Prolactin and estrogen up-regulate carboxypeptidase-d to promote nitric oxide production and survival of mcf-7 breast cancer cells

Endocrinology. 2008 Oct;149(10):4821-8. doi: 10.1210/en.2008-0145. Epub 2008 Jun 5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Mercaptopropionic Acid / analogs & derivatives
  • 3-Mercaptopropionic Acid / pharmacology
  • Apoptosis / drug effects
  • Apoptosis / physiology
  • Arginine / pharmacology
  • Breast Neoplasms / pathology
  • Breast Neoplasms / physiopathology*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Dipeptides / pharmacology
  • Estradiol / pharmacology*
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Enzymologic / physiology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Expression Regulation, Neoplastic / physiology
  • Humans
  • Nitric Oxide / metabolism*
  • Prolactin / pharmacology*
  • Protease Inhibitors / pharmacology
  • Proteins / genetics*
  • Proteins / metabolism*
  • RNA, Messenger / metabolism
  • Transfection
  • Up-Regulation / drug effects
  • Up-Regulation / physiology

Substances

  • Dipeptides
  • Protease Inhibitors
  • Proteins
  • RNA, Messenger
  • Nitric Oxide
  • Estradiol
  • furylacryloylalanylarginine
  • 2-mercaptomethyl-3-guanidinoethylthiopropionic acid
  • Prolactin
  • Arginine
  • 3-Mercaptopropionic Acid
  • metallocarboxypeptidase D