Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration.
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.
Enomoto
H
H
Bone Center, Cedars-Sinai Medical Center/University of California, Los Angeles 90048.
Hendy
G N
GN
Andrews
G K
GK
Clemens
T L
TL
D000818:Animals; D015152:Blotting, Northern; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002478:Cells, Cultured; D002645:Chickens; D003513:Cycloheximide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014808:Vitamin D Deficiency / Q000378:metabolism
D064026:Calbindins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D012313:RNA; D003513:Cycloheximide; D002117:Calcitriol; D002118:Calcium