Regulation of bradykinin B2 receptors by the ras oncogene: evidence for multiple mechanisms.
The objective of this study was to probe the molecular mechanisms underlying the increase in sensitivity of cells to bradykinin (BK) following expression of a transforming Ha-ras oncogene. We used native NIH3T3 fibroblast (3T3) cells and 3T3 cells transfected with a glucocorticoid sensitive oncogenic Ha-ras construct (DT cells). DT cells incubated in the presence of 1 microM dexamethasone (DEX) for 24 hr expressed a relatively high level of membrane-bound Ha-Ras protein, BK B2 receptor mRNA, and B2 receptor binding as determined by Western blotting with anti-Ha-Ras antibodies, reverse transcriptase-polymerase chain reaction using B2 receptor-specific primers, and specific [3H]BK binding, respectively. BK also stimulated a significant B2 receptor-mediated increase in [3H]thymidine incorporation in the cells both alone and in synergy with epidermal growth factor. In the absence of DEX, the DT cells expressed a considerably lower but yet clearly significant level of Ha-Ras. Under this condition, receptor mRNA and receptor binding remained maximally expressed. On the other hand, BK was unable to stimulate any increase in [3H]thymidine incorporation. In contrast to DT cells, no Ha-Ras, receptor mRNA, receptor binding, or BK-stimulated, B2 receptor-mediated [3H]thymidine incorporation was detected in 3T3 cells (+/- DEX). However, BK stimulated a transient increase in the level of intracellular free Ca2+ in the 3T3 cells indicating that these cells express a small number of functional B2 receptors. In all, these results show that oncogenic Ha-Ras regulates the sensitivity of 3T3 cells to BK through at least two different mechanisms. One mechanism occurs at a relatively low level of Ha-Ras and involves an increase in B2 receptor mRNA and expressed B2 receptor levels, and another mechanism occurs at a relatively high level of Ha-Ras and involves an increase in B2 receptor-mediated mitogenic signaling.
Hembree
T N
TN
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284, USA.
Leeb-Lundberg
L M
LM
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics
D017931:DNA Primers; D012333:RNA, Messenger; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; C012258:mevastatin; D003907:Dexamethasone; D008148:Lovastatin; D001920:Bradykinin; D002118:Calcium