Connective tissue growth factor-mediated upregulation of neuromedin U expression in trabecular meshwork cells and its role in homeostasis of aqueous humor outflow

Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated the effects of Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes in gene express...

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Bibliographic Details
Published in:Investigative ophthalmology & visual science 2012-07, Vol.53 (8), p.4952-4962
Main Authors: Iyer, Padma, Maddala, Rupalatha, Pattabiraman, Padmanabhan P, Rao, Ponugoti Vasantha
Format: Article
Language:English
Subjects:
Animals
Aqueous Humor - physiology
Connective Tissue Growth Factor - metabolism
Extracellular Matrix Proteins - genetics
Extracellular Matrix Proteins - metabolism
Gene Expression Profiling
Homeostasis - physiology
Neuropeptides - metabolism
Phosphorylation
rhoA GTP-Binding Protein - genetics
rhoA GTP-Binding Protein - metabolism
Swine
Trabecular Meshwork - cytology
Trabecular Meshwork - metabolism
Up-Regulation
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Summary:Connective tissue growth factor (CTGF) is a matricellular protein presumed to be involved in the pathobiology of various fibrotic diseases, including glaucoma. We investigated the effects of Rho GTPase-dependent actin cytoskeletal integrity on CTGF expression and CTGF-induced changes in gene expression profile in human trabecular meshwork (HTM) cells. CTGF levels were quantified by immunoblotting and ELISA. CTGF-induced changes in gene expression, actin cytoskeleton, myosin light chain (MLC) phosphorylation, and extracellular matrix (ECM) proteins were evaluated in trabecular meshwork (TM) cells by cDNA microarray, q-PCR, fluorescence microscopy, and immunoblot analyses. The effects of neuromedin U (NMU) on aqueous humor (AH) outflow were determined in enucleated porcine eyes. Expression of a constitutively active form of RhoA (RhoAV14), activation of Rho GTPase by bacterial toxin, or inhibition of Rho kinase by Y-27632 in HTM cells led to significant but contrasting changes in CTGF protein levels that were detectable in cell lysates and cell culture medium. Stimulation of HTM cells with CTGF for 24 hours induced actin stress fiber formation, and increased MLC phosphorylation, fibronectin, and laminin levels, and NMU expression. NMU independently induced actin stress fibers and MLC phosphorylation in TM cells, and decreased AH outflow facility in perfused porcine eyes. These data revealed that CTGF influences ECM synthesis, actin cytoskeletal dynamics, and contractile properties in TM cells, and that the expression of CTGF is regulated closely by Rho GTPase. Moreover, NMU, whose expression is induced in response to CTGF, partially mimics the effects of CTGF on actomyosin organization in TM cells, and decreases AH outflow facility, revealing a potentially important role for this neuropeptide in the homeostasis of AH drainage.
ISSN:1552-5783
0146-0404
1552-5783
DOI:10.1167/iovs.12-9681