Abstract
Purpose::
The characteristic cupping of the optic nerve head (ONH) in glaucoma is associated with elevated TGF-ß2 expression and altered synthesis and deposition of extracellular matrix (ECM) proteins. Bone morphogenetic proteins (BMP) have been reported to selectively inhibit the fibrotic action of TGF-ß2. We hypothesize that in glaucoma, elevated TGF-ß2 levels in the ONH induce expression of gremlin, a secreted BMP antagonist, which selectively blocks BMP antagonism of TGF-ß2 signaling. This antagonism would result in increased ECM synthesis and deposition. The purpose of this initial study was to demonstrate that A) human ONH astrocytes and lamina cribrosa (LC) cells express and secrete gremlin, B) TGF-ß2 treatment upregulates gremlin expression in ONH astrocytes and LC cells, and C) gremlin inhibition of BMP-4 increases TGF-ß2- stimulated fibronectin (FN) production in cultured human ONH astrocytes and LC cells.
Methods::
Human ONH astrocytes (N=4) and LC cells (N=5) were utilized. RT-PCR, Affimatrix gene chip, western blot, and immunostaining were used to demonstrate mRNA expression and protein levels of gremlin. Western blot was used to demonstrate gremlin protein in human ONH tissues. The effect of TGF-ß2 (5ng/ml) on gremlin secretion by ONH astrocytes and LC cells was examined by western blot analysis of conditioned media. To examine BMP-4 inhibition of TGF-ß2 signaling, ONH astrocytes and LC cells were treated exogenously with a) TGF-ß2 (5ng/ml) or BMP-4 (10ng/ml), b) TGF-ß2 and BMP-4, c) TGF-ß2, BMP-4 and gremlin (300ng/ml), and FN content in conditioned media and cell lysate was assessed by western blot and ELISA.
Results::
ONH astrocytes and LC cells express, synthesize and secrete gremlin. Western blot analysis demonstrated that gremlin protein is present in ONH tissues and that exogenous TGF-ß2 treatment significantly increased gremlin secretion in conditioned media of ONH astrocytes and LC cells. Western blot analysis also demonstrated that TGF-ß2 significantly increased FN synthesis and secretion. Co-incubation of BMP-4 and TGF-ß2 reduced TGF-ß2 stimulated synthesis and secretion of FN, and addition of gremlin to this regime increased synthesis and secretion of FN.
Conclusions::
Elevated TGF-ß2 in the glaucomatous ONH may induce the secretion of the BMP antagonist gremlin that blocks the inhibitory action of BMP-4 leading to increased ECM deposition. ECM modulation by gremlin provides a novel therapeutic target for glaucoma.
Keywords: extracellular matrix • lamina cribrosa • growth factors/growth factor receptors