Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat.
Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.
Laboratoire Peau Humaine et Immunité, Unité INSERM 346, Lyon, France.
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Serres
D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002999:Clone Cells; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D011905:Genes, ras / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D039201:Integrin alpha3beta1; D016023:Integrins / Q000096:biosynthesis; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D038981:Receptors, Collagen; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D039201:Integrin alpha3beta1; D016023:Integrins; D061965:Matrix Metalloproteinase Inhibitors; D038981:Receptors, Collagen; D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; C100342:marimastat; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9