibroblasts and proGelA activation in the presence of Con A. (A) Northern blot  analysis. 10 μg of total RNA obtained from RAT1 (lanes 1 and 2)  or FR3T3 (lanes 3 and 4) rat fibroblasts cultured in the absence  (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml)  were used in each lane. Blots were hybridized with 32P-labeled  rat MT1-MMP and GelA cDNA probes. Blots were reprobed  with the 36B4 cDNA used as a loading control (Masiakowski et al.,  1982). (B) Western blot analysis. Crude cell membrane extracts  corresponding to half a culture plate of subconfluent RAT1 (lanes  1 and 2) or FR3T3 (lanes 3 and 4) fibroblasts cultured in the  absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50  μg/ml) were analyzed. Proteins were detected using anti-rat  MT1-MMP mouse monoclonal antibody, 1MMP-1C1. (C) Gelatin zymography. 20 μl of medium conditioned by COS-1 cells  transiently transfected with pTL1GelA (lane 1), or pTL1GelA  and pTL1MT1-MMP (lane 2), and 20 μl of medium conditioned  by RAT1 (lanes 3 and 4) and FR3T3 (lanes 5 and 6) rat fibroblasts cultured in the absence (lanes 3 and 5) or presence (lanes 4  and 6) of Con A (50 μg/ml), were used.
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Figure 5: MT1-MMP gene expression in rat fibroblasts and proGelA activation in the presence of Con A. (A) Northern blot analysis. 10 μg of total RNA obtained from RAT1 (lanes 1 and 2) or FR3T3 (lanes 3 and 4) rat fibroblasts cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml) were used in each lane. Blots were hybridized with 32P-labeled rat MT1-MMP and GelA cDNA probes. Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982). (B) Western blot analysis. Crude cell membrane extracts corresponding to half a culture plate of subconfluent RAT1 (lanes 1 and 2) or FR3T3 (lanes 3 and 4) fibroblasts cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 μg/ml) were analyzed. Proteins were detected using anti-rat MT1-MMP mouse monoclonal antibody, 1MMP-1C1. (C) Gelatin zymography. 20 μl of medium conditioned by COS-1 cells transiently transfected with pTL1GelA (lane 1), or pTL1GelA and pTL1MT1-MMP (lane 2), and 20 μl of medium conditioned by RAT1 (lanes 3 and 4) and FR3T3 (lanes 5 and 6) rat fibroblasts cultured in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of Con A (50 μg/ml), were used.
Mentions: During rat skin wound healing, MT1-MMP and GelA transcripts were specifically found in wound stromal cells (Fig. 3, B, C, I, and J). This finding prompted us to examine MT1-MMP and GelA gene expression in two different rat fibroblast cell lines, RAT1 and FR3T3. Both cell lines were found to express high levels of MT1-MMP and GelA transcripts (Fig. 5 A, lanes 1 and 3). However, the MT1-MMP protein could not be detected either in crude cell membrane extracts or in cytosolic fractions by Western blot analysis (Fig. 5 B, lanes 1 and 3, and data not shown). Consistently, media conditioned by these cells contained mainly pro-GelA, as shown by gelatin zymography (Fig. 5 C, lanes 3 and 5). Since pro-GelA activation has been demonstrated to be induced by Con A treatment in human fibroblasts (Overall and Sodek, 1990), we then examined whether treatment with con A of these rat fibroblasts was associated with pro-GelA activation. Although both cell lines stimulated by Con A showed only a weak increase in MT1-MMP and GelA transcript levels (Fig. 5 A, lanes 2 and 4), the MT1-MMP protein could now be easily detected by Western blot analysis of crude cell membrane fractions prepared from the Con A–treated cells (Fig. 5 B, lanes 2 and 4). Furthermore, as shown by gelatin zymography, the mature form of GelA was found to be the major GelA species represented in media conditioned by Con A–treated cells (Fig. 5 C, lanes 4 and 6).

Bottom Line: Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T.Shinagawa, E.Seiki. 1994.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, C.U. de Strasbourg, France.

ABSTRACT
Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.

Show MeSH
Related in: MedlinePlus