Fibroblast Contractility without an Increase in Basal Myosin Light Chain Phosphorylation in Wild Type Cells and Cells Expressing the Catalytic Domain of Myosin Light Chain Kinase

Abstract

We investigated the role of myosin light chain (MLC20) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC-P is 0.96 MOL of PO4/mol of MOL20 in cell expressing tMK compared to 0.20 mol of PO4/mol of MLC20 in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers.

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

“…In vitro phosphorylation of MRLC at Thr18 and Ser19 stimulates the actin-activated ATPase of myosin II and filament assembly [15]. However, while manipulating the phosphorylation state of MRLC by overexpression of Thr18/Ser19 mutants has some effects on cell migration [16,17,18], other studies with pharmacological agents suggest that phosphorylation of MRLC is not necessary for migration [19]. The analysis is complicated by the involvement of multiple Ca2+ dependent and Ca2+ independent pathways in regulating MRLC phosphorylation at Thr18/Ser19; the former is mediated by the myosin light chain kinase (MLCK) downstream of Ca2+-calmodulin, while the latter may involve the Rhodependent kinase (ROCK), which may act directly on MRLC or through the myosin light chain phosphatase [20].…”

Horse Chestnut Extract Induces Contraction Force Generation in Fibroblasts through Activation of Rho/Rho Kinase

“…Obara et al also reported that stress fiber formation is important in fibroblasts to generate contraction force. 53) Kolodney and Wysolmerski further reported that phosphorylation of MLC plays a critical role in the force generation of fibroblasts induced by antagonists such as thrombin since the time course of the increase of MLC phosphorylation in response to thrombin closely paralleled the increase in isometric force. 5) In contrast, Obara et al demonstrated that increase of MLC phosphorylation is independent of the contraction force generation of fibroblasts induced by serum.…”

“…5) In contrast, Obara et al demonstrated that increase of MLC phosphorylation is independent of the contraction force generation of fibroblasts induced by serum. 53) Nobe et al investigated the mechanism of contraction force generation focusing on stress fiber formation, MLC phosphorylations, and force generation using a force measuring system similar to ours. 54) They reported that the Rho kinase inhibitor Y27632 inhibits force generation in fibroblasts and prevents stress fiber formation induced by serum, but does not affect MLC phosphorylation.…”

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

“…In addition to Rho kinase, the spatial and temporal pattern of MLC phosphorylation in fibroblasts is controlled by . Other mechanisms of force generation independent of MLC phosphorylation also have been described (18,19), even in smooth muscle cells (20,21) where MLC phosphorylation generally is thought to be the key regulator of contractile activity (22).The current studies were carried out to learn more about the molecular motors responsible for LPA-and PDGF-stimulated fibroblast-collagen matrix contraction. We found that neither PDGF-nor LPA-dependent contractile mechanisms require MLC kinase or increased phosphorylation of MLC (measured as diphosphorylation).…”

“…In addition to Rho kinase, the spatial and temporal pattern of MLC phosphorylation in fibroblasts is controlled by . Other mechanisms of force generation independent of MLC phosphorylation also have been described (18,19), even in smooth muscle cells (20,21) where MLC phosphorylation generally is thought to be the key regulator of contractile activity (22).…”