Melatonin-induced calbindin-D9k expression reduces hydrogen peroxide-mediated cell death in rat pituitary GH3 cells.

In this study, we investigated whether calbindin-D9k (CaBP-9k) expression was regulated by melatonin during hydrogen peroxide (H(2)O(2))-induced cell death in rat pituitary GH3 cells. CaBP-9k expression was increased by melatonin in a dose- and time-dependent manner, indicating that CaBP-9k expression is regulated by melatonin. Cell survival was increased approximately 27-30% where H(2)O(2)-treated cells (0.25 or 0.5 mm) were also incubated with 1 mm melatonin, when compared with H(2)O(2) alone or H(2)O(2) plus 0.5 mm melatonin. This result was consistent with 4,6-diamidino-2-phenylindole staining. CaBP-9k expression was also augmented by co-treatment with H(2)O(2) and 1 mm melatonin, suggesting a functional relationship between increased cell death and melatonin-induced CaBP-9k expression during H(2)O(2)-mediated apoptosis. Bcl-2-associated protein expression increased following treatment with H(2)O(2) alone, whereas Bcl-2 expression was elevated following treatment with melatonin alone, or H(2)O(2) plus melatonin. The expression of p53 was depressed by treatment with melatonin alone, or co-treatment with H(2)O(2) plus melatonin. These results correlated with CaBP-9k expression levels and activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway. Knockdown of CaBP-9k expression using a small inhibitory RNA resulted in an elevation of H(2)O(2)-induced cell death, whereas cell survival was increased in cells that overexpressed CaBP-9k, providing additional evidence that the induction of CaBP-9k expression may be associated with survival signaling during H(2)O(2)-mediated oxidative cell death. CaBP-9k appears to interact with p53, suggesting a possible role for this interaction in cell proliferation and cell cycle progression.