Different Molecular Motors Mediate Platelet-derived Growth Factor and Lysophosphatidic Acid-stimulated Floating Collagen Matrix Contraction

Abstract

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase, whereas plateletderived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA-and PDGF-stimulated contraction. We found that neither PDGF nor LPA-dependent contractile mechanisms require myosin II regulatory light chain kinase or increased phosphorylation of myosin II regulatory light chain (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF-and LPAstimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent, Rho kinase-independent force generation also was detected during fibroblast spreading on collagen-coated coverslips.Form and function of multicellular organisms depend on tissue-specific programs of cell motility. Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. The motile mechanisms that fibroblasts use to remodel the extracellular matrix during these morphogenetic processes have been studied by using cells cultured in three-dimensional collagen matrices (1, 2).As fibroblasts exert force on and move collagen fibrils of the matrix, collagen concentration around the cells increases, and the corresponding decrease in matrix volume (typically referred to as contraction) can be measured as a decrease in the diameter of free matrices or a decrease in height of restrained matrices. During contraction of restrained matrices, collagen fibrils become oriented in the same plane as restraint, and mechanical loading develops. In floating matrices, on the other hand, contraction occurs without collagen fibrils developing a particular orientation, and the matrix remains mechanically unloaded (1-5).The signaling mechanisms used by fibroblasts to regulate collagen matrix contraction depend on whether the cells are mechanically loaded or unloaded at the time that contraction is initiated as well as on the growth factor used to initiate contraction. For instance, stimulation of fibroblasts by lysophosphatidic acid (LPA) 1 but not by platelet-derived growth factor (PDGF) causes robust force generation in restrained matrices (6), whereas LPA and PDGF stimulate floating matrix contraction equally well (7,8).Floating matrix contraction has presented something of an enigma because LPA stimulation of fibroblasts in floating matrices causes activation of the small G protein Rho (GTP loading) (9), but blocking Rho kinase with the pharmacological reagent Y27632 does not inhibit contraction (10). Conversely, P...

Collagen Fibril Flow and Tissue Translocation Coupled to Fibroblast Migration in 3D Collagen Matrices

“…Human fibroblasts in collagen lack detectable Rho activation upon PDGF stimulation . Therefore, basal rather than agoniststimulated Rho kinase probably is required for cell migration and contraction, perhaps by maintaining basal levels of myosin light chain phosphorylation (Abe et al, 2003;Knock et al, 2008).…”

Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of α2β1 integrin

“…Based on pharmacological approaches, previous works suggested involvement of Rho GTPases in collagen gel contraction by fibroblasts (Abe et al, 2003;Parizi et al, 2000). Our recent studies show more directly that a tight regulation of RhoA activation controls collagen gel contraction and distribution of focal adhesions in fibroblasts .…”

Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase

“…The involvement of myosin II also appeared to be supported by morphological/behavior responses of cells to the potent non-muscle myosin II inhibitor blebbistatin [8], including the inhibition of fibroblasts to remodel collagen fibers [9], invade the matrices [10] and contract floating matrices [11]. However these effects could *Corresponding Author: Yu-li Wang, University of Massachusetts Medical School, 377 Plantation, Suite 327, Worcester, MA 01605, Tel: 508-856-8782, Fax: 508-856-8774, e-mail: yuli.wang@umassmed.edu.…”