1,25(OH)₂D₃‐dependent regulation of calbindin‐D_28k mRNA requires ongoing protein synthesis in chick duodenal organ culture

Organ culture of 19‐day‐old chick embryo duodena was utilized to evaluate the mechanism of 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃)‐dependent calbindin‐D_28k (CaBP) expression. Duodenal CaBP and 1,25(OH)₂D₃ receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northen blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)₂D₃ resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [³H‐1β]1α,25(OH)₂D₃ in duodenal chromatin is rapid (⩽ 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)₂D₃. The inclusion of 1.6 μM actinomycin D in the organ culture partially inhibited the 1,25(OH)₂D₃‐regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre‐mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 μM cycloheximide 1 h prior to 1,25(OH)₂D₃ addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady‐state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)₂D₃‐mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)₂D₃‐dependent, is necessary for 1,25(OH)₂D₃‐mediated CaBP gene transcription in chick duodena.