Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation
Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
10/2005; 280(38):33083-95. DOI: 10.1074/jbc.M503173200
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 microm forskolin, 40 microm 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.
Available from: Haim Breitbart
- "Naseem's group showed that PKA inhibits ROCK phosphorylation and activation by phosphorylation/ inhibition of RhoA on serine-188 (Aburima et al. 2013). Other studies support this idea by showing that elevated cAMP levels may indirectly lead to cofilin dephosphorylation (Goeckeler and Wysolmerski 2005; Meberg et al. 1998). On the other hand, another study showed that LIMK is directly activated by PKA through phosphorylation on serine-323 and -596 (Nadella et al. 2009). "
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ABSTRACT: The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.
Cell and Tissue Research 06/2015; DOI:10.1007/s00441-015-2229-1 · 3.57 Impact Factor
Available from: Keith H. K. Wong
- "It is well known from studies of endothelial cells that endothelial contractility results from activation of the actomyosin filament . Here, the second messenger cyclic AMP (cAMP) serves to " relax " endothelial cells by activating protein kinase A, which phosphorylates the GTPase RhoA, and thus inhibits generation of tension ; nonclassical relaxation mechanisms that bypass protein kinase A also exist  . We have thus investigated to what extent elevation of cAMP levels could stabilize vessels in microfluidic scaffolds . "
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ABSTRACT: This chapter describes methods to form, vascularize, and optimize microfluidic hydrogels for applications in tissue engineering. Methods to form such scaffolds are based on removal of a sacrificial material or on the bonding of gels. Long-term stable vascularization of microfluidic gels appears to require leakproof vessels. Perfusion introduces chemical and mechanical signals that play complementary roles in controlling the stability of the vessel wall. Computational models enable optimization of microfluidic designs for which perfusion through the vessels is stable and sufficient to oxygenate surrounding tissue. Modulation of scaffold pressures with the use of drainage channels is an important strategy to promote vascular stability. We propose a straightforward algorithm for the design of microfluidic scaffolds for perfusion of vascularized, engineered tissues.
Microfluidic Cell Culture Systems, Edited by Christopher Bettinger, Jeffrey T. Borenstein, Sarah L. Tao, 12/2012: chapter 8: pages 205-221; Elsevier., ISBN: 978-1-4377-3459-1
Available from: Lester R Drewes
- "The identity of the phosphatase remains unknown, although, a number of prominent phosphor-serine,-threonine protein phosphatases and a phosphotyrosine phosphatase are known to be activated by PKA in endothelial cells, including alkaline phosphatase and myosin light chain phosphatase. The latter plays an important role in cytoskeleton remodeling and the breakdown in vascular barrier permeability during disease (Aslam et al., 2010; Beuckmann et al., 1995; Goeckeler and Wysolmerski, 2005). Therefore, PKAdependent dephosphorylation of Mct1 may be a component of such pathological processes and it will be important to identify the phosphatase in future studies. "
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ABSTRACT: In the cerebrovascular endothelium, monocarboxylic acid transporter 1 (Mct1) controls blood-brain transport of short chain monocarboxylic and keto acids, including pyruvate and lactate, to support brain energy metabolism. Mct1 function is acutely decreased in rat brain cerebrovascular endothelial cells by β-adrenergic signaling through cyclic adenosine monophosphate (cAMP); however, the mechanism for this acute reduction in transport capacity is unknown. In this report, we demonstrate that cAMP induces the dephosphorylation and internalization of Mct1 from the plasma membrane into caveolae and early endosomes in the RBE4 rat brain cerebrovascular endothelial cell line. Additionally, we provide evidence that Mct1 constitutively cycles through clathrin vesicles and recycling endosomes in a pathway that is not dependent upon cAMP signaling in these cells. Our results are important because they show for the first time the regulated and unregulated vesicular trafficking of Mct1 in cerebrovascular endothelial cells; processes which have significance for better understanding normal brain energy metabolism, and the etiology and potential therapeutic approaches to treating brain diseases, such as stroke, in which lactic acidosis is a key component.
Brain research 08/2012; 1480:1-11. DOI:10.1016/j.brainres.2012.08.026 · 2.84 Impact Factor
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