The myristoylated alanine-rich C kinase substrate differentially regulates kinase interacting with stathmin in vascular smooth muscle and endothelial cells and potentiates intimal hyperplasia formation.

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 µM 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.

Prognostic Value of Upregulation of Myristoylated Alanine-Rich C-Kinase Substrate in Gastric Cancer.

BACKGROUND Accumulating evidence suggests a connection of Myristoylated alanine-rich C-kinase substrate (MARCKS) with several physiological and pathological processes. However, the relevance of MARCKS in gastric cancer (GC) needs to be elucidated. MATERIAL AND METHODS The abundance of MARCKS in GC tissues was assessed using techniques of immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). Moreover, the MARCKS expression profile in the TCGA database was analyzed through an online website analysis. We also investigated MARCKS function using cell wounding and Matrigel invasion assays. RESULTS TCGA analysis and our data suggest that transcript abundance and protein level of MARCKS was higher in GC tumor samples compared with peri-tumor tissues. There was a remarkable association of upregulated MARCKS with the cell differentiation (P<0.001), T stage (P=0.034), and N stage (P=0.002) followed by advanced TNM phase (P=0.008). Furthermore, it was predicted that higher expression of MARCKS is linked to poor overall survival (P=0.015) and disease-free survival (P=0.020), and that high levels of MARCKS function as an independent prognostic marker, as shown by multivariate Cox regression analysis in prediction of poor overall (HR=0.408; 95% confidence interval=0.247-0.674; P<0.001) and disease-free survival rates (HR=0.525; 95% confidence interval=0.216-0.584; P<0.001). GC cells showed significant reduction in cell migration and invasion upon depletion of MARCKS as noted through Matrigel invasion and cell wounding assays. Further analyses showed that silencing MARCKS impeded the epithelial-mesenchymal transition (EMT). CONCLUSIONS Our study indicates that elevated expression of MARCKS is significantly associated with metastatic capability of GC cells, and MARCKS overexpression can serve as a biomarker of GC poor prognosis.

MARCKS phosphorylation by PKC strongly impairs cell polarity in the chick neural plate.

Neurulation involves a complex coordination of cellular movements that are in great part based on the modulation of the actin cytoskeleton. MARCKS, an F-actin-binding protein and the major substrate for PKC, is necessary for gastrulation and neurulation morphogenetic movements in mice, frogs, and fish. We previously showed that this protein accumulates at the apical region of the closing neural plate in chick embryos, and here further explore its role in this process and how it is regulated by PKC phosphorylation. PKC activation by PMA caused extensive neural tube closure defects in cultured chick embryos, together with MARCKS phosphorylation and redistribution to the cytoplasm. This was concomitant with an evident disruption of neural plate cell polarity and extensive apical cell extrusion. This effect was not due to actomyosin hypercontractility, but it was reproduced upon MARCKS knockdown. Interestingly, the overexpression of a nonphosphorylatable form of MARCKS was able to revert the cellular defects observed in the neural plate after PKC activation. Altogether, these results suggest that MARCKS function during neurulation would be to maintain neuroepithelial polarity through the stabilization of subapical F-actin, a function that appears to be counteracted by PKC activation.