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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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Gelatinolytic activities during rat skin wound  healing. Tissues were microdissected from cryostat sections, incubated with SDS  sample buffer, and subjected  to gelatin zymography. (A)  Normal skin containing both  epidermis and dermis (lane  1), and healing skin wounds  at days 1, 3, 5, 7, 10, and 14  after cutaneous incision  (lanes 2–7). (B) Normal skin  adjacent to the wound tissue  and containing both epidermis and dermis (N) (lane 1),  and granulation tissue (G)  (lanes 2 and 4) or proliferative epithelial layer (E) (lanes 3 and 5) of healing skin wounds at days 3 (lanes 2 and 3) and 5 (lanes 4 and 5)  after cutaneous incision. In A and B, proteins extracted from ∼1 mg of fresh tissue were analyzed in each lane.
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Figure 6: Gelatinolytic activities during rat skin wound healing. Tissues were microdissected from cryostat sections, incubated with SDS sample buffer, and subjected to gelatin zymography. (A) Normal skin containing both epidermis and dermis (lane 1), and healing skin wounds at days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7). (B) Normal skin adjacent to the wound tissue and containing both epidermis and dermis (N) (lane 1), and granulation tissue (G) (lanes 2 and 4) or proliferative epithelial layer (E) (lanes 3 and 5) of healing skin wounds at days 3 (lanes 2 and 3) and 5 (lanes 4 and 5) after cutaneous incision. In A and B, proteins extracted from ∼1 mg of fresh tissue were analyzed in each lane.

Mentions: To directly evaluate pro-GelA activation during rat skin wound healing, we analyzed microdissected tissue samples from frozen sections by gelatin zymography. These samples were collected at different times after cutaneous incision. In wound tissues containing both the stroma and epithelium, protein species corresponding to pro-GelA/GelA were detected from days 1–14, and pro-GelB or pro-GelB/ GelB were detected from days 1–7 (Fig. 6 A). The level of pro-GelA was maximal on day 5, when the granulation tissue was most active, and pro-GelB expression was elevated all through from days 1–5. However, the mature form of GelB was only detected on days 1 and 3, while the mature form of GelA was observed, along with its corresponding pro-form, in all the tissue samples examined (Fig. 6). When the granulation tissue and the proliferative epithelial cell layer from healing wounds on days 3 and 5 were analyzed separately, pro-GelA/GelA was almost exclusively detected in the granulation tissue (Fig. 6 B). ProGelB/GelB was also predominantly detected in the granulation tissue, with only low levels associated with the proliferative epithelial cell layer. These observations indicate that the wound stroma is the predominant site of action for both GelA and GelB during rat skin wound healing.


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)

Gelatinolytic activities during rat skin wound  healing. Tissues were microdissected from cryostat sections, incubated with SDS  sample buffer, and subjected  to gelatin zymography. (A)  Normal skin containing both  epidermis and dermis (lane  1), and healing skin wounds  at days 1, 3, 5, 7, 10, and 14  after cutaneous incision  (lanes 2–7). (B) Normal skin  adjacent to the wound tissue  and containing both epidermis and dermis (N) (lane 1),  and granulation tissue (G)  (lanes 2 and 4) or proliferative epithelial layer (E) (lanes 3 and 5) of healing skin wounds at days 3 (lanes 2 and 3) and 5 (lanes 4 and 5)  after cutaneous incision. In A and B, proteins extracted from ∼1 mg of fresh tissue were analyzed in each lane.
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Figure 6: Gelatinolytic activities during rat skin wound healing. Tissues were microdissected from cryostat sections, incubated with SDS sample buffer, and subjected to gelatin zymography. (A) Normal skin containing both epidermis and dermis (lane 1), and healing skin wounds at days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2–7). (B) Normal skin adjacent to the wound tissue and containing both epidermis and dermis (N) (lane 1), and granulation tissue (G) (lanes 2 and 4) or proliferative epithelial layer (E) (lanes 3 and 5) of healing skin wounds at days 3 (lanes 2 and 3) and 5 (lanes 4 and 5) after cutaneous incision. In A and B, proteins extracted from ∼1 mg of fresh tissue were analyzed in each lane.
Mentions: To directly evaluate pro-GelA activation during rat skin wound healing, we analyzed microdissected tissue samples from frozen sections by gelatin zymography. These samples were collected at different times after cutaneous incision. In wound tissues containing both the stroma and epithelium, protein species corresponding to pro-GelA/GelA were detected from days 1–14, and pro-GelB or pro-GelB/ GelB were detected from days 1–7 (Fig. 6 A). The level of pro-GelA was maximal on day 5, when the granulation tissue was most active, and pro-GelB expression was elevated all through from days 1–5. However, the mature form of GelB was only detected on days 1 and 3, while the mature form of GelA was observed, along with its corresponding pro-form, in all the tissue samples examined (Fig. 6). When the granulation tissue and the proliferative epithelial cell layer from healing wounds on days 3 and 5 were analyzed separately, pro-GelA/GelA was almost exclusively detected in the granulation tissue (Fig. 6 B). ProGelB/GelB was also predominantly detected in the granulation tissue, with only low levels associated with the proliferative epithelial cell layer. These observations indicate that the wound stroma is the predominant site of action for both GelA and GelB during rat skin wound healing.

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