Prolactin and estrogen up-regulate carboxypeptidase-d to promote nitric oxide production and survival of mcf-7 breast cancer cells.
Carboxypeptidase-D (CPD) and carboxypeptidase-M (CPM) release C-terminal arginine (Arg) from polypeptides, and both are present in the plasma membrane. Cell-surface CPD increases intracellular Arg, which is converted to nitric oxide (NO). We have reported that prolactin (PRL) regulated CPD mRNA levels in MCF-7 breast cancer cells. This study examined PRL/17beta-estradiol (E2) regulation of CPD/CPM expression, and the role of CPD in NO production for survival of MCF-7 cells. We showed that PRL or E2 up-regulated CPD mRNA and protein expression. PRL/E2 increased CPD mRNA levels by 3- to 5-fold but had no effect on CPM. In Arg-free DMEM, exogenous L-Arg or substrate furylacryloyl-Ala-Arg (Fa-Ala-Arg) increased NO levels and cell survival, measured using 4,5-diaminofluorescein diacetate and the MTS assay, respectively. In the presence of Fa-Ala-Arg, NO production was enhanced by PRL and/or E2 but inhibited by CPD/CPM-specific inhibitor, 2-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MGTA). MGTA also decreased MCF-7 cell survival. In Arg-free medium, annexin-V staining showed that apoptotic MCF-7 cells (approximately 60%) were rescued by Fa-Ala-Arg (25%) or diethylamine/NO (10%). Finally, CPD or CPM gene expression was knocked down with small interfering (si) CPD or siCPM, respectively, with nontargeting siNT as controls. In Arg-free DMEM, the stimulatory effect of Fa-Ala-Arg on NO production was inhibited by siCPD only, showing that CPD depletion inhibited Fa-Ala-Arg cleavage. Furthermore, more than 60% of siCPD-transfectants were apoptotic, and L-Arg, not Fa-Ala-Arg, significantly decreased apoptosis to 32% (P<or=0.05). Thus, CPD gene knockdown did not affect L-Arg uptake, which protected cells from apoptosis. In summary, PRL/E2-induced cell-surface CPD released Arg from extracellular substrates, increased intracellular NO, promoted survival and inhibited apoptosis of MCF-7 cells.
Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5.
Salma A
SA
Abdelmagid
Catherine K L
CK
Too
D015097:3-Mercaptopropionic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001120:Arginine / Q000494:pharmacology; D001943:Breast Neoplasms / Q000473:pathology / Q000503:physiopathology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004151:Dipeptides / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D011388:Prolactin / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D004151:Dipeptides; D011480:Protease Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D004958:Estradiol; C028060:furylacryloylalanylarginine; C029729:2-mercaptomethyl-3-guanidinoethylthiopropionic acid; D011388:Prolactin; D001120:Arginine; D015097:3-Mercaptopropionic Acid; C094315:metallocarboxypeptidase D