Regulation of intestinal calbindin-D28K gene expression: a solution hybridization study.
A solution hybridization assay employing specific synthetic oligodeoxynucleotide probes was developed to study the regulation of intestinal calbindin-D28K mRNA. The technique is rapid and quantitative and eliminates the need for sample transfer, blotting, autoradiography, and densitometry. Following validation of the assay, chick intestinal calbindin-D and calbindin-D mRNA levels were compared under conditions known to stimulate intestinal calcium (Ca) transport. Both the protein and its mRNA were undetectable in 25-day-old vitamin D-deficient chicks. Following acute administration of vitamin D3, calbindin-D mRNA levels increased somewhat more rapidly than calbindin-D protein, but overall, the correlation was excellent. Chicks fed a nutritionally adequate diet for 14 days and then changed to a low Ca (0.1%) diet responded with increased calbindin-D and calbindin-D mRNA levels. Again the correlation was excellent over the ensuing 14-day experimental period. The combined effects of vitamin D repletion and dietary Ca status were also investigated with respect to calbindin D and its mRNA. Fourteen-day-old vitamin D-deficient chicks were changed to diets containing vitamin D and either adequate (1.2%) or low (0.3%) in Ca. The intestinal responses were measured at intervals up to 14 days. In the normal Ca situation, there were initial increases in both calbindin mRNA levels, which peaked at between 4 and 7 days, and calbindin protein levels, which peaked at 7 days. Both values subsequently declined during the remaining 7 days of the experimental period. In the low Ca situation, there were similar increases in calbindin mRNA and protein levels through 4 and 7 days respectively, but these levels remained high for the remainder of the 14-day experimental period. The present results demonstrate that intestinal tissue levels of calbindin D and its mRNA respond similarly to vitamin D repletion and dietary Ca restriction as well as the combination of these stimuli. There is no evidence to support significant post-transcriptional regulation of calbindin-D by Ca.
Fullmer
C S
CS
Department of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D064026:Calbindins; D002645:Chickens; D015342:DNA Probes; D004386:Duodenum / Q000378:metabolism*; D005786:Gene Expression Regulation; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D064030:S100 Calcium Binding Protein G / Q000235:genetics*
D064026:Calbindins; D015342:DNA Probes; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G