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H. Yang, X. Yang, E. Chaum; A Reassessment of IGF Binding Protein Gene Expression in the Human Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2280.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Recent experiments in our laboratory suggest that there is a broader IGF binding protein (IGFBP) gene expression profile in the RPE than has previously been recognized. In these studies we quantified the expression profile of the IGFBP gene family in human RPE cells using sensitive RT-PCR and ELISA techniques. Methods: RPE cells were isolated from donor eyes of ten patients and maintained in DMEM medium. Primary explants were cultured at confluence for 72 hours. Media was collected for ELISA assays and the cells were lysed for RNA isolation. RT-PCR was performed with gene-specific primers for IGFBP-1 through -6. IGFBP-2 and IGFBP-3 secretion into the media was quantified by ELISA and correlated with gene-specific transcription levels. Results: Our studies showed that recently explanted RPE cells can express all of the IGFBPs. IGFBP-3, -5, and -6 were expressed in all RPE cell lines. IGFBP-1 was expressed at low levels in three cell lines. IGFBP-2 expression was seen in five cell lines and showed a pattern of co-expression with IGFBP-4. In later passages in vitro there was a trend toward increased transcription of IGFBP-2, -5, and -6. ELISA assays demonstrated IGFBP-3 secretion by all RPE cell lines (range, 29±3 to 56±6 ng/ml). Similarly, IGFBP-2 protein synthesis and secretion (range, 7±0.3 to 39±6 ng/ml) was directly correlated with the level of IGFBP-2 transcription. Conclusions: IGFBP family genes are ubiquitously expressed in explanted RPE cells. IGFBP-3, -5, and -6 are constitutively expressed, but RPE cells have the ability to express all six IGFBPs. The expression profile of IGFBP-1, -2, and -4 appears to be variable. ELISA studies substantiated a quantitative correlation between gene-specific transcription and protein synthesis. Further correlations of IGFBP family transcription and protein synthesis are in progress.
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