Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration

Abstract

Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.