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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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Northern blot analysis of MMP and TIMP RNAs during rat skin wound healing. Total RNA (10 μg) from normal skin (lanes  1) and skin wounds on days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7), were electrophoresed, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for rat MT1-MMP, GelA, ST3, ST1, GelB, Col3 (A); TIMP1, TIMP2, TIMP3 (B).  Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982).
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Figure 1: Northern blot analysis of MMP and TIMP RNAs during rat skin wound healing. Total RNA (10 μg) from normal skin (lanes 1) and skin wounds on days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7), were electrophoresed, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for rat MT1-MMP, GelA, ST3, ST1, GelB, Col3 (A); TIMP1, TIMP2, TIMP3 (B). Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982).

Mentions: The next step was to determine whether other MMPs were expressed during rat skin wound healing. We constructed a second cDNA library in the Uni-ZAP™XR vector (Stratagene, La Jolla, CA) using RNA prepared from healing wounds on days 3 and 5 after cutaneous incision, which corresponded to the period of concomitant expression of the MMP genes already identified (Fig. 1 A). For screening this second library, a probe was designed in such a way that any known or novel members of the MMP family would be identified. We amplified 3- and 5-d rat skin wound RNA by RT-PCR, using a set of degenerate primers derived from nucleotide sequences corresponding to the highly conserved cysteine-switch and zinc-binding domains of the six rat MMPs known at that time (ST3, GelA, GelB, ST1, ST2, and Col3) (Fig. 2).


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)

Northern blot analysis of MMP and TIMP RNAs during rat skin wound healing. Total RNA (10 μg) from normal skin (lanes  1) and skin wounds on days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7), were electrophoresed, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for rat MT1-MMP, GelA, ST3, ST1, GelB, Col3 (A); TIMP1, TIMP2, TIMP3 (B).  Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139851&req=5

Figure 1: Northern blot analysis of MMP and TIMP RNAs during rat skin wound healing. Total RNA (10 μg) from normal skin (lanes 1) and skin wounds on days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7), were electrophoresed, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for rat MT1-MMP, GelA, ST3, ST1, GelB, Col3 (A); TIMP1, TIMP2, TIMP3 (B). Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982).
Mentions: The next step was to determine whether other MMPs were expressed during rat skin wound healing. We constructed a second cDNA library in the Uni-ZAP™XR vector (Stratagene, La Jolla, CA) using RNA prepared from healing wounds on days 3 and 5 after cutaneous incision, which corresponded to the period of concomitant expression of the MMP genes already identified (Fig. 1 A). For screening this second library, a probe was designed in such a way that any known or novel members of the MMP family would be identified. We amplified 3- and 5-d rat skin wound RNA by RT-PCR, using a set of degenerate primers derived from nucleotide sequences corresponding to the highly conserved cysteine-switch and zinc-binding domains of the six rat MMPs known at that time (ST3, GelA, GelB, ST1, ST2, and Col3) (Fig. 2).

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