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1,25(OH) 2 D 3 ‐dependent regulation of calbindin‐D 28k mRNA requires ongoing protein synthesis in chick duodenal organ culture
Author(s) -
Meyer Julia,
Galligan Michael A.,
Jones Glenville,
Komm Barry S.,
Haussler Carol A.,
Haussler Mark R.
Publication year - 1995
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240580306
Subject(s) - cycloheximide , messenger rna , calcitriol receptor , northern blot , microbiology and biotechnology , biology , gene expression , western blot , medicine , endocrinology , protein biosynthesis , vitamin d and neurology , gene , biochemistry
Organ culture of 19‐day‐old chick embryo duodena was utilized to evaluate the mechanism of 1,25‐dihydroxyvitamin D 3 (1,25(OH) 2 D 3 )‐dependent calbindin‐D 28k (CaBP) expression. Duodenal CaBP and 1,25(OH) 2 D 3 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) 2 D 3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [ 3 H‐1β]1α,25(OH) 2 D 3 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) 2 D 3 . The inclusion of 1.6 μM actinomycin D in the organ culture partially inhibited the 1,25(OH) 2 D 3 ‐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) 2 D 3 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) 2 D 3 ‐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) 2 D 3 ‐dependent, is necessary for 1,25(OH) 2 D 3 ‐mediated CaBP gene transcription in chick duodena.