Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines.
Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.
Nova
D
D
Inserm U 346, HEH, Lyon, France.
Le Griel
C
C
Holvoet
S
S
Gentilhomme
E
E
Vincent
C
C
Staquet
M J
MJ
Schmitt
D
D
Serres
M
M
D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002461:Cell Line, Transformed; D003094:Collagen / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D011905:Genes, ras; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology*; D020782:Matrix Metalloproteinases / Q000096:biosynthesis / Q000378:metabolism*; D008954:Models, Biological; D012867:Skin / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D003094:Collagen; D020782:Matrix Metalloproteinases