Abstract
Lens epithelium-cell derived growth factor (LEDGF) is a transcriptional activator. It protects the cells by binding to cis-stress response ((A/T)GGGG(T/A)), and heat shock (HSE; nGAAn) elements in the stress genes and activating their transcription. Transforming growth factor-beta (TGF-beta) has been implicated in the control of tissue homeostasis, terminal differentiation, and apoptosis. Here we provide evidence that TGF-beta1 down-regulates LEDGF expression and diminishes its affinity for DNA during TGF-beta1-induced phenotypic changes and apoptosis in human lens epithelial cells. Surprisingly, TGF-beta1 treatment for 48 h markedly decreased the LEDGF, Hsp27, and alphaB-crystallin promoter activities with the decrease of abundance of LEDGF mRNA and protein. Deletion mutants of the LEDGF promoter showed that one TGF-beta1 inhibitory element (TIE) like sequence nnnTTGGnnn (-444 to -433) contributed to this negative regulation. Mutation of TIE (TTGG to TATT) abolished the down-regulation of the LEDGF promoter. Gel mobility and supershift assays showed that LEDGF in the nuclear extracts of TGF-beta1-treated human lens epithelial cells did not bind to stress-response elements and HSE. The TGF-beta1-induced down-regulation of LEDGF, Hsp27, and alphaB-crystallin promoters activity was reversed by cotransfection with a plasmid expressing LEDGF. Because overexpression of LEDGF was able to relieve TGF-beta1 and/or stress-induced changes, it would be a candidate molecule to postpone age-related degenerating disorders.
Highlights
Transforming growth factor  (TGF-)1 is a multifunctional regulator of cell growth and differentiation
We demonstrated that TGF-1 induced phenotypic changes, expression of ␣ smooth muscle actin (␣ sm-actin), suppression of Hsps in hLECs, and apoptosis, and that these changes were associated with the repression of transcription and reduction of protein of Lens epithelium-cell derived growth factor (LEDGF)
TGF-1 Treatment Induced the Phenotypic Changes, Apoptosis, and Expression of ␣ sm-actin—HLECs were exposed for 48 h to the various concentrations (5.0, 1.0, 0.5, and 0.25 ng/ml) of TGF-1 to induce the alterations in these cells, and MTS assay was performed
Summary
To perform RT-PCR, either total RNA or mRNA was extracted from hLECs after treatment with or without TGF-1 (1 ng/ml). Constructs of different sizes (Fig. 6A, construct B, Ϫ1461, C; Ϫ1285, D; Ϫ482, E; Ϫ315 to ϩ35) of LEDGF promoter were prepared with appropriate sense primers bearing SacI or MluI and antisense with NheI (see Table I) and ligated into pCAT-Basic vector (Promega, Madison, WI) using appropriate restriction enzymes. The forward primer having a SacI site (5Ј-CTCTCTTCCAAGAGCTCACAAAG-3Ј) and the reverse primer having an XhoI site (5Ј-ATGGTGGCTACTCGAGAGTGA-3Ј) were used to generate the above fragment and cloned between EcoRI sites of the TA vector These Hsp and ␣B-crystallin/TA constructs were digested with SacI and XhoI (Ϫ214 to ϩ21), and the promoter fragments were ligated with pCAT-Basic vector (Promega, Madison, WI) using appropriate restriction enzymes. To examine the effect of TGF-, transfected and untransfected cells were treated with TGF-1 at 1 ng/ml for 48 h
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