期刊
ENDOCRINOLOGY
卷 149, 期 10, 页码 4821-4828出版社
ENDOCRINE SOC
DOI: 10.1210/en.2008-0145
关键词
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资金
- Cancer Research Training Program (CRTP)
- Nova Scotia Health Research Foundation
- Canadian Institutes of Health Research [MOP12895, ROP84154]
- Canada Breast Cancer Foundation
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/17 beta-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-guanidinoethylthiopropanoic acid (MGTA). MGTA also decreased MCF-7 cell survival. In Arg-free medium, annexin-V staining showed that apoptotic MCF-7 cells (similar to 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 <= 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.
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