The myristoylated alanine-rich C kinase substrate (MARCKS) differentially regulates kinase interacting with stathmin (KIS) 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 anti-proliferative 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 p27 kip1 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 transection with small interfering RNA (siRNA). Messenger RNA (mRNA) was quantitated with the real-time reverse transcription polymerase chain reaction (qPCR). 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 siRNA 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 mRNA expression in either cell type. In the presence of cyclohexamide, 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 ECs. MARCKS knockdown significantly increased the level of ubiquitinated KIS in VSMCs, but not 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 four 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. Abstract This basic research study demonstrates that MARCKS depletion decreases KIS expression through increased ubiquitination in smooth muscle cells but not endothelial cells in vitro, and results in decreased KIS expression, decreased intimal hyperplasia formation, and more rapid reendothelialization in vivo. This study suggests that both MARCKS and KIS are potential targets for therapy to prevent intimal hyperplasia formation. Key Findings: MARCKS differentially regulates smooth muscle cell and endothelial cell proliferation. MARCKS knockdown increases KIS ubiquitination and decreases KIS expression in smooth muscle cells but not endothelial cells. In vivo, MARCKS knockdown decreases KIS expression, decreases intimal hyperplasia and increases the rate of reendothelialization. Take home Message: MARCKS and KIS are both potential targets for therapy to prevent intimal hyperplasia formation. and ECs. Cells were grown in culture and transfected with either siMARCKS or non-targeting, siControl. The cells were then treated with MG-132 to block the 26s proteasome function and prevent the degradation of ubiquitinated protein. Total cell lysates were prepared immunoprecipitated with an Ubiquitin Enrichment Kit (Pierce). The precipitate was blotted with anti-KIS (IP). To confirm equal target loading, the total cell lysate (TL) was blotted for GAPDH. D . MARCKS knockdown resulted in increased KIS ubiquitination in VSMCs, but paradoxically decreased KIS ubiquitination in ECs. Data are presented as means ± standard deviations of three separate experiments. Statistical significance was determined by the two-tail Student’s t-test.