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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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Amino acid sequence alignments of rat MMPs. Amino acids are represented using the one-letter code. The predicted amino  acid sequences of MT1-MMP, ST3, GelA, GelB, and ST1 were derived from cDNAs cloned from two distinct rat skin wound libraries  (Okada et al., 1994, and the present study). One amino acid residue of ST1 (isoleucine 189) differs from the corresponding residue (threonine) in the sequence reported by Matrisian et al. (1985). The sequences of ST2, Col3, and matrilysin were obtained from the PIR-protein  data bank. These sequences were aligned using the CLUSTAL program of the Wisconsin program package. Identical amino acid residues  in all MMPs are indicated by asterisks below the sequences. The cysteine-switch region (P-/LRCGV/NPD), and the zinc-binding domain  (XVAXHXHEL/FGHXL/MGLXHS/T) are in the boxed regions. The two regions selected for designing the degenerated oligoprimers  used for RT-PCR amplification are indicated by arrows above the sequences. The putative transmembrane segment (amino acid 539563) of rat MT1-MMP is overlined.
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Figure 2: Amino acid sequence alignments of rat MMPs. Amino acids are represented using the one-letter code. The predicted amino acid sequences of MT1-MMP, ST3, GelA, GelB, and ST1 were derived from cDNAs cloned from two distinct rat skin wound libraries (Okada et al., 1994, and the present study). One amino acid residue of ST1 (isoleucine 189) differs from the corresponding residue (threonine) in the sequence reported by Matrisian et al. (1985). The sequences of ST2, Col3, and matrilysin were obtained from the PIR-protein data bank. These sequences were aligned using the CLUSTAL program of the Wisconsin program package. Identical amino acid residues in all MMPs are indicated by asterisks below the sequences. The cysteine-switch region (P-/LRCGV/NPD), and the zinc-binding domain (XVAXHXHEL/FGHXL/MGLXHS/T) are in the boxed regions. The two regions selected for designing the degenerated oligoprimers used for RT-PCR amplification are indicated by arrows above the sequences. The putative transmembrane segment (amino acid 539563) of rat MT1-MMP is overlined.

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)

Amino acid sequence alignments of rat MMPs. Amino acids are represented using the one-letter code. The predicted amino  acid sequences of MT1-MMP, ST3, GelA, GelB, and ST1 were derived from cDNAs cloned from two distinct rat skin wound libraries  (Okada et al., 1994, and the present study). One amino acid residue of ST1 (isoleucine 189) differs from the corresponding residue (threonine) in the sequence reported by Matrisian et al. (1985). The sequences of ST2, Col3, and matrilysin were obtained from the PIR-protein  data bank. These sequences were aligned using the CLUSTAL program of the Wisconsin program package. Identical amino acid residues  in all MMPs are indicated by asterisks below the sequences. The cysteine-switch region (P-/LRCGV/NPD), and the zinc-binding domain  (XVAXHXHEL/FGHXL/MGLXHS/T) are in the boxed regions. The two regions selected for designing the degenerated oligoprimers  used for RT-PCR amplification are indicated by arrows above the sequences. The putative transmembrane segment (amino acid 539563) of rat MT1-MMP is overlined.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Amino acid sequence alignments of rat MMPs. Amino acids are represented using the one-letter code. The predicted amino acid sequences of MT1-MMP, ST3, GelA, GelB, and ST1 were derived from cDNAs cloned from two distinct rat skin wound libraries (Okada et al., 1994, and the present study). One amino acid residue of ST1 (isoleucine 189) differs from the corresponding residue (threonine) in the sequence reported by Matrisian et al. (1985). The sequences of ST2, Col3, and matrilysin were obtained from the PIR-protein data bank. These sequences were aligned using the CLUSTAL program of the Wisconsin program package. Identical amino acid residues in all MMPs are indicated by asterisks below the sequences. The cysteine-switch region (P-/LRCGV/NPD), and the zinc-binding domain (XVAXHXHEL/FGHXL/MGLXHS/T) are in the boxed regions. The two regions selected for designing the degenerated oligoprimers used for RT-PCR amplification are indicated by arrows above the sequences. The putative transmembrane segment (amino acid 539563) of rat MT1-MMP is overlined.
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