In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum (original) (raw)
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Biochimie, 1986
-Cholecalciferol (calcitriol) the active hormonal form of vitamin D induces the synthesis of at least two intracellular calcium-binding proteins (K a =: 10 6 M-I), the cholecalcins (CaBP) in mammals. We used the synthesis of these proteins to study the genomic steroid-like action of vitamin D. The 9 kDa CaBP is mainly concentrated in the duodenum while 28 kDa CaBP is located in the kidney and cerebellum. Complementary DNA copies of rat intestinal 9 kDa CaBP mRNA were cloned in E. coli. The deduced amino acid sequence for 9 kDa CaBP contains tw o 'EF hand' domains corresponding to calcium-binding sites I and II. The homology observed suggests, after comparison with the structures of other intracellular CaBPs, that rat 9 kDa CaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure seen in 28 kDa CaBP from kidney and cerebellum of rat. Northern blots showed that the eDNA sequence hybridizes to a homogeneous 500-600 nucleotide mRNA species from rat duodenum. Larger mRNA species encoding 28 kDa CaBP were undetectable in rat kidney and cerebellum even under Jow stringency conditions. These findings demonstrate that there is no cross-hybridization between 9 kDa and 28 kDa CaBP mRNAs, and Southern analysis indicates that there are distinct genes coding for each rat cholecalcin. The cDNA probe was used to analyze the specific 9 kDa CaBP gene expression along the intestine of growing rats and during gestation and fetal development. In situ hybridization histochemistry using par la sonde tout le long de l'intestin. Chez les rats recevant de la vitamine D, l'ARNm de la CaBP 9 kDa mesure par hybridation de type «dot blot» est present en grande quantite dans Ie duodenum, Ie jejunum proximal et le caecum. Ces differences dans le taux d'expression du gene de la CaBP 9 kDa et de la concentration de la proteine sont arapprocher des sites d'absorption du calcium. Son taux decroit fortement chez les animaux prives d'apport en vitamine D pendant 4 semaines. Une injection unique de 1,25 (OH)2D3 retablit un taux normal de CaBP dans le duodenum et supranormal dans l'intestin distal et Ie caecum. L 'augmentation precoce du taux d'ARNm et de leur activite traductionnelle 1 heure apres l'injection de l'hormone est en faveur d'un role direct du 1,25 (OH)2D3 sur la transcription du gene de la cholecalcine. L 'hybridation in situ montre que les ARNm de La CaBP, visualisespar les grains d'argent, sont presents dans Les eel/ules prismatiques hautes de l'epithelium de revetement du duodenum tandis que les eellules calciformes (ou amucus) sont depourvues de radioactivite. Les grains d'argent sont principalement localises dans le cytoplasme des cel/ules marquees assezfrequemment dans la regionperinucleaire mais sont absents du placenta strie. Le marquage est plus important dans les cellules de Ia partie haute des villosites qtc'd la base des espaces intervilleux ou couche profonde de la muqueuse. Les autres structures du duodenum ne montrent pas de marquage specifique. Dans Ie placenta, les ARNm sont presents dans la zone du labyrinthe. Une concentration tres importante de grains d'argent est observee dans Ie cytoplasme des cellules de l'epithelium trophoblastique. Le criblage d'une banque genomique de rat a perm is d'isoler 7 clones, recouvrant 15 kb, repartis de part et d'autre du site Eco RI de l'ADNc. La sequence et l'organisation du gene sont en cours d'analyse.
Rat duodenal calcium-binding protein messenger rna: Induction by 1,25-dihydroxyvitamin D3
Journal of Steroid Biochemistry, 1983
To extend our previous observations on the regulation of CaBP biosynthesis by 1.25 dihydroxyvitamin D,, we have studied the specific mRNA encoding this protein in vitamin D-deficient and in vitamin D-repleted rats as well as the rate of its induction after a single injection of 1 .25(OH),D3 to vitamin D-deficient animals. The CaBP-mRNA was quantified by translation in a cell-free reticulocyte lysate system. CaBP-mRNA activity and cytoplasmic CaBP (measured by radioimmunoassay), dramatically decreased in rats previously fed a vitamin D-free diet for 5 weeks but neither parameter was zero. In vitamin D-deficient rats, a single injection of 1,25(OH)2D, led to an increase in CaBP-mRNA activity within 2 h. This CaBP-mRNA activity peaked at about 4-6 h and thereafter declined to low value by 48 h. and the changes in mRNA activity always preceded the changes in cytosolic CaBP concentration. These results indicate that the induction of CaBP biosynthesis results from a 1,25(0H)?D,-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by l,25(OH-zD,. This study also shows that the production of many other proteins seem to be under the control of vitamin D1.
Biosynthesis and compartmentalization of rat-intestinal vitamin-D-dependent calcium-binding protein
European Journal of Biochemistry, 1984
We have purified the primary translation product of rat intestinal vitamin-D-dependent calcium-binding protein mRNA from wheat germ and ascites cell-free systems. We show that calcium-binding protein is neither synthesized as a larger precursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH,-terminally labeled with f~rmyl[~~S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co-and post-translational cleavage assays in vitro.
1983
We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and 11) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site 111-like structure and that low M, ICaBP could result in early termination of the translation of a larger molecule containing four sites. Vitamin D-dependent CaBPs' (Wasserman and Taylor, 1966; Wasserman et al., 1978) belong to the large family of intracellular proteins which bind calcium with affinity constants in the range 10-s-10-5 M (Kretsinger, 1976). Based upon the number of their calcium-binding sites, two classes of such vitamin D-dependent proteins have been identified in mammals. A M , = 28,000 molecule found in kidney and cerebellum has four calcium-binding sites, whereas a M , =
Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes
Archives of Biochemistry and Biophysics, 1980
The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D,, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg*+ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D, was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 cLgimg vs kidney, 0.9 pg/mg). Also, after giving D,, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH),D3 in both the intestine and kidney is determined by similar control mechanisms. 809
Oral calcium transiently increases calbindin9k gene expression in adult rat duodena
Calcified Tissue International, 1997
In rat intestine, the 9 kilodalton calbindin (CaBP 9 d is significantly increased in vivo by 1,2Sdihydroxyvitamin D3 (l,2S(OH)zD 3) through a vitamin D (D) response element located in the S'-flanking region of the gene. However, in vitro calcium has also been reported to increase CaBP 9K gene expression in fetal duodenal culture preparations. The aim of the studies was to investigate whether calcium feeding alone can influence CaBP 9K gene expression in vivo in adult rat duodena by evaluating the pattern of expression of its mRNA following short-or longterm exposure to oral calcium, comparing the data to exposure to the known inducer of the gene, 1,2S(OH)zD 3. Hypocalcemic D-depleted rats were acutely or chronically supplemented with calcium per os, or with 1,2S(OH)zD 3 in the presence or absence of oral calcium. Short-term calcium feeding was shown to significantly increase the expression of the CaBP 9K gene to a level similar to that observed in 1,2S(OH)zD r treated rats but no additive effect between oral calcium and 1,2S(OH)zD 3 on the level of its mRNA was observed. Moreover, the calcium effect on CaBP 9K gene expression was shown to be independent of the circulating ionized calcium concentration and, contrary to the effect of 1,2S(OH)zD 3 , not sustained following long-term exposure. Our data clearly indicate that oral calcium alone has a significant but only transient effect of the expression of the adult rat intestinal CaBP 9K gene in vivo and that maintenance of its expression requires normalization of the D endocrine system.
Journal of Biological Chemistry
The purification and properties of vitamin D dependent calcium binding proteins were studied in rat intestinal mucosa. Two different vitamin D de pendent calcium binding proteins were fractionated by the DEAE-cellulose column chromatography. Their properties were analyzed with the polyacryl amide gel disc electrophoresis and the sucrose density gradient ultracentrifuga tion. One of them is having a molecular weight about 24,000 representing rapid migration on acrylamide gel electrophoresis and seems similar to the calcium binding protein obtained from chick intestinal mucosa in respect of its size and electrophoretic mobility. The other protein is a larger molecule (mol. wt. 145,000) which is eluted in first fraction from DEAE cellulose chromatogra phy. The smaller molecule of binding protein associates with one atom of calcium per one molecule of protein and the larger molecule would associate with more than 2 atoms of calcium.
Endocrinology, 2003
We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D9k expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] injection strongly regulated duodenal calbindin D9k and CaT1 mRNA. Following a single dose of 1,25(OH)2D3, induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 ± 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)2D3 injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)2D3 in ...