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L. Tong, J.-K. Siak, K. G. Samivelu, S.-H. Yeo, J. Chew, R. W. Beuerman; The Effect of Transglutaminase-2 Over-Expression in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5194.
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Currently, we aim to investigate the effects of over-expressing transglutaminase (TGM)-2 in corneal epithelial cells. Previous studies have shown that TGM-2, an important multi-functional enzyme, is critically involved in Sjogren syndrome, Meibomian gland disease, allergic conjunctivitis and pterygium; and has an important role in corneal wound healing. We have shown that osmotically stressed corneal epithelial cells up-regulated TGM-2 protein and crosslinking activity, and that TGM-2 also played a role in UVB-induced cell death.
Human corneal epithelial cells (HCE-T) were cultured and transfected with a cDNA plasmid that expressed full length human TGM-2 protein. Confirmation of over-expression was using relative quantitative RT-PCR, Western blot and immunofluoresecent staining. Alteration of transamidase activity was quantified using a microtiter plate assay based on cross-linking of the CBZ-Gln-Gly substrate to biotin cadaverine (Covalab). Global gene expression was profiled using the Affymetrix human exon array, performed on triplicate samples. Analysis was performed using the Genespring GX 10.0 and the Partek Genomics suite softwares.
Cells transfected for 24 hours were verified to show increased TGM-2 expression relative to vector control. Increased TGM activity was seen in cells over-expressing TGM-2. Expression signals in the various exons were averaged to obtain whole transcript signals, which were then normalized to the median chip signals. Out of 17881 transcripts, 14972 passed the quality control step (based on signal strength criteria). Hierarchical clustering on the expression of 14972 genes revealed clusters where TGM-2 over-expression up-regulated specific genes. A high proportion of genes in these clusters were encoding for extracellular proteins, membrane proteins, ubiquitin pathway regulators and elements of the interferon pathway. These clusters were examined in detail for statistically significant changes of individual transcripts. The late envelope protein 1D was noted to be up-regulated 2.4 fold. Other up-regulated genes include dynamin-3, retinoic acid early transcript 1L and the retinoic acid receptor responder (tarzarotene induced)-3.
Over-expression of TGM-2 in corneal epithelial cells was sufficient to increase cellular transamidase activity. A gene signature pattern of increased protein processing, structural protein and extracellular protein was notable in cells over-expressing TGM-2. TGM-2 may be a useful target for addressing extracellular matrix diseases in the ocular surface.
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