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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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In situ hybridization of rat MMP RNAs in cutaneous wounds. Serial frozen sections of rat skin wounds on day 3 (A–G), or day  5 (H–N) after cutaneous incision, were hybridized with 35S-labeled antisense RNA probes derived from cDNA templates for rat MT1MMP (B and I), GelA (C and J), ST3 (D and K), ST1 (E and L), GelB (F and M), Col3 (G and N), or stained with hematoxylin and eosin  (A and H). Note that the hair follicle which is observed on sections B and C, is only partially visible on  A (arrows). In D and E, arrows  indicate transcripts expressed in stromal cells surrounding a hair follicle and juxtaposed to the proliferative epithelial cell layer, respectively. No signal above background could be detected with the corresponding 35S-labeled sense RNA probes (data not shown). gt, granulation tissue; e, epithelial layer; hf, hair follicle; s, scar tissue. Bar, 100 μm.
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Figure 3: In situ hybridization of rat MMP RNAs in cutaneous wounds. Serial frozen sections of rat skin wounds on day 3 (A–G), or day 5 (H–N) after cutaneous incision, were hybridized with 35S-labeled antisense RNA probes derived from cDNA templates for rat MT1MMP (B and I), GelA (C and J), ST3 (D and K), ST1 (E and L), GelB (F and M), Col3 (G and N), or stained with hematoxylin and eosin (A and H). Note that the hair follicle which is observed on sections B and C, is only partially visible on A (arrows). In D and E, arrows indicate transcripts expressed in stromal cells surrounding a hair follicle and juxtaposed to the proliferative epithelial cell layer, respectively. No signal above background could be detected with the corresponding 35S-labeled sense RNA probes (data not shown). gt, granulation tissue; e, epithelial layer; hf, hair follicle; s, scar tissue. Bar, 100 μm.

Mentions: We then examined MMP transcript distribution by in situ hybridization on normal skin sections and skin wound sections from days 1–14 after cutaneous incision. Each wound was examined at its extreme and median portions. The results of analyses performed on days 3 and 5 are presented in Fig. 3. On day 3, various stages of granulation tissue and immature capillary lumens were observed, together with spurs of epithelial cells migrating from both edges of wounds beneath the surface scab (Fig. 3 A and data not shown). MT1-MMP transcripts were detected in cells of the granulation tissue and those of the superficial dermis juxtaposed to the proliferative epithelial cell layer (Fig. 3 B). The transcripts were also observed in stromal cells surrounding hair follicles and especially in the dermal papilla. Most cells expressing MT1-MMP transcripts were of the fibroblastic type, thus likely corresponding to fibroblasts or myofibroblasts. However, some cells were round or oval, suggesting that other types of stromal cells, including endothelial cells or macrophages, may also express the MT1-MMP gene. While it is clear that additional studies are required to better define the precise nature of these stromal cells, MT1-MMP transcripts could not be detected in any epithelial cell. The localization of GelA transcripts was similar to that observed for MT1-MMP, although the expression levels were higher for GelA than for MT1MMP (Fig. 3 C). ST3 transcripts were undetectable, except in a few stromal cells surrounding hair follicles (Fig. 3 D). ST1 transcripts were detected in some proliferative epithelial basal cells, and in a few stromal cells juxtaposed to the proliferative epithelial layer (Fig. 3 E). GelB and Col3 RNAs were highly expressed in the epithelial basal cell layer, but not in the dermis (Fig. 3, F and G). On day 5, though granulation tissue was still observed in the median portion of incision (data not shown), reepithelization had been completed at the wound extremities, where thickened epidermis, scar tissue and tissue contraction were observed (Fig. 3 H). MT1-MMP, GelA, and ST3 transcripts were detected in cells of the scar tissue, below the thickened epithelial cell layer (Fig. 3, I–K). The distribution of MT1-MMP transcripts (Fig. 3 I) was more narrow than that of GelA (Fig. 3 J), but broader than that of ST3 (Fig. 3 K). MT1-MMP, GelA, and ST3 transcripts were not detected in epithelial cells. ST1 transcripts were not detected (Fig. 3 L). GelB and Col3 transcripts were intensely expressed in some basal cells of the thickened epidermis, and in some cells of the hair follicle sheath (Fig. 3, M and N).


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)

In situ hybridization of rat MMP RNAs in cutaneous wounds. Serial frozen sections of rat skin wounds on day 3 (A–G), or day  5 (H–N) after cutaneous incision, were hybridized with 35S-labeled antisense RNA probes derived from cDNA templates for rat MT1MMP (B and I), GelA (C and J), ST3 (D and K), ST1 (E and L), GelB (F and M), Col3 (G and N), or stained with hematoxylin and eosin  (A and H). Note that the hair follicle which is observed on sections B and C, is only partially visible on  A (arrows). In D and E, arrows  indicate transcripts expressed in stromal cells surrounding a hair follicle and juxtaposed to the proliferative epithelial cell layer, respectively. No signal above background could be detected with the corresponding 35S-labeled sense RNA probes (data not shown). gt, granulation tissue; e, epithelial layer; hf, hair follicle; s, scar tissue. Bar, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139851&req=5

Figure 3: In situ hybridization of rat MMP RNAs in cutaneous wounds. Serial frozen sections of rat skin wounds on day 3 (A–G), or day 5 (H–N) after cutaneous incision, were hybridized with 35S-labeled antisense RNA probes derived from cDNA templates for rat MT1MMP (B and I), GelA (C and J), ST3 (D and K), ST1 (E and L), GelB (F and M), Col3 (G and N), or stained with hematoxylin and eosin (A and H). Note that the hair follicle which is observed on sections B and C, is only partially visible on A (arrows). In D and E, arrows indicate transcripts expressed in stromal cells surrounding a hair follicle and juxtaposed to the proliferative epithelial cell layer, respectively. No signal above background could be detected with the corresponding 35S-labeled sense RNA probes (data not shown). gt, granulation tissue; e, epithelial layer; hf, hair follicle; s, scar tissue. Bar, 100 μm.
Mentions: We then examined MMP transcript distribution by in situ hybridization on normal skin sections and skin wound sections from days 1–14 after cutaneous incision. Each wound was examined at its extreme and median portions. The results of analyses performed on days 3 and 5 are presented in Fig. 3. On day 3, various stages of granulation tissue and immature capillary lumens were observed, together with spurs of epithelial cells migrating from both edges of wounds beneath the surface scab (Fig. 3 A and data not shown). MT1-MMP transcripts were detected in cells of the granulation tissue and those of the superficial dermis juxtaposed to the proliferative epithelial cell layer (Fig. 3 B). The transcripts were also observed in stromal cells surrounding hair follicles and especially in the dermal papilla. Most cells expressing MT1-MMP transcripts were of the fibroblastic type, thus likely corresponding to fibroblasts or myofibroblasts. However, some cells were round or oval, suggesting that other types of stromal cells, including endothelial cells or macrophages, may also express the MT1-MMP gene. While it is clear that additional studies are required to better define the precise nature of these stromal cells, MT1-MMP transcripts could not be detected in any epithelial cell. The localization of GelA transcripts was similar to that observed for MT1-MMP, although the expression levels were higher for GelA than for MT1MMP (Fig. 3 C). ST3 transcripts were undetectable, except in a few stromal cells surrounding hair follicles (Fig. 3 D). ST1 transcripts were detected in some proliferative epithelial basal cells, and in a few stromal cells juxtaposed to the proliferative epithelial layer (Fig. 3 E). GelB and Col3 RNAs were highly expressed in the epithelial basal cell layer, but not in the dermis (Fig. 3, F and G). On day 5, though granulation tissue was still observed in the median portion of incision (data not shown), reepithelization had been completed at the wound extremities, where thickened epidermis, scar tissue and tissue contraction were observed (Fig. 3 H). MT1-MMP, GelA, and ST3 transcripts were detected in cells of the scar tissue, below the thickened epithelial cell layer (Fig. 3, I–K). The distribution of MT1-MMP transcripts (Fig. 3 I) was more narrow than that of GelA (Fig. 3 J), but broader than that of ST3 (Fig. 3 K). MT1-MMP, GelA, and ST3 transcripts were not detected in epithelial cells. ST1 transcripts were not detected (Fig. 3 L). GelB and Col3 transcripts were intensely expressed in some basal cells of the thickened epidermis, and in some cells of the hair follicle sheath (Fig. 3, M and N).

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