Gulab S Zode1, Abbot F Clark, Robert J Wordinger. 1. Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. gzode@hsc.unt.edu
Abstract
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.
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.