INTERACTION OF NONMUSCLE MYOSIN II WITH PLASMA MEMBRANES

Heart, Lung, And Blood Institute

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Abstract

The availability of the human genome sequence has facilitated the recognition of a third mammalian nonmuscle myosin isoform, NMHC-IIC, which has a widespread distribution in human and mouse tissues. Using an antibody specific for NMHC-IIC, we have explored the expression of NMHC-IIC in neuro-2a neuroblastoma cells. Non-stimulated neuro-2a cells express NMHC-IIA and -IIB, but not NMHC-IIC. Differentiation of the cells with dbcAMP, which induces neurite formation, leads to expression of NMHC-IIC. However, other agents which cause neurite outgrowth, including forskolin and 8-bromo-cAMP, did not induce NMHC-IIC, suggesting that butyrate derived from breakdown of dbcAMP might mediate induction. Treatment of neuro-2a cells with sodium butyrate resulted in the dose-dependent expression of NMHC-IIC, with significant induction at 0.2mM butyrate, while expression of -IIA and -IIB was unaffected. NMHC-IIC mRNA was detectable by RT-PCR in non-stimulated cells, but was increased by 9 hour stimulation with 2mM butyrate and was maximal at 48 hours. NMHC-IIC protein expression was detectable by 18 hours and maximal at 3 days. Removal of butyrate after 3 days resulted in a decrease in NMHC-IIC protein expression, showing induction to be reversible. Sodium butyrate is known to inhibit histone deacetylase and the effects of butyrate on NMHC-IIC induction were mimicked by trichostatin A, another histone deacetylase inhibitor. Dose-dependent induction of NMHC-IIC was also found in RAW264.7 mouse macrophages treated with butyrate and induction of NMHC-IIC coincided with onset of apoptosis in RAW264.7 cells treated with 10mM butyrate. Induction of NMHC-IIC is not, however, a general response to butyrate, since butyrate-treated HeLa cells did not express NMHC-IIC despite demonstrating a more differentiated phenotype. In summary, NMHC-IIC expression is induced by sodium butyrate in neuro-2a and RAW264.7 cells undergoing differentiation or apoptosis. Further studies are in progress to elucidate the function of the induced NMHC-IIC.