202 INDUCTION OF CALBINDIN-D9k EXPRESSION BY MELATONIN APPEARS TO BE INVOLVED IN HYDROGEN PEROXIDE-INDUCED CELL DEATH IN RAT PITUITARY GH3 CELLS

Abstract

Calcium-binding protein-D9k (CaBP-9k) is a 9-kDa polypeptide, with 2 calcium binding sites, found in mammalian intestine, placenta, uterus, kidney, pituitary gland, and growth cartilage. However, the regulation of CaBP-9k by endocrine factors (i.e. melatonin) in pituitary gland is not known yet. Melatonin, the major secretory product of the pineal gland, has been known to interact with the neuroendocrine axis, circadian rhythms, and apoptotic pathways in various cell types. Thus, in this study, we investigated whether CaBP-9k gene is regulated by melatonin in association with hydrogen peroxide (H₂O₂)-induced cell death or apoptosis in rat pituitary GH3 cells. We examined the expression levels of CaBP-9k mRNA by RT-PCR and immunoblot analysis, and further measured protein levels of p53, Bax, and Bcl-2 by immunoblot analysis, and kit-8 cell survival marker by cell counting following mRNA or protein isolation by Trizol reagent or Proprep solution, respectively. CaBP-9k expression was increased by melatonin in a dose-dependent (0, 0.1, 0.25, 0.5, and 1 mm) and time-dependent (1 to 48 h) manner, indicating that CaBP-9k expression may be related to melatonin receptor-mediated signal transduction in these cells. Cell death increased about 10% in H₂O₂ (0.25 mm) plus melatonin (1 mm) compared with treatment with only H₂O₂ (0.25 mm) or H₂O₂ (0.25 mm) plus melatonin (0.5 mm), and CaBP-9k expression was augmented by this treatment with H₂O₂ (0.25 mm) plus melatonin (1 mm). This suggests that the mechanism between the increase in cell death and melatonin-induced CaBP-9k expression may be involved in apoptosis by hydrogen peroxide. Bax as a cell death marker was detected in cell death condition of H₂O₂ (0.25 mm) or H₂O₂ (0.25 mm) plus melatonin (0.5 or 1 mm), whereas Bcl-2 as a survival marker was identified in the culture condition with melatonin (0.5 or 1 mm) or H₂O₂ (0.25 mm) plus melatonin (1 mm). p53 as a stress marker mediated with calcium change was decreased in the melatonin only treatment or H₂O₂ plus melatonin. This result is in agreement with CaBP-9k expression level. Knockdown of CaBP-9k using its siRNA (5′GAGCG CTAAG AAATC TCCCG AAGAA3′, Invitrogen Life Technologies) showed that the reduction of H₂O₂-induced cell death, suggesting that induction of CaBP-9k expression caused by melatonin is associated with H₂O₂-induced cell death in rat pituitary GH3 cells.