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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 increases KIS expression and cell proliferation in endothelial cells.A. Human coronary artery ECs were treated with 20 nM non-targeting, control or MARCKS siRNA. Four days after transfection, MARCKS knockdown increased both KIS and pSer10-p27kip1 protein expression. B. Endothelial cells were cultured at subconfluence and they remained subconfluent for the duration of the experiment. MARCKS knockdown also resulted in 25±6% (p<0.05) more proliferation at five days after transfection. C. Proliferation was confirmed by assessing levels of proliferating cell nuclear antigen (PCNA). Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
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pone.0141397.g006: MARCKS knockdown increases KIS expression and cell proliferation in endothelial cells.A. Human coronary artery ECs were treated with 20 nM non-targeting, control or MARCKS siRNA. Four days after transfection, MARCKS knockdown increased both KIS and pSer10-p27kip1 protein expression. B. Endothelial cells were cultured at subconfluence and they remained subconfluent for the duration of the experiment. MARCKS knockdown also resulted in 25±6% (p<0.05) more proliferation at five days after transfection. C. Proliferation was confirmed by assessing levels of proliferating cell nuclear antigen (PCNA). Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.

Mentions: In contrast to observations in VSMCs, siRNA-mediated MARCKS knockdown resulted in a significant increase of KIS expression in human CAECs. MARCKS knockdown also resulted in increased pSer10-p27kip1 and decreased levels of p27kip1 in CAECs (Fig 6A). Furthermore, in subconfluently cultured CAECs, MARCKS knockdown increased EC proliferation. Five days after MARCKS siRNA treatment, EC proliferated 25±6% (p<0.05) faster than EC treated with non-targeting siRNA (Fig 6B). Proliferation was confirmed by assessing levels of PCNA (Fig 6C).


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 increases KIS expression and cell proliferation in endothelial cells.A. Human coronary artery ECs were treated with 20 nM non-targeting, control or MARCKS siRNA. Four days after transfection, MARCKS knockdown increased both KIS and pSer10-p27kip1 protein expression. B. Endothelial cells were cultured at subconfluence and they remained subconfluent for the duration of the experiment. MARCKS knockdown also resulted in 25±6% (p<0.05) more proliferation at five days after transfection. C. Proliferation was confirmed by assessing levels of proliferating cell nuclear antigen (PCNA). Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631550&req=5

pone.0141397.g006: MARCKS knockdown increases KIS expression and cell proliferation in endothelial cells.A. Human coronary artery ECs were treated with 20 nM non-targeting, control or MARCKS siRNA. Four days after transfection, MARCKS knockdown increased both KIS and pSer10-p27kip1 protein expression. B. Endothelial cells were cultured at subconfluence and they remained subconfluent for the duration of the experiment. MARCKS knockdown also resulted in 25±6% (p<0.05) more proliferation at five days after transfection. C. Proliferation was confirmed by assessing levels of proliferating cell nuclear antigen (PCNA). Statistical significance was determined by the two-tailed Student’s t-test. * denotes p<0.05.
Mentions: In contrast to observations in VSMCs, siRNA-mediated MARCKS knockdown resulted in a significant increase of KIS expression in human CAECs. MARCKS knockdown also resulted in increased pSer10-p27kip1 and decreased levels of p27kip1 in CAECs (Fig 6A). Furthermore, in subconfluently cultured CAECs, MARCKS knockdown increased EC proliferation. Five days after MARCKS siRNA treatment, EC proliferated 25±6% (p<0.05) faster than EC treated with non-targeting siRNA (Fig 6B). Proliferation was confirmed by assessing levels of PCNA (Fig 6C).

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