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Myristoylated Alanine-Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation.

ABSTRACT: Transcription of the myristoylated alanine-rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS-mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo.MARCKS knockdown blocked platelet-derived growth factor (PDGF)-induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP2, restored membrane-associated PIP2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation.MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP2 at the plasma membrane. This study delineates a novel mechanism for MARCKS-mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

SUBMITTER: Yu D

PROVIDER: S-EPMC4845127 | biostudies-other | 2015 Oct

REPOSITORIES: biostudies-other

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Project description:The myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been at the crossroads of multiple signaling pathways that govern several critical operations in normal and malignant cellular physiology. Functioning as a target of protein kinase C, MARCKS shuttles between the phosphorylated cytosolic form and the unphosphorylated plasma membrane-bound states whilst regulating several molecular partners including, but not limited to calmodulin, actin, phosphatidylinositol-4,5-bisphosphate, and phosphoinositide-3-kinase. As a result of these interactions, MARCKS directly or indirectly modulates a host of cellular functions, primarily including cytoskeletal reorganization, membrane trafficking, cell secretion, inflammatory response, cell migration, and mitosis. Recent evidence indicates that dysregulated expression of MARCKS is associated with the development and progression of hematological cancers. While it is understood that MARCKS impacts the overall carcinogenesis as well as plays a part in determining the disease outcome in blood cancers, we are still at an early stage of interpreting the pathophysiological roles of MARCKS in neoplastic disease. The situation is further complicated by contradictory reports regarding the role of phosphorylated versus an unphosphorylated form of MARCKS as an oncogene versus tumor suppressor in blood cancers. In this review, we will investigate the current body of knowledge and evolving concepts of the physical properties, molecular network, functional attributes, and the likely pathogenic roles of MARCKS in hematological malignancies. Key emphasis will also be laid upon understanding the novel mechanisms by which MARCKS determines the overall disease prognosis by playing a vital role in the induction of therapeutic resistance. Additionally, we will highlight the importance of MARCKS as a valuable therapeutic target in blood cancers and will discuss the potential of existing strategies available to tackle MARCKS-driven blood cancers.

Project description:OBJECTIVE:Restenosis limits the durability of all cardiovascular reconstructions. Vascular smooth muscle cell (VSMC) proliferation drives this process, but an intact, functional endothelium is necessary for vessel patency. Current strategies to prevent restenosis employ antiproliferative agents that affect both VSMCs and endothelial cells (ECs). Knockdown of the myristoylated alanine-rich C kinase substrate (MARCKS) arrests VSMC proliferation and paradoxically potentiates EC proliferation. MARCKS knockdown decreases expression of the kinase interacting with stathmin (KIS), increasing p27kip1 expression, arresting VSMC proliferation. Here, we seek to determine how MARCKS influences KIS protein expression in these two cell types. METHODS:Primary human coronary artery VSMCs and ECs were used for in vitro experiments. MARCKS was depleted by transfection with small interfering RNA. Messenger RNA was quantitated with the real-time reverse transcription polymerase chain reaction. Protein expression was determined by Western blot analysis. Ubiquitination was determined with immunoprecipitation. MARCKS and KIS binding was assessed with co-immunoprecipitation. Intimal hyperplasia was induced in CL57/B6 mice with a femoral artery wire injury. MARCKS was knocked down in vivo by application of 10 ?M of small interfering RNA targeting MARCKS suspended in 30% Pluronic F-127 gel. Intimal hyperplasia formation was assessed by measurement of the intimal thickness on cross sections of the injured artery. Re-endothelialization was determined by quantitating the binding of Evans blue dye to the injured artery. RESULTS:MARCKS knockdown did not affect KIS messenger RNA expression in either cell type. In the presence of cycloheximide, MARCKS knockdown in VSMCs decreased KIS protein stability but had no effect in ECs. The effect of MARCKS knockdown on KIS stability was abrogated by the 26s proteasome inhibitor MG-132. MARCKS binds to KIS in VSMCs but not in ECs. MARCKS knockdown significantly increased the level of ubiquitinated KIS in VSMCs but not in ECs. MARCKS knockdown in vivo resulted in decreased KIS expression. Furthermore, MARCKS knockdown in vivo resulted in decreased 5-ethynyl-2'-deoxyuridine integration and significantly reduced intimal thickening. MARCKS knockdown enhanced endothelial barrier function recovery 4 days after injury. CONCLUSIONS:MARCKS differentially regulates the KIS protein stability in VSMCs and ECs. The difference in stability is due to differential ubiquitination of KIS in these two cell types. The differential interaction of MARCKS and KIS provides a possible explanation for the observed difference in ubiquitination. The effect of MARCKS knockdown on KIS expression persists in vivo, potentiates recovery of the endothelium, and abrogates intimal hyperplasia formation.

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Myristoylated Alanine-Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation.

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