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T.O. Tovar, A.F. Clark, R.J. Wordinger; Cultured Human Trabecular Meshwork Cells Respond to Exogenous BMP–4 via the Smad Signaling Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1855.
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The bone morphogenetic proteins (BMP) are members of the TGF–ß superfamily of growth factors. We have previously demonstrated that trabecular meshwork cells (TM) are capable of secreting BMPs and that BMP–4 selectively counteracts the action of TGF–ß2 in TM cells with respect to synthesis of extracellular matrix proteins. BMPs can signal via the Smad canonical pathway or via non–canonical pathways (e.g. p38). In the canonical pathway, signaling is activated upon ligand binding through serine/threonine kinase type I and type II receptors. This signal then recruits intracellular Smad proteins. Following the recruitment of common Smad 4, the signaling complex translocates into the nucleus to activate gene expression. The purpose of this study was to determine if cultured human TM cells (a) express Smad pathway proteins and (b) respond to exogenous BMP–4 via the Smad pathway.
Human TM cells (N=3) were grown until approximately 80% confluent and treated in a time dependent manner or at 48 hours with BMP–4 (20ng/ml) in serum free media. Untreated cell lines acted as controls. Western Blot analysis was used to demonstrate the presence of phosphorylated Smad1, total Smad1, Smad4, and Smad 5. Intracellular localization of Smad5, Smad6, Smad7, Smad4, phosphorylated Smad1 (pSmad1), phosphorylated Smad1,5,8 (pSmad1,5,8) was studied via immunocytochemistry.
Western blot analysis demonstrated the presence of all Smad signaling proteins in human TM cells. Immunocytochemistry demonstrated the presence and increased expression of Smad7 and pSmad1,5,8 following 48 hours of BMP–4 treatment. Immunocytochemistry also demonstrated the presence of pSmad1, total Smad1, Smad4, and Smad5.
These studies demonstrate that human TM cells express proteins for the canonical Smad pathway and respond to exogenous BMP4. Since we have previously demonstrated that human TM cells secrete BMP–2, BMP–4 and BMP–5, it is possible that secreted BMPs act on TM cells via an autocrine signaling mechanism.
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