Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton.

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.

Cellular contraction of collagen lattices is inhibited by nonenzymatic glycation.

High glucose concentrations associated with diabetes have been shown to cause the nonenzymatic modification of proteins. Reducing sugars covalently bind to free amine groups, undergo Amadori rearrangements, and crosslink with other glucose-modified proteins. Crosslinking of type I collagen by incubation with different concentrations of glucose 6-phosphate for up to 5 days resulted in a nondeformable collagen lattice as assayed by physical compaction analysis. Nonglycated collagen was fully compactible. Fibroblasts cultured on nonglycated collagen lattices were able to contract the lattice over a 5-day period, while fibroblasts on collagen glycated with 50 mM or more glucose 6-phosphate were unable to do this. Cells on both nonglycated and glycated collagen lattices initially lacked organized bundles of actin microfilaments or stress fibers. Over time, the cells on glycated lattices formed stress fibers, suggesting that they were still exerting mechanical force on a nondeformable matrix. These results suggest that crosslinking of collagen fibrils by nonenzymatic glycation alters the physical properties of the extracellular matrix, resulting in changes in the organization of the intracellular actin cytoskeleton.