INTERACTION OF NONMUSCLE MYOSIN II WITH PLASMA MEMBRANES

人类基因组序列的获得促进了对第三种哺乳动物非肌肉肌球蛋白亚型NMHC-IIC的识别，NMHC-IIC在人类和小鼠组织中广泛分布。利用一种针对NMHC-IIC的抗体，我们研究了NMHC-IIC在神经母细胞瘤细胞中的表达。未受刺激的神经-2a细胞表达NMHC-IIA和-IIB，但不表达NMHC-IIC。DBcAMP诱导细胞分化，诱导轴突形成，导致NMHC-IIC表达。然而，包括Forskolin和8-bromo-cAMP在内的其他引起神经突起生长的物质不能诱导NMHC-IIC，提示DBcAMP降解产生的丁酸可能参与了诱导。丁酸钠作用于Neu-2a细胞后，NMHC-IIC的表达呈剂量依赖性，0.2 mM丁酸钠可显著诱导NMHC-IIC的表达，而对-IIA和-IIB的表达无明显影响。RT-PCR检测到NMHC-IIC mRNA在未刺激的细胞中表达，2 mM丁酸盐刺激9h后NMHC-IIC基因表达增强，48h最高。NMHC-IIC蛋白在培养18小时即可检测到，3天达高峰。去除丁酸3天后，NMHC-IIC蛋白表达下降，表明诱导是可逆的。丁酸钠可以抑制组蛋白去乙酰化酶，其对NMHC-IIC诱导的影响可被另一种组蛋白去乙酰化酶抑制剂曲古抑素A所模拟。丁酸盐对RAW264.7小鼠巨噬细胞NMHC-IIC的诱导具有剂量依赖性，10 mM丁酸盐诱导RAW264.7细胞发生凋亡与诱导NMHC-IIC一致。然而，NMHC-IIC的诱导并不是丁酸盐的普遍反应，因为丁酸处理的HeLa细胞不表达NMHC-IIC，尽管表现出更多的分化表型。总之，NMHC-IIC的表达是由丁酸钠诱导分化或凋亡的Neuro-2a和RAW264.7细胞所致。进一步的研究正在阐明诱导的NMHC-IIC的功能。

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.