Browsing by Subject "TGFb2"
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Item BMP4 INDUCED ID PROTEIN PROTECTS TM FROM GLAUCOMATOUS EFFECTS OF TGFβ-2(2014-03) Mody, Avani A.; Wordinger, Robert J.; Clark, Abbot F.Insight into the BMP4 pathways in various disease models and different cell types has shown BMP4 to be a potent inducer of inhibitor of DNA binding proteins (ID1 and ID3). ID1 and ID3 are negative regulators of basic Helix loop Helix (bHLH) transcription factors and are known to control specific gene expression, including extracellular cellular matrix (ECM) genes. We previously have shown that BMP4 attenuates the pathogenic effects of TGFβ2 in the TM, an ocular tissue involved in regulation of intraocular pressure in glaucoma. We hypothesize that BMP-4 attenuates the effects TGFβ2 in the TM by inducing ID1 and ID3 expression. In our current study, we show that BMP4 induces ID1 and ID3 expression in TM cells. Over-expression and knockdown of ID1 and ID3 in TM cells show that ID1 and ID3 play a crucial role in attenuating the profibrotic effects of TGFβ2 in TM cells. Purpose (a): Increased aqueous humor (AH) outflow resistance causes high intraocular pressure (IOP), which is a critical risk factor in primary open-angle glaucoma. Elevated transforming growth factor b2 (TGFβ2) in the AH of glaucoma patients increases extracellular matrix (ECM) protein deposition in the trabecular meshwork (TM), thereby elevating IOP. Bone morphogenetic protein 4 (BMP4) inhibits the pathogenic effects of TGFβ2 in the TM. However, the underlying molecular mechanism for this BMP4 inhibition remains unknown. BMP4 regulates various cellular processes by induction of inhibitors of DNA binding proteins (ID1, ID3), which are transcriptional regulators that bind specific transcription factors and suppress their functions. This study will determine whether ID1/ID3 are downstream targets of BMP4, attenuating the TGFb-2 effects on TM cells. Methods (b): Cultured primary human TM cells and the GTM3 cell line were treated with BMP4 (5-10ng/ml) for 1-48 hrs. Q-PCR and western immunoblotting were performed to determine ID1 and ID3 expression. GTM3 and primary TM cells were transfected with ID1 and ID3 expression plasmids vectors or ID1 and ID3 siRNA to determine the effects of ID1 and ID3 on TGFβ2 induced extracellular matrix (ECM) proteins. The expression of fibronectin and plasminogen activator inhibitor-1 (PAI-1) was studied by western immunoblotting. Results (c): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma. Conclusions (d): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma.Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(2017-12-01) Hernandez, Humberto; McDowell, Colleen; Clark, Abbot F.; Pang, Iok-HouThe trabecular meshwork (TM) is the main site of outflow resistance in primary-open angle glaucoma (POAG) patients. In these patients, aqueous humor outflow resistance increases, subsequently leading to a rise in intraocular pressure (IOP). The rise in IOP ultimately damages the optic nerve and leads to blindness. Accumulation of extracellular matrix (ECM) at the TM has been shown by our laboratory and many others to be responsible for the increase in outflow resistance. The molecular mechanisms underlying the pathology are beginning to be elucidated. The pro-fibrotic cytokine, transforming growth factor beta-2 (TGFβ2), has been shown to be elevated in the aqueous humor of POAG patients. Mice injected with adenovirus encoding active TGFβ2 develop ocular hypertension and ECM deposition at the TM. Recently, toll-like receptor 4 (TLR4) signaling has been linked to the development of fibrosis. In our studies, we evaluated the crosstalk between TGFβ2 and TLR4 in the TM. We utilized in vitro and in vivo models to evaluate the role of TLR4 on the production of ECM and development of ocular hypertension. We also utilized a conditional knockout in vitro and in vivo adenovirus delivery system to study BMP and Activin Membrane Bound Inhibitor (BAMBI), a critical molecule in the crosstalk between TGFβ2 and TLR4. Our studies reveal a novel pathway involved in the development of TM damage and potential targets to lower IOP.Item TGF Beta 2 and Gremlin Signaling Pathways Regulate Extracellular Matrix Changes in TM Cells: Implications for Glaucoma(2011-05-01) Sethi, Anirudh; Clark, Abbot F.Purpose: Glaucoma is a leading cause of blindness worldwide. The leading risk factor is elevated intraocular pressure (IOP) due to fibrotic changes in the trabecular meshwork (TM) tissue. The profibrotic cytokine TGFβ2 is elevated in the aqueous humor and TM of glaucomatous eyes. TGFβ2-mediated extracellular matrix (ECM) deposition in the TM appears to be responsible for increased IOP in ex vivo and in vivo models. Bone morphogenetic proteins (BMPs) inhibit TGFβ regulation of ECM, and elevated levels of the BMP antagonist gremlin in the glaucomatous TM restores the fibrotic response of TGFβ2. Lysyl oxidase (LOX) is a collagen and elastin polymer crosslinking enzyme, and recent genome wide association studies showed that SNPs in LOXL1, a LOX family member, significantly increased the risk of developing exfoliation glaucoma. The overall aim of my work is to delineate the signaling pathways involved in TGFβ2 and gremlin alteration of the TM ECM and to evaluate the potential role of the LOX family of cross-linking enzymes in this TM ECM remodeling. Methods: Human TM cells were cultured in the presence or absence of recombinant human TGFβ (0.1-10 ng/ml) or mouse gremlin (100-5000 ng/ml) for 1-72 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 qRT-PCR and western immunoblotting, respectively. TGFβ2- and gremlin-treated TM cells were also examined for Smad2/3, p38, and JNK activation using western immunoblotting. Cells were treated with ii Smad3 inhibitor SIS3 (5 uM), TGFβ receptor inhibitors LY364947 and SB431542 (5 uM), JNK inhibitor SP600125 (10 uM), and AP-1 inhibitor SR11342 (5 uM) with or without TGFβ2/gremlin, to examine the involvement of the TGFβ/Smad signaling pathway. Results: TGFβ2 and gremlin induced expression of each other in TM cells. TGFβ2 activated both Smad and non-Smad pathways and strongly induced mRNA and protein expression of all 5 LOX genes. Using a novel LOX activity assay, we observed greater ECM crosslinking in TGFβ- treated cells. Gremlin elevated ECM gene and protein expression via the Smad signaling pathway. Conclusions: The TGFβ induction of LOXs and gremlin-induction of ECM proteins highlight the complex interplay of Smad and the non-Smad signaling in regulating the TGFβ response. TGFβ2 and gremlin are profibrotic in a feed forward loop. The TGFβ response in TM cells involves evasion of BMP inhibition by gremlin induction and also ECM crosslinking through the LOX enzymes.