Faculty Bibliography

The regulation of calcitonin (CT) secretion by calcium was studied by measuring CT mRNA extracted from thyroids of normal rats subjected to acute calcium stimulation in vivo. The 15000-Mr primary translation product of CT mRNA was identified by immunoprecipitation using specific antibodies. While total mRNA and total radioactivity incorporated after translation of total mRNA remained unaffected by the calcium stimulation, a fourfold increase in radioactivity incorporated in CT primary translation product occurred as early as 2 min after calcium administration. This peak coincided with a rise in plasma levels of the hormone and preceded a detectable decrease in tissue stores. These results suggest that calcium ion, either directly or indirectly via its action on intracellular stores of the hormone or its precursors, causes a rapid increase in cell levels of translatable CT mRNA. In view of the extremely short time (2 min) in which this increase occurs, the action is probably at the post-transcriptional level as no increase in CT mRNA levels could be detected by hybridization assay using a specific cDNA probe for human CT mRNA.

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 II) 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 III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

To extend our previous observations on the regulation of CaBP biosynthesis by 1,25-dihydroxyvitamin D3, 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)2D3 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)2D3 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(OH)2D3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH)2D3. This study also shows that the production of many other proteins seem to be under the control of vitamin D3.

The primary step of calcitonin biosynthesis was studied in a normal organ: chicken ultimobranchial gland, a tissue particularly rich in calcitonin secretory cells. Poly(A)-rich RNA was extracted and purified from ultimobranchial organs and translated in a reticulocyte lysate in the presence of labelled methionine. Polyacrylamide gel electrophoresis of specific immunoprecipitates revealed a major band of Mr 14 500 and a band of Mr 13 300. Thus, in chicken the precursor of calcitonin is a Mr 14 500 polypeptide. The minor component of Mr 13 300 could represent limited processing by the reticulocyte lysate.

The mRNA coding for rat intestinal calcium-binding protein, a vitamin D3-induced protein (Mr 7500), has been partially purified from growing rat duodenum. Double-stranded DNA synthesized from the purified mRNA preparation was inserted into the PstI site of pBR322, using the oligo(dG-dC) tailing procedure. Clones containing DNA complementary to vitamin D-dependent calcium-binding protein mRNA were selected by differential colony hybridization with [32P] cDNA synthesized from enriched or low vitamin D-dependent calcium-binding protein mRNA preparations. Plasmid DNAs from the selected clones were each verified by both a solution hybrid-arrest assay and a filter hybrid-selection assay. Four recombinant clones showed identical endonuclease restriction maps and contained inserts ranging from 250 to 380 base pairs.