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

Abstract

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...

Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells

“…Recent research paid more interest in the role of myosin regulatory light chain 2 in actin reorganization. 29,30) Therefore, in the present paper, we only focused on MRLC2, in our experiment, we found that MRLC2 was up-regulated in the treatment of low doses of ouabain and the western blot assay obtained results similar to 2-DE, the expression of MRLC2 increased in the low doses of ouabain-treated HUVEC compared with control group, and the unregulated MRLC2 played an important role in the HUVEC proliferation without the inhibition of NKA. Further work is underway.…”

“…26) MRLC2, 27,28) which was found to be up regulated in the present study, has important effect on cell contraction and signaling pathway. Recent research paid more interest in the role of myosin regulatory light chain 2 in actin reorganization.…”

Prohibitin and Cofilin Are Intracellular Effectors of Transforming Growth Factor β Signaling in Human Prostate Cancer Cells

“…In insulin-stimulated cells, phosphatidylinositol 3-kinase and phosphatidylinositol-3,4,5-triphosphate are involved in cofilin dephosphorylation (48). In response to extracellular signals that elevate intracellular Ca 2+ levels, calciuminduced cofilin dephosphorylation is mediated by Slingshot via calcineurin in HeLa cells (49), whereas in other cells, cyclic AMP and protein kinase C signals stimulate cofilin dephosphorylation (50). Considering the significance of changes in the intracellular Ca 2+ in apoptosis induction, one could argue that cofilin dephosphorylation in response to TGF-h might be indirectly mediated via transient changes in Ca 2+ levels.…”

Cerebrospinal fluid-derived Semaphorin3B orients neuroepithelial cell divisions in the apicobasal axis

“…3312 Cell Signaling Technology, 1/1,000), a rabbit anti-phospho-CRMP2 antibody directed against the phosphorylated Thr514 (ref. 63; Abcam, 1/1,000) and a rabbit anti-CRMP2 (total) antibody 63 (no. 9393 Cell Signaling Technology, 1/1,000).…”