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Vasanth Rao, Padma Iyer, Padmanabhan Pattabiraman, Rupalatha Maddala; Connective Tissue Growth Factor -Mediated Upregulation of Neuromedin U Expression in Trabecular Meshwork Cells and its Role in Homeostasis of Aqueous Humor Outflow. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3233.
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Connective Tissue Growth Factor (CTGF), a matricellular protein is presumed to be involved in the pathobiology of various fibrotic diseases including glaucoma. Since actin cytoskeletal integrity has been shown to influence the expression of CTGF, here we investigated the possible influence of Rho GTPase-dependent regulation of actin cytoskeletal integrity on CTGF expression in trabecular meshwork (TM) cells.
CTGF expression was detected and quantitated by immunoblotting and enzyme-linked ELISA. CTGF-induced effects on gene expression, cytoskeletal reorganization, myosin light chain phosphorylation and extracellular matrix (ECM) protein profiles were evaluated in human TM (HTM) cells using cDNA microarray, q-PCR, immunofluorescence and immunoblot analyses, respectively. The effects of Neuromedin U (NMU) on aqueous humor (AH) outflow facility were determined by perfusing enucleated porcine eyes with NMU.
Expression of a constitutively active RhoA (RhoAV14) in HTM cells led to significant increases in CTGF protein levels, both in cell lysates and cell-conditioned media. Consistent with these observations, shRNA-based suppression of G-actin-regulated serum response factor (SRF) and inhibition of Rho kinase activity by Y-27632 caused measurable decreases (P<0.05, n=4) in CTGF levels in HTM cells. Stimulation of serum starved HTM cells with CTGF for 24 hrs resulted in induction of actin stress fiber formation and increased myosin light chain (MLC) phosphorylation, along with significantly elevated levels of fibronectin and laminin. Expression of NMU, the NMU receptor (NMU-R1), Interleukin 6 and Interleukin 32 was also noted to exhibit significant increases under these conditions in HTM cells. NMU was also noted to stimulate the formation of actin stress fibers and MLC phosphorylation in porcine TM cells, and to cause a significant but transient decrease in AH outflow facility in perfused eyes.
These data reveal that CTGF influences ECM synthesis, actin cytoskeletal dynamics and contractile properties in TM cells and that the expression of this matricellular protein is closely regulated by the Rho GTPase/SRF pathway. Moreover, NMU, whose expression is upregulated by CTGF, was found to mimic the effects of CTGF on actomyosin contractile activity in TM cells, and decrease aqueous humor outflow, revealing a potentially important role for this neuropeptide in the homeostasis of aqueous humor drainage.
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