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D.P. Singh, P. Sharma, T. Shinohara, L.T. Chylack Jr, N. Fatma; LEDGF Regulates Expression of SPARC, a Gene Having Important Biological Function in Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4499.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: LEDGF is a transcriptional survival factor and exerts its cellular survival function against stress by binding to stress response (STRE; A/TGGGG/TA) and heat shock (nGAAn) elements in the stress response genes and activating transcription. LEDGF is present in lens. SPARC has been implicated in cataractogenesis, development, remodeling tissue repair and homeostasis. We investigated the regulation of SPARC expression by LEDGF in human lens epithelial cells (hLECs). Methods: Differential-display (DD-PCR) was carried out to identify differential gene expression in normal hLECs and cells over-expressing LEDGF. RT-PCR and western blot was used to validate expression levels. 5’-flanking region of SPARC gene promoter (-638 to + 42) containing LEDGF binding sites was generated from genomic mouse DNA linked to pCAT Basic vector (Promega) and promoter activity was monitored with a transfection system. Specificity of trans-activation was confirmed with LEDGF-antisense. Gel-Shift and super-shift assays were used to assess LEDGF-DNA binding. A GFP-LEDGF cDNA construct was used to over expressed hLECs. Results: Two transcripts of SPARC gene were up-regulated in LEDGF over-expressing cells. RT-PCR confirmed over-expression of the transcripts. Western blot with specific anti-SPARC antibody showed the increased SPARC protein in cell extracts and in culture medium. Analysis of the promoter region of the SPARC gene revealed multiple LEDGF binding sites (nGAAn and A/TGGGGA/T). Gel-shift and CAT-ELISA assays confirmed binding and trans-activation of SPARC gene by LEDGF. Down-regulation of promoter activity in cells transfected with LEDGF-antisense confirmed the functional significance of LEDGF binding. Conclusions: The present study provides evidence that LEDGF regulated SPARC expression in lens epithelial cells, that may be vital for lens homeostasis and transparency.
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