Increased Myosin Light Chain Phosphorylation Is Not Required for Growth Factor Stimulation of Collagen Matrix Contraction

Skuta, Gabriella; Ho, Cheryl; Grinnell, Frederick

doi:10.1074/jbc.274.42.30163

Cited by 27 publications

(25 citation statements)

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“…PDGF-dependent floating matrix contraction required myosin II judging from its sensitivity to inhibition by blebbistatin and the Rho kinase inhibitor. MLC kinase did not appear to be required, however, and no stimulation of MLC-diphosphorylation was detected, consistent with previous observations of PDGF-treated cells on planar surfaces (21,23). Because PDGF treatment of fibroblasts in collagen matrices activates Rac and not Rho (9), as has been shown for cells on planar surfaces (42,43), Rho activity necessary for PDGF-stimulated contraction likely is provided by a mechanism other than growth factor stimulation, e.g.…”

Section: Figsupporting

confidence: 77%

“…Previously, we showed by using the ureaglycerol method that LPA but not PDGF stimulated both monoand diphosphorylation of fibroblasts on collagen-coated coverslips, but for technical reasons the urea-glycerol method could not be used for fibroblasts in collagen matrices (23). Therefore, MLC phosphorylation in fibroblasts in collagen matrices was assessed using antibodies specific to (di)phosphorylated MLC with total MLC as a loading control.…”

Section: Fibroblast-collagen Matrix Remodelingmentioning

confidence: 99%

“…SDS-PAGE and Immunoblotting-SDS-PAGE and immunoblotting were performed as described previously (8,23). Briefly, cells were extracted in extraction buffer (150 mM NaCl, 6 mM Na 2 HPO 4 , 4 mM NaH 2 PO 4 , 2 mM EDTA, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 g/ml leupeptin, 1 g/ml pepstatin A, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 50 mM NaF, 1 mM Na 3 VO 4 , and 1 mM ammonium molybdate tetrahydrate, pH 7.0) (100 l per matrix and 150 l per 35-mm tissue-culture dish) by homogenization using 100 strokes with a Dounce homogenizer (pestle B; Wheaton Scientific, Millville, NJ).…”

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confidence: 99%

“…Collagen matrix cultures were prepared using Vitrogen 100 collagen as described previously (8,23). Briefly, neutralized collagen solution (1.5 mg/ml) containing harvested cells (10 6 cells/ml unless indicated otherwise) was prewarmed to 37°C for 4 min, and then aliquots (200 l) were placed on an area outlined by a 12-mm-diameter circular score within a well of 24-well culture plates (Greiner Bio-one, Frickenhausen, Germany) and allowed to polymerize for 60 min at 37°C in 5% CO 2 -humidified incubator.…”

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confidence: 99%

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

Abe¹,

Ho²,

Kamm

et al. 2003

Journal of Biological Chemistry

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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...

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Section: Figsupporting

confidence: 77%

Section: Fibroblast-collagen Matrix Remodelingmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Abe¹,

Ho²,

Kamm

et al. 2003

Journal of Biological Chemistry

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“…In nonmuscle cells, elevated cAMP has been shown to cause the following: 1) alterations in cell morphology; 2) inhibition of cell motility; 3) disruption of actin filaments; 4) pericyte relaxation in a silicone rubber wrinkling assay; and 5) fibroblast relaxation in a collagen gel assay (1,(25)(26)(27)(28)(29). In endothelial cells, preliminary studies have shown that elevations in cAMP correlate with an increase in MLCK phosphorylation (13,30) and a decrease in RLC phosphorylation.…”

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confidence: 99%

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Goeckeler

Wysolmerski

2005

Journal of Biological Chemistry

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This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 M forskolin, 40 M 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.Endothelial cells lining blood vessels form a continuous layer that constrains proteins and blood elements to the vascular lumen. Disruption of the continuous endothelial barrier leads to an increase in permeability and development of edema, a hallmark of acute and chronic inflammation. Chemical or inflammatory mediators activate signaling pathways that cause endothelial cells to contract forming gaps between adjacent cells leading to an increase in vascular permeability. Several laboratories studying methods to inhibit endothelial cell contraction and vascular permeability have demonstrated that elevation of intracellular cAMP augments barrier function and prevents increases in permeability to a wide range of inflammatory agonists. The primary pathway implicated in preventing increases in permeability is believed to occur through the activation of adenylate cyclase, accumulation of intracellular cAMP, and activation of PKA.2 Even though there are numerous reports (1-5) implicating cAMP/PKA in enhancing barrier function and inhibiting endothelial cell contraction, the mechanism by which cAMP/PKA protects vascular integrity is poorly understood.cAMP is a primary mediator of the biolo...

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Arachidonic acid signaling to the cytoskeleton: The role of cyclooxygenase and cyclic AMP‐dependent protein kinase in actin bundling

Glenn

Jacobson

2002

Cell Motil. Cytoskeleton

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Cell adhesion to the extracellular matrix via integrins is a primary regulatory mechanism for numerous aspects of normal cellular function. However, disruption of this interaction can result in pathology. For example, one characteristic of transformed cells is loss of adhesion dependence for viability. Adhesion also is a necessary step in tumor metastasis. It has been shown previously, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. We now demonstrate by inhibitor studies and biochemical analysis, a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. This work elucidates a system of interacting signals in which arachidonic acid functions at a branch point in cytoskeletal signaling. The lipoxygenase branch provides polymerized actin; these actin filaments act as a substrate for the cylooxygenase branch to generate actin bundles.

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Increased Myosin Light Chain Phosphorylation Is Not Required for Growth Factor Stimulation of Collagen Matrix Contraction

Cited by 27 publications

References 50 publications

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

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

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

Arachidonic acid signaling to the cytoskeleton: The role of cyclooxygenase and cyclic AMP‐dependent protein kinase in actin bundling

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