Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension

The aim of the work was to analyze, on a comparative basis, the signaling p athways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR p rocedure using a synthetic RNA as internal standard. In monolayer, most MMP s were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulat ion of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant tip-regulation o f these MMPs was also observed although to a lower extent than in CD-treate d monolayers. Among the investigated MMPs, the MMP-8 and -11 were not repro ducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The. CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibi ting the tyrosine-kinases of the src family by herbimycin A for all MMPs, e xcept MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyr osine kinases-dependent for MMP-3 and MMP-13, as previously described for M MP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP -13, which was strongly induced. The processing of MMP-2 was tyrosine kinas es-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kina ses pathways diversely affected the MMPs expression. Inhibiting the Rho-kin ase activity by Y-27632 was inactive. These results point to the potent reg ulation operated by the status of the cytoskeleton on the cell phenotype, a nd to distinct regulatory pathways involved in the control of different MMP s expression. (C) 2001 Elsevier Science B.V./International Society of Matri x Biology. All rights reserved.