Abstract
Gelatinase A (GL-A) is a matrix metalloproteinase (MMP) involved in both connective tissue remodeling and tumor invasion. GL-A activation is mediated by a membrane-type MMP (MT-MMP) that cleaves the GL-A propeptide. In this study, we examined the role of the actin cytoskeleton in regulating GL-A activation and MT-MMP-1 expression. Human palmar fascia fibroblasts and human fetal lung fibroblasts were cultured on a planar substratum or within different types of collagen lattices. Fibroblasts that formed stress fibers, either on a planar substratum or within an attached collagen lattice, showed reduced GL-A activation compared with fibroblasts lacking stress fibers, within either floating or stress-released collagen lattices. To determine whether changes in the organization of the actin cytoskeleton could promote GL-A activation, fibroblasts with stress fibers were treated with cytochalasin D. Within 24 h after treatment, GL-A activation was dramatically increased. Associated with this GL-A activation was an increase in MT-MMP-1 mRNA as determined by Northern blot analysis. Treatment with nocodazole, which induced microtubule depolymerization and cell shape changes without affecting stress fibers, did not promote GL-A activation. These results suggest that the extracellular matrix and the actin cytoskeleton transduce signals that modulate GL-A activation and regulate tissue remodeling.
Highlights
MMPs1 are among the key extracellular enzymes involved in turnover of the ECM during normal and pathological processes
In this study, using collagen lattice models and cyto D treatment, we demonstrate an inter-relationship between the mechanical properties of the ECM and actin cytoskeleton organization in the regulation of Gelatinase A (GL-A) activation and expression of MT-matrix metalloproteinase (MMP)-1
Changes in the Organization of the Actin Cytoskeleton Regulate GL-A and membrane-type MMP (MT-MMP)-1 Expression— we evaluated whether the steady state levels of MT-MMP-1 and GL-A mRNA increased after cyto D treatment, conditions which disrupt the actin cytoskeleton
Summary
Cell Culture—Human palmar fascia fibroblasts were obtained and cultured, as described previously [13, 14, 16], in complete medium M-199 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA), 2 mM glutamine, and 1% antibiotic-antimycotic solution (Life Technologies, Inc.). Cultures were briefly washed with serum-free medium and treated with 8 M cyto D (Sigma), 2 g/ml nocodazole (Sigma), or 25 nM GM6001 for 24 h, after which conditioned medium or total RNA was collected. For experiments in collagen lattices, stabilized collagen lattices were cultured for 5 days, washed briefly with serum-free medium, and treated as described above. Floating collagen lattices were prepared by placing a 250-l drop of the collagen-cell suspension on 35-mm Petri dishes (Falcon). Stabilized collagen lattices were prepared by the identical protocol with the exception that the collagen/cell suspension was placed on a 35-mm tissue culture dish (Falcon). Thin layer collagen substrata were prepared by incubating 50 g of collagen/0.25 ml per 24-well culture dish well for 1 h at 37 °C, followed by a wash with phosphate-buffered saline. Fluorescence Staining for Actin Filaments—Fibroblasts were cultured within floating, stabilized, or released collagen lattices, as described above.
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