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C. Cai, M. A. Fields, L. V. Del Priore; CD46 Transfection Leads to Changes in the Expression of the Critical Transcription Factor MITF in RPE. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1678.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies in our lab reveal transcription factors in RPE important for retina development; artificially introducing MITF (microphthalmia-associated transcription factor) and RARG (retinoic acid receptor, gamma) into stem cells may direct pluripotent stem cells to differentiate into RPE cells. In addition CD46, a complement regulatory protein located on the basal surface of the retinal pigment epithelium, is known to be important to regulating RPE functions. Herein we studied the effect of over-expression of CD46 in RPE on gene expressions of other important transcription factor genes in RPE.
Immortalized ARPE-19 cells were cultured until 50% confluence and mammalian expression construct of CD46, MITF or ARAG was transfected into ARPE19 for 48-60 hours. ARPE19 cells then were collected and total RNA were isolated using Qiagen’s RNAeasy Kit. Oligo primers for CD46, MITF and ARAG were designed with LC Primer Design software. Real-time quantitative polymerase chain reaction (Roche LightCycler) was used to study the expression levels of CD46, MITF and ARAG genes and RT-PCR data were analyzed with Lightcycler3 Data Analysis software.
48 hours after CD46 transfections, MITF mRNA are expressed in 102 higher level in RPE compared with untransfected control group in RPE while ARAG mRNA expression was not affected by CD46 transfection. With RARG gene transfection, MITF mRNA was express at 103 times higher level compared with untransfected RPE cells. Apparently MITF transfection does not affect RARG gene expression levels in RPE cells.
The results suggest that transcription factor MITF and ARAG interact with each other to regulate their gene expression levels. To our knowledge it is the first report that CD46 in RPE cells may play an important role to regulate transcription factor MITF.
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