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A. Sethi, R. J. Wordinger, A. F. Clark; Gremlin Alters the Trabecular Meshwork Extracellular Matrix via TGFβ/Smad Signaling. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5836.
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Transforming growth factor beta 2 (TGFβ2) is elevated in glaucomatous aqueous humor (AH). TGFβ2 increases AH outflow resistance and increases extracellular matrix (ECM) deposition in the TM. BMP4 and BMP7 inhibit TGFβ2-mediated ECM deposition in cultured TM cells. Expression of the BMP antagonist gremlin is higher in glaucomatous TM cells. Gremlin blocks the BMP4 inhibition of TGFβ2 and also increases IOP in perfusion organ cultured eyes. The purpose of the study is to determine whether gremlin alone can influence TM cell ECM or signal transduction pathways.
Human TM cells were cultured in the presence or absence of recombinant mouse gremlin (100-5000 ng/mL) for 1-48 hours, and total RNA or protein lysates and conditioned medium were harvested from the cells. Effects of gremlin treatment on ECM gene and protein expression were assayed by RT-PCR and western immunoblotting, respectively. To determine effects of TGFβ2 on gremlin cells were treated with TGFβ2 (0.1-10 ng/mL) for 48 hours, and total RNA and protein lysates were harvested for RT-PCR and western immunoblotting. Gremlin-treated TM cells were also examined for Smad2/3, p38, and JNK activation using western immunoblotting. Cells were treated with Smad3 inhibitor SIS3 (5 uM) and TGFβ receptor inhibitors, LY364947 and SB431542 (5 uM), with or without gremlin, to examine the involvement of the TGFβ/Smad signaling pathway.
TGFβ2 induced the expression of gremlin protein in a dose-dependent manner. Gremlin also induced TGFβ2 mRNA. Gremlin induced the protein and mRNA expression of ECM components like fibronectin, collagen-1, PAI-1 and tropoelastin in a dose- and time-dependent manner. Gremlin also induced mRNA expression of the ECM components. Gremlin time dependently increased Smad2/3 phosphorylation, but did not activate p38 or JNK. Treatment with Smad3 and TGFβ receptor inhibitors blocked the gremlin induction of ECM proteins.
Gremlin increased the expression of ECM proteins in the TM. Gremlin and TGFβ2 appeared to induce the expression of each other in a feed forward loop. Gremlin also activated the canonical TGFβ/Smad signaling in TM. Moreover, gremlin’s effects are inhibited upon blocking the TGFβ/Smad signaling. These data imply that gremlin functions not only blocking endogenous BMPs but also enhancing the effect of endogenously expressed TGFβ2 in cultured TM cells.
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