Abstract 197: MARCKS Signaling Modulates Vascular Smooth Muscle Cell Proliferation
Background: The myrisolated alanine rich C-kinase substrate (MARCKS) is over expressed in animal models of intimal hyperplasia (IH) and in several human neoplasms. Further it has been demonstrated that MARCKS knock-down in vivo arrests vascular smooth muscle cell (VSMC) proliferation. We hypothesize that MARCKS arrests VSMC proliferation through a p27 dependent mechanism.
Materials and Methods: Human coronary artery VSMC (Lonza) were grown in culture. Cell proliferation was determined by the AlamarBlue assay. Protein expression was determined by Western Blot analysis. MARCKS signaling was stimulated by treatment with the phorbol ester PMA, and MARCKS signaling was inhibited by either siRNA mediated knock-down or with one of two peptides that act as selective competitive antagonists.
Results: Stimulation of VSMC in culture with PMA for 30 minutes resulted in and increased fraction of phosphor-MARCKS and complete knock-down of p27 protein. Treatment of VSMC in culture with siRNA resulted in arrest of cell proliferation, an increase in p27 expression with no change in p21 expression. Similarly, the peptide designed to specifically inhibit the functions of the N-terminal domain also resulted in similar effects in proliferation, p27, and p21 expression. The same results were noted when a separate peptide designed to inhibit the functions of the C-terminus domain was used to treat VSMC in culture.
Conclusion: MARCKS signaling results in decreased p27 expression, which possibly explains proliferative VSMC phenotype observed in animal models of IH. Blockade of MARCKS signaling by siRNA mediated knock-down results in decreased cell proliferation and increased p27 protein expression. Antagonists to either the N- and C-terminus domains yield similar results in proliferation and p27 expression. VSMC proliferation requires both functional domains of MARCKS.
- © 2012 by American Heart Association, Inc.