The effect of transforming growth factor beta–1 (TGF–ß1) on gene expression profiles in lamina cribrosa cells from the human optic nerve head

Abstract

Abstract: : Purpose:In primary open angle glaucoma (POAG) the extracellular matrix (ECM) of the lamina cribrosa (LC) is extensively degraded and remodeled. Elevated TGF–ß1 has been shown in lamina cribrosa tissue of monkeys with experimental glaucoma and elevated TGF–ß2 has been shown in lamina cribrosa tissue of glaucomatous humans. LC cells may respond to elevated TGF–ß in the glaucomatous lamina cribrosa and play a role in ECM synthesis and degradation. We, therefore, investigated the effect of TGF–ß1 on global gene expression, matrix metalloproteinase–2 (MMP2) activity and tissue inhibitor of matrix metalloproteinase–2 (TIMP2) release in LC cells in–vitro. Methods:Primary cultures of LC cells were generated from the optic nerve head tissue of three normal human donors. Confluent cells (n=3) were treated for 24 hours with human TGF–ß1 (10ng/ml) or vehicle (4mM HCL/1%BSA). Gene expression profiles were determined using Affymetrix^TM U133A microarrays and confirmed in independent experiments using quantitative TaqMan^TM PCR and ELISA. MMP2 protein activity in LC cell media was determined using gelatin zymography and TIMP2 release was measured using ELISA. Promoter sequences of the human and mouse homologues of TGF–ß1 responsive genes in our system were aligned using Genomatix DiAlign^TM and transcription factor binding sites were determined using Genomatix MatInspector^TM software. Results:TGF–ß1 induced differential expression of 2817 genes. Prominent among the up–regulated genes were those involved in regulation of transcription (Jun–B, STAT–5), cell proliferation (VEGF, PDGF–A, bFGF), ECM accumulation (TGF–ß2, CTGF, fibronectin, collagen VI) and inflammation (IL–8, IL–11). TGF–ß1 induced significant increases in MMP2 activity (p<0.01) and TIMP2 release (p<0.01). Up–regulated genes with potentially novel TGF–ß1 response elements included ADAMTS1 and MMP16. We identified putative transcription factor binding sites (Sp1, PAX) in evolutionary conserved sequences in the promoters of these genes. Conclusion:Exposure of LC cells to TGF–ß1 induces expression and release of ECM components, which we believe facilitate matrix remodeling in the lamina cribrosa. We have also identified potentially novel TGF–ß1 responsive genes that degrade the ECM. Targeted inhibition of TGF–ß1 mediated pathways in LC cells may reduce ECM remodeling in the glaucomatous lamina cribrosa.

Keywords: extracellular matrix • lamina cribrosa • growth factors/growth factor receptors