181 THE POTENTIAL ROLE OF NON-GENOMIC PATHWAYS AND THEIR EFFECTS ON THE REGULATION OF CALBINDIN-D9k IN VITRO

Abstract

Calbindin-D9k (CaBP-9k), a cytosolic protein, is one of the members of the family of vitamin D-dependent calcium-binding proteins with high affinity for calcium. The previous in vitro studies indicated that this gene is controlled by 17β-estradiol (E2), a physiological estrogen, via both genomic (through its classical nuclear receptors) and non-genomic (through different cypoplasmic signals) mechanisms. In order to provide a better understanding in molecular events by which E2 exerts its actions in the regulation of CaBP-9k, we employed GH3 cells as an in vitro model to examine the possible non-genomic effects of E2 on the induction of CaBP-9k. GH3 cells were treated dose-dependently (10^–5, 10^–6, 10^–7, 10^–8, and 10^–9 m) with E2-BSA, a membrane-impermeable E2 conjugated with BSA, for 24 h. To examine the time dependency, the cells were also exposed to a high concentration (10^–6 m) of E2-BSA and harvested at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h). Furthermore, in order to determine the potential involvement of non-genomic signaling pathways in E2-BSA-induced expression of CaBP-9k, several inhibitors also were employed, including ICI 182 780 for membrane estrogen receptor (ER) pathway, pertussis toxin (PTX) for G protein signaling, U0126 for ERK pathway, and wortmannin for Akt pathway. The non-genomic effects of E2-BSA on the induction of CaBP-9k mRNA and protein were determined by semi-quantitative RT-PCR and Western blotting, respectively. In a dose-dependent manner, administration with E2-BSA (10^–6 m) induced the highest response of CaBP-9k at transcriptional (mRNA) level, whereas protein level of CaBP-9k peaked at E2-BSA concentration (10^–7 m) at 24 h. In a time course, E2-BSA (10^–6 m) exposure caused a significant increase in both CaBP-9k mRNA and protein expressions as early as 15 min and peaked at 24 h. Co-treatment with ICI 182 780 and PTX completely inhibited E2-BSA-induced CaBP-9k mRNA and protein expressions. Interestingly, although co-treatments with U0126 and/or wortmannin alone failed to attenuate the effects of E2-BSA, a combination of 2 inhibitors completely reversed E2-BSA-induced CaBP-9k expressions at both transcriptional (mRNA) and translational (protein) levels, suggesting their involvement in the regulation of CaBP-9k in GH3 cells. Taken together, these results demonstrate that various signaling pathways may be involved in E2-induced regulation of CaBP-9k in which membrane ER and G protein signaling pathways play a central role in non-genomic responses. Further in vitro experiments are required to elucidate additional details of the interaction of ERK and Akt pathways in the regulation of CaBP-9k in these cells, offering a new insight into the mode of E2 action in the pituitary gland of human and wildlife.