Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway.
The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450(SCC)), 17alpha-Hydroxylase/17,20-lyase (P450(c17)) and 3beta-Hydroxysteroid dehydrogenase (3betaHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450(scc) P450(c17) and 3betaHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21(ras) function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway.
Wu
C-H
CH
Graduate Institute of Medicine, Kaohsiung Medical University Kaohsiung, 80317, Taiwan.
Chen
Y-F
YF
Wang
J-Y
JY
Hsieh
M-C
MC
Yeh
C-S
CS
Lian
S-T
ST
Shin
S-J
SJ
Lin
S-R
SR
D000302:Adrenal Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011905:Genes, ras / Q000502:physiology*; D006801:Humans; D006854:Hydrocortisone / Q000096:biosynthesis*; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D007544:Isopropyl Thiogalactoside; D006913:Hydroxysteroid Dehydrogenases; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D006854:Hydrocortisone