1,25(OH)2D3-dependent regulation of calbindin-D(28k) mRNA requires ongoing protein synthesis in chick duodenal organ culture

J. Meyer, M. A. Galligan, G. Jones, B. S. Komm, C. A. Haussler, Mark R Haussler

Research output: Contribution to journalArticle

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Abstract

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 Northern 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.

Original languageEnglish (US)
Pages (from-to)315-327
Number of pages13
JournalJournal of Cellular Biochemistry
Volume58
Issue number3
DOIs
StatePublished - 1995

Fingerprint

Calbindins
Organ Culture Techniques
Messenger RNA
Proteins
Transcription
Genes
Calcitriol
Polymerase chain reaction
RNA Precursors
Dactinomycin
Chick Embryo
Cycloheximide
Cell culture
Northern Blotting
Chromatin
Western Blotting

Keywords

  • 1,25-dihydroxyvitamin D
  • calbindin-D(28k)
  • messenger RNA
  • organ culture
  • polymerase chain reaction

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

1,25(OH)2D3-dependent regulation of calbindin-D(28k) mRNA requires ongoing protein synthesis in chick duodenal organ culture. / Meyer, J.; Galligan, M. A.; Jones, G.; Komm, B. S.; Haussler, C. A.; Haussler, Mark R.

In: Journal of Cellular Biochemistry, Vol. 58, No. 3, 1995, p. 315-327.

Research output: Contribution to journalArticle

Meyer, J. ; Galligan, M. A. ; Jones, G. ; Komm, B. S. ; Haussler, C. A. ; Haussler, Mark R. / 1,25(OH)2D3-dependent regulation of calbindin-D(28k) mRNA requires ongoing protein synthesis in chick duodenal organ culture. In: Journal of Cellular Biochemistry. 1995 ; Vol. 58, No. 3. pp. 315-327.
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AU - Jones, G.

AU - Komm, B. S.

AU - Haussler, C. A.

AU - Haussler, Mark R

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N2 - 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 Northern 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.

AB - 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 Northern 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.

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