Article

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

Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Investigative ophthalmology & visual science (impact factor: 3.43). 07/2012; 53(8):4952-62. DOI:10.1167/iovs.12-9681 pp.4952-62
Source: PubMed

ABSTRACT 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.

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Keywords

actin cytoskeletal dynamics
 
actin cytoskeleton
 
Connective tissue growth factor
 
contractile properties
 
CTGF influences ECM synthesis
 
CTGF levels
 
CTGF protein levels
 
enucleated porcine eyes
 
extracellular matrix
 
gene expression profile
 
HTM cells
 
human trabecular meshwork
 
immunoblot analyses
 
laminin levels
 
MLC phosphorylation
 
myosin light chain
 
perfused porcine eyes
 
TM cells
 
trabecular meshwork
 
various fibrotic diseases