98th Annual Meeting DOG 2000

P 10

The expression of the protein "Regulator of G-Protein Signaling-5" is upregulated in cultured human RPE cells

N. Kociok, P. Esser, G. Thumann, A. Hueber, R. Krott, U. Schraermeyer

Introduction: Human retinal pigment epithelial cells (RPE) dedifferentiate and lose their epithelial characteristics in pathological situations e.g. proliferative vitreoretinopathy (PVR) as well as during cell culture. To learn more about this dedifferentiation process we monitored changes in gene expression of cultured RPE cells by differential expressed mRNA analysis (DEmRNA-PCR).

Methods: Total RNA of human RPE cells of passage 0 (P0) and P3 was isolated and analyzed by DEmRNA-PCR. A band with an enhanced level of expression in P3 was cut off the gel, the PCR fragment was reamplified, sequenced, and identified by homology search in the gene data bank. The sequence of the identified protein was used to select gene-specific PCR-primers. Using this primers the mRNA expression of the protein in RPE cells directly prepared from a donor eye, and from P0, and further passages was analyzed.

Results: DEmRNA-PCR analysis revealed an enhanced expression of a 400 bp PCR fragment in RPE cells from P3 compared to P0. A homology search with 243 sequenced base pairs in the gene data bank resulted in a 100% identity with the 3’-end of the coding sequence for a novel G-protein signaling regulator gene 5 (RGS5). RT-PCR with gene-specific primers confirmed the expression of RGS5 in human RPE cells, that were prepared directly from donor eyes, and from P0, P4, and P8. The mRNA expression were semi-quantified by comparing RGS5 expression with the expression of the house-keeping genes glyceraldehyde-3-phosphate dehydrogenase (GADPH) and ß2-microglobulin.

Conclusion: This is the first demonstration of RGS5 mRNA expression in RPE cells. By its GTPase-activating function RGS5 may be an important factor in the regulation of signal transduction through the G-protein pathway in proliferating RPE cells. This function was proposed for RGS5 and other members of the RGS family1.

1Seki N et al. J Hum Genet (1998) 433:202-205
Universitäts-Augenklinik, Joseph-Stelzmann-Str. 9, D-50931 Köln



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