Zode Gulab S, Clark Abbot F, Wordinger Robert J
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Invest Ophthalmol Vis Sci. 2007 Nov;48(11):5058-67. doi: 10.1167/iovs.07-0127.
PURPOSE: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-beta superfamily that controls multiple functions in a variety of cells. We have previously shown that human optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells express BMP and BMP receptor mRNA and proteins. The purpose of the present study was to determine whether human ONH tissues express the canonical BMP signaling pathway and whether ONH cells secrete BMP-4 and respond to exogenous BMP-4 through this pathway. METHODS: Well-characterized human ONH astrocytes (N = 2) and LC cells (N = 3) were treated with exogenous BMP-4 (20 ng/mL) for various times. Western immunoblot analysis was used to detect secreted BMP-4 in serum-free conditioned media of ONH cells and in human ONH tissues (N = 4) and Smad proteins in total cell lysate of ONH cells and tissues. Intracellular colocalization of p-R-Smad1 with Co-Smad4 and localization of inhibitory Smads (e.g., I-Smad6 and I-Smad7) were studied through immunocytochemistry. In addition, coimmunoprecipitation was used to verify the interaction of p-R-Smad1 with Co-Smad4. RESULTS: ONH astrocytes and LC cells secrete BMP-4 and synthesize R-Smad1, R-Smad5, I-Smad6, I-Smad7, and Co-Smad4 proteins. Exposure to BMP-4 for either 10 or 60 minutes resulted in increased p-R-Smad1 and p-R-Smad1/5/8 protein levels that declined after 12 hours of treatment. Immunocytochemistry and coimmunoprecipitation studies revealed that p-R-Smad1/5/8 and Co-Smad4 interact and colocalize in the nucleus. BMP-4 treatment resulted in increased coprecipitation of p-R-Smad1/5/ 8 and Co-Smad4. I-Smad6 and I-Smad7 are localized in the nucleus and cytoplasm of ONH astrocytes and LC cells. Proteins for BMP-4, p-R-Smad1/5/8, R-Smad1, R-Smad5, R-Smad8, and Co-Smad4 are present in human ONH tissues. In addition, phosphorylated Smad1 and Smad5 colocalize with Smad4 in the nuclei of ONH tissues. CONCLUSIONS: These results indicate that BMP-4 and Smad signaling proteins are present in human ONH tissues, isolated ONH astrocytes, and LC cells. In addition, exogenous BMP-4 treatment of ONH astrocytes and LC cells results in downstream signaling through the canonical Smad pathway. Thus, cells within the human ONH may respond to locally released BMP through paracrine or autocrine mechanisms.
目的：骨形态发生蛋白（BMPs）是转化生长因子（TGF）-β超家族的成员，可控制多种细胞中的多种功能。我们之前已经表明，人视神经乳头（ONH）星形胶质细胞和筛板（LC）细胞表达BMP和BMP受体mRNA及蛋白。本研究的目的是确定人ONH组织是否表达经典的BMP信号通路，以及ONH细胞是否分泌BMP-4并通过该通路对外源性BMP-4作出反应。 方法：用外源性BMP-4（20 ng/mL）处理特征明确的人ONH星形胶质细胞（N = 2）和LC细胞（N = 3）不同时间。采用蛋白质免疫印迹分析检测ONH细胞无血清条件培养基和人ONH组织（N = 4）中分泌的BMP-4，以及ONH细胞和组织总细胞裂解物中的Smad蛋白。通过免疫细胞化学研究磷酸化的R-Smad1与共同Smad4的细胞内共定位以及抑制性Smad（如I-Smad6和I-Smad7）的定位。此外，采用免疫共沉淀法验证磷酸化的R-Smad1与共同Smad4的相互作用。 结果：ONH星形胶质细胞和LC细胞分泌BMP-4并合成R-Smad1、R-Smad5、I-Smad6、I-Smad7和共同Smad4蛋白。用BMP-4处理10分钟或60分钟后，磷酸化的R-Smad1和磷酸化的R-Smad1/5/8蛋白水平升高，处理12小时后下降。免疫细胞化学和免疫共沉淀研究表明，磷酸化的R-Smad1/5/8与共同Smad4在细胞核中相互作用并共定位。BMP-4处理导致磷酸化的R-Smad1/5/8与共同Smad4的共沉淀增加。I-Smad6和I-Smad7定位于ONH星形胶质细胞和LC细胞的细胞核和细胞质中。人ONH组织中存在BMP-4、磷酸化的R-Smad1/5/8、R-Smad1、R-Smad5、R-Smad8和共同Smad4的蛋白。此外，磷酸化的Smad1和Smad5在ONH组织细胞核中与Smad4共定位。 结论：这些结果表明，BMP-4和Smad信号蛋白存在于人ONH组织、分离的ONH星形胶质细胞和LC细胞中。此外，用外源性BMP-4处理ONH星形胶质细胞和LC细胞会导致通过经典Smad途径的下游信号传导。因此，人ONH内的细胞可能通过旁分泌或自分泌机制对局部释放的BMP作出反应。
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