Stimulation of calbindin-D9K (CaBP9K) gene expression by calcium and 1,25-dihydroxycholecalciferol in fetal rat duodenal organ culture.
We have used organ cultures of 18-day fetal rat duodena to study the regulation of calbindin-D9K (CaBP9K) gene expression by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and calcium. The epithelium of the fetal rat duodena maintained in culture for 10 days in a defined serum-free medium with no exogenous 1,25(OH)2D3 contains differentiated enterocytes. Treatment with 10(-8) M 1,25(OH)2D3 resulted in a 2-fold increase in CaBP9K messenger RNA (mRNA) after 1 h; CaBP9K content was increased 2.5-fold in 24 h. Simultaneous treatment of cultures with 1,25(OH)2D3 and 1 microM actinomycin D selectively blocked 1,25(OH)2D3-stimulated CaBP9K mRNA and protein synthesis. Fifty micromolars of cycloheximide significantly decreased CaBP9K protein accumulation but not mRNA. These findings indicate that 1,25(OH)2D3 controls CaBP9K gene transcription. Enterocytes exhibit some CaBP9K gene expression after 10 days under basal culture conditions, suggesting that CaBP9K synthesis is not dependent on 1,25(OH)2D3 alone. This basal expression drops after 24 h in a calcium free medium with 1 mM EGTA, suggesting that extracellular calcium is involved in the control of this gene. Increasing the calcium concentration to 1.2 mM resulted in a 6-fold increase in CaBP9K mRNA after 3 h and a 10-fold increase in CaBP9K protein content after 24 h. Calcium may increase CaBP9K gene transcription, since simultaneous addition of 1 microM actinomycin D and calcium blocked the calcium effect on CaBP9K mRNA. Therefore, this in vitro study demonstrates that both calcium and 1,25(OH)2D3 stimulate CaBP9K gene expression in the rat duodenum.
Brehier
A
A
INSERM U120, Alliée CNRS, Le Vésinet, France.
Thomasset
M
M
D000818:Animals; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004386:Duodenum / Q000187:drug effects / Q000378:metabolism*; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009924:Organ Culture Techniques; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D064030:S100 Calcium Binding Protein G / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D064026:Calbindins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; C576508:S100g protein, rat; D003609:Dactinomycin; D012313:RNA; D003513:Cycloheximide; D002117:Calcitriol; D002118:Calcium