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MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

Yu D, Makkar G, Dong T, Strickland DK, Sarkar R, Monahan TS - PLoS ONE (2015)

Bottom Line: MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation.MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent.MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT

Background: Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and results: siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions: MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

No MeSH data available.


Related in: MedlinePlus

MARCKS knockdown decreases p27kip1, pSer10-p27kip1, KIS, cyclin D1, and ubiquitin ligase E3 Skp2, but does not affect cyclin E1 protein expression.A. The cyclin-dependent kinase inhibitor p27kip1 protein expression is regulated in a multistep process by degradation by the 26s proteasome. B. Human coronary artery smooth muscle cells (CASMCs) were treated with MARCKS siRNA or non-targeting siRNA (Control). Protein expression was determined by Western Blot analysis. C. Protein expression was normalized to β-actin and compared with densitometry. Expression of pSer10-p27kip1, pThr187-p27kip1, KIS, cyclin D1, and the E3 ubiquitin protein ligase all decreased significantly with MARCKS knockdown. Only Cylcin E did not change significantly as a result of MARCKS knockdown. KIS and pSer10-p27kip1 had the greatest decrease in protein expression as a result of MARCKS knockdown. All experiments were performed in triplicate. Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
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pone.0141397.g003: MARCKS knockdown decreases p27kip1, pSer10-p27kip1, KIS, cyclin D1, and ubiquitin ligase E3 Skp2, but does not affect cyclin E1 protein expression.A. The cyclin-dependent kinase inhibitor p27kip1 protein expression is regulated in a multistep process by degradation by the 26s proteasome. B. Human coronary artery smooth muscle cells (CASMCs) were treated with MARCKS siRNA or non-targeting siRNA (Control). Protein expression was determined by Western Blot analysis. C. Protein expression was normalized to β-actin and compared with densitometry. Expression of pSer10-p27kip1, pThr187-p27kip1, KIS, cyclin D1, and the E3 ubiquitin protein ligase all decreased significantly with MARCKS knockdown. Only Cylcin E did not change significantly as a result of MARCKS knockdown. KIS and pSer10-p27kip1 had the greatest decrease in protein expression as a result of MARCKS knockdown. All experiments were performed in triplicate. Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.

Mentions: Protein levels of p27kip1 fluctuate during the cell cycle, with the highest level occurring at G0/G1 and subsequently decreases through proteolysis allowing cells to progress from phase G0/G1 to S [30]. Canonically, p27kip1 degradation is a multistep process involving several key regulators of p27kip1 phosphorylation: first, p27kip1 is phosphorylated at serine 10 (pSer10-p27kip1) by kinase interacting with stathmin (KIS) [31], which allows its export from the nucleus in association with cyclin D. In the cytoplasm p27kip1 is further phosphorylated at threonine 187 (pThr187-p27kip1) by CDK2, then ubiquinated by the ubiquitin ligase E3 Skp2, and ultimately degraded by the 26s proteasome (Fig 3A) [32]. After treatment of human CASMCs with MARCKS siRNA, there was a significant decrease in the protein expression of pSer10-p27kip1, pThr187-p27kip1, and ubiquitin ligase E3 Skp2 (Fig 3B and 3C, * denotes p<0.05, n = 3). Additionally, there was a significant decrease (60.7%±5.7%) in KIS expression. No change was observed in expression of Cyclin E (Fig 3B, p = NS). The change in pSer10-p27kip1 and KIS expression were greatest in magnitude, a 61.3% and 60.7% decrease respectively, among intermediate products and enzymes studied (Fig 3C).


MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

Yu D, Makkar G, Dong T, Strickland DK, Sarkar R, Monahan TS - PLoS ONE (2015)

MARCKS knockdown decreases p27kip1, pSer10-p27kip1, KIS, cyclin D1, and ubiquitin ligase E3 Skp2, but does not affect cyclin E1 protein expression.A. The cyclin-dependent kinase inhibitor p27kip1 protein expression is regulated in a multistep process by degradation by the 26s proteasome. B. Human coronary artery smooth muscle cells (CASMCs) were treated with MARCKS siRNA or non-targeting siRNA (Control). Protein expression was determined by Western Blot analysis. C. Protein expression was normalized to β-actin and compared with densitometry. Expression of pSer10-p27kip1, pThr187-p27kip1, KIS, cyclin D1, and the E3 ubiquitin protein ligase all decreased significantly with MARCKS knockdown. Only Cylcin E did not change significantly as a result of MARCKS knockdown. KIS and pSer10-p27kip1 had the greatest decrease in protein expression as a result of MARCKS knockdown. All experiments were performed in triplicate. Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
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pone.0141397.g003: MARCKS knockdown decreases p27kip1, pSer10-p27kip1, KIS, cyclin D1, and ubiquitin ligase E3 Skp2, but does not affect cyclin E1 protein expression.A. The cyclin-dependent kinase inhibitor p27kip1 protein expression is regulated in a multistep process by degradation by the 26s proteasome. B. Human coronary artery smooth muscle cells (CASMCs) were treated with MARCKS siRNA or non-targeting siRNA (Control). Protein expression was determined by Western Blot analysis. C. Protein expression was normalized to β-actin and compared with densitometry. Expression of pSer10-p27kip1, pThr187-p27kip1, KIS, cyclin D1, and the E3 ubiquitin protein ligase all decreased significantly with MARCKS knockdown. Only Cylcin E did not change significantly as a result of MARCKS knockdown. KIS and pSer10-p27kip1 had the greatest decrease in protein expression as a result of MARCKS knockdown. All experiments were performed in triplicate. Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
Mentions: Protein levels of p27kip1 fluctuate during the cell cycle, with the highest level occurring at G0/G1 and subsequently decreases through proteolysis allowing cells to progress from phase G0/G1 to S [30]. Canonically, p27kip1 degradation is a multistep process involving several key regulators of p27kip1 phosphorylation: first, p27kip1 is phosphorylated at serine 10 (pSer10-p27kip1) by kinase interacting with stathmin (KIS) [31], which allows its export from the nucleus in association with cyclin D. In the cytoplasm p27kip1 is further phosphorylated at threonine 187 (pThr187-p27kip1) by CDK2, then ubiquinated by the ubiquitin ligase E3 Skp2, and ultimately degraded by the 26s proteasome (Fig 3A) [32]. After treatment of human CASMCs with MARCKS siRNA, there was a significant decrease in the protein expression of pSer10-p27kip1, pThr187-p27kip1, and ubiquitin ligase E3 Skp2 (Fig 3B and 3C, * denotes p<0.05, n = 3). Additionally, there was a significant decrease (60.7%±5.7%) in KIS expression. No change was observed in expression of Cyclin E (Fig 3B, p = NS). The change in pSer10-p27kip1 and KIS expression were greatest in magnitude, a 61.3% and 60.7% decrease respectively, among intermediate products and enzymes studied (Fig 3C).

Bottom Line: MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation.MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent.MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, United States of America; Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT

Background: Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and results: siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions: MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

No MeSH data available.


Related in: MedlinePlus