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G. Zode, A.F. Clark, R.J. Wordinger; Expression of BMP–Related Smad Signaling Proteins in Cells Isolated From the Human Optic Nerve Head . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1249.
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Purpose: Bone morphogenetic proteins (BMP) are members of the TGFß family of growth factors and initiate signaling via binding to cell surface type I and type II serine/threonine kinase receptors. The type II BMP receptor is a constitutively active kinase that transphosphorylates the type I BMP receptor upon ligand binding. Smad proteins are involved in downstream signaling following BMP binding. Receptor regulated Smads (Smad1, Smad5, Smad8) transiently associate with the type I BMP receptor and undergo direct phosphorylation. Subsequently, the phosphorylated receptor Smad associates with a common Co–Smad4 and the heteromeric complex translocates to the nucleus to regulate target genes. We have previously demonstrated that cells isolated from the human optic nerve head (ONH) express mRNA and protein for BMP receptors. The purpose of this study was to determine if ONH cells express both receptor and common Smad(s) and respond to exogenous BMP–4. Methods: Well–characterized human ONH astrocytes (N=5) and lamina cribrosa (LC) cells (N=5) were utilized. Intracellular localization of receptor Smad1 and Co–Smad4 proteins in ONH astrocytes and LC cells was studied via immunocytochemistry. In addition, cells were exposed to exogenous BMP–4 (10 ng/ml) for various times. Western blot analysis was used to determine total Smad1, Co–Smad4 phosphorylated Smad1 (pSmad1) and phosphorylated Smad1,5,8 (p–Smad1,5,8). Results: Immunocytochemistry demonstrated cytoplasmic localization of Smad1 in both ONH astrocytes and LC cells. Immunocytochemistry also demonstrated cytoplasmic and nuclear localization of Co–Smad4 in ONH astrocytes and LC cells. Western blot analysis demonstrated Smad1 and Co–Smad4 in cell lysates of both ONH astrocytes and LC cells. There was no significant upregulation of Smad1 or Smad 4 following BMP–4 treatment for 10 minutes or 60 minutes. However, exposure to BMP–4 for 10 minutes or 60 minutes caused an increase in p–Smad1 protein levels. Likewise, exposure to BMP–4 for 20 minutes and 12 hours caused an increase in p–Smad1,5,8 protein levels. Conclusions: These studies demonstrate that ONH astrocytes express Smad1 and Smad4 and are capable of responding to exogenous BMP–4 via receptor Smads. Thus, cells of the ONH may be targets for and respond to locally released BMP.
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