Calbindin D 28K Regulation in Precociously Matured Chick Egg Shell Gland in Vitro

Abstract

Egg shell calcification in the hen uterus (egg shell gland, ESG) depends primarily on intestinal absorption of dietary Ca 2+ as well as ESG Ca 2+ transport into the shell. Intestinal Ca 2+ absorption is linked to vitamin D-induced calbindin D 28K (D28K) concentration. The ESG also contains D28K, and Ca 2+ transport into the shell appears to be linked to D28K gene expression, but until this report, there was no direct proof that ESG D28K was or was not vitamin D-dependent. To address this issue, highly developed ESG from estradiol (E 2)injected, severely vitamin D-depleted chicks were cultured in serum-free medium with excellent viability. Addition of the vitamin D-hormone, 1,25(OH) 2 vitamin D 3 (1,25), to the culture medium increased ESG D28K levels as much as 70%. E 2 alone had no effect, but E 2 plus 1,25 further increased ESG D28K levels up to 160%. By contrast, progesterone (P 4) prevented the 1,25-stimulated increase in D28K, while having no effect on basal D28K level. Of considerable interest, thapsigargin (THAPS), which increases intracellular Ca 2+ concentration ([Ca 2+]i) in many cell types, stimulated D28K synthesis in a concentration-dependent manner in the complete absence of 1,25 and independent of the [Ca 2+] of the medium. These results are the first direct evidence that ESG D28K is under direct control of 1,25 and that both gonadal steroid hormones, E 2 and P 4, may be coregulators. Further, the effects of THAPS suggest that [Ca 2+]i itself may also regulate D28K. This new in vitro model clearly represents a unique opportunity to study the regulation of the ESG calcium transport mechanism under stringently defined conditions.