EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase.
The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.
Lopes
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Disciplina de Biologia Molecular, Universidade Federal de São Paulo, Rua Três de Maio 100, CEP 04044-020 São Paulo, SP, Brazil.
Toma
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Pinhal
M A S
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Porcionatto
M A
MA
Sogayar
M C
MC
Dietrich
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CP
Nader
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HB
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D005434:Flow Cytometry; D016193:G1 Phase; D019812:Heparan Sulfate Proteoglycans / Q000096:biosynthesis; D006801:Humans; D015513:Oncogene Proteins / Q000378:metabolism*; D011817:Rabbits; D016196:S Phase; D015398:Signal Transduction; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D053671:Syndecan-4 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D019812:Heparan Sulfate Proteoglycans; D015513:Oncogene Proteins; D053671:Syndecan-4; C077183:human oncogene protein EJ-ras; D015238:Sulfotransferases; C021350:heparitin sulfotransferase; D002238:Carbohydrate Epimerases; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase; D001973:Bromodeoxyuridine