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C. N. Nagineni, V. K. Kommineni, K. V. Chalam, R. Raju, B. Detrick, J. J. Hooks; TGF-ß Upregulates Extracellular Matrix Protein Anosmin (KAL-1) Expression in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2525.
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Transforming growth factor-beta (TGF-ß) plays a critical role in extracellular matrix (ECM) synthesis, degradation and reconstitution. ECM proteins and proteoglycans are critical in maintaining choroid- retinal pigment epithelium (RPE)- retinal barrier and retinal adhesion. Therefore, we studied the effects of TGF-ßin the regulation of ECM molecule expression by human retinal pigment epithelial (HRPE) cells.
HRPE cells were prepared from human donor eyes. RNA prepared from HRPE cells treated with TGF-ßfor 8 hr was used for global gene expression analysis with Affymetrix GeneChip. The results of the expression of selected genes were validated by RT-PCR and Real-Time PCR methods. HRPE and other human cells were treated with TGF-ßin serum free medium for 24 hr and concentrated culture supernatants were used for Western blot analysis of secreted proteins.
Microarray analysis of TGF-ßtreated HRPE cell mRNA revealed several fold increase in the expression of genes related to matrix metalloproteases and growth factors such as VEGF, PDGF and CTGF. ECM proteins, chondroitin sulfate proteoglycan, fibronectin, collagen, thrombospondin and integrin beta 6 were upregulated significantly. In addition, we found that TGF-ßincreased the expression of a less known ECM gene, anosmin (KAL-1), by four-fold in HRPE. Increased expression of anosmin mRNA was validated by Real-Time PCR analysis. Anosmin protein was detected in culture supernatants of HRPE and corneal cells treated with TGF-ß The addition of heparin sulfate to the cell cultures resulted in increased quantity of anosmin in the culture medium suggesting its adhesion to cell surface after secretion by the cells.
Microarray analysis identified expression of mRNA for anosmin, an ECM protein, in HRPE cells. This is the first report to demonstrate the expression of anosmin in human ocular tissues. Our results show, for the first time, that TGF-ßregulates mRNA and protein expression of anosmin. The role of anosmin, an ECM protein with adhesion function, in corneal and retinal structure, function and path-physiology remains to be investigated.
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