Induction of calcitonin and calcitonin receptor expression in rat mammary tissue during pregnancy.

Human breast milk samples at early time points after parturition contain high levels of calcitonin (CT) in both normal and thyroidectomized mothers, suggesting that mammary tissue produces CT. Using blot hybridization and reverse-transcriptase polymerase chain (RT-PCR) analysis of rat mammary RNA we found that CT messenger RNA is induced at midpregnancy (day 12), remains elevated through late pregnancy (day 19) but then decreases before the day of birth. RIA of mammary CT revealed that levels increase from 0.3 ng/g tissue in nonpregnant animals to peak at 1.6 ng/g on day 19 and then decline after that, paralleling messenger RNA expression. Dilution profiles for extracted mammary CT showed close parallelism with monomeric rat CT. Plasma samples from thyroparathyroidectomized rats contained 10-20 pg/ml CT that did not increase during pregnancy, suggesting that mammary CT is not released into plasma but functions locally. Consistent with this, RT-PCR detected that the CT receptor C1a isoform is expressed in rat mammary tissues during both pregnancy and lactation. This is the first report that mammary tissue expresses both CT and the CT receptor during pregnancy, suggesting that CT may have a paracrine regulatory role in the mammary gland.

Retinoic acid repression of cell-specific helix-loop-helix-octamer activation of the calcitonin/calcitonin gene-related peptide enhancer.

We have investigated the mechanism underlying repression of calcitonin/calcitonin gene-related peptide (CT/CGRP) gene expression by retinoic acid. Retinoic acid treatment of the CA77 thyroid C-cell line decreased CT/CGRP promoter activity two- to threefold, which correlates well with the decrease in calcitonin and CGRP mRNA levels. Repression is mediated through the nuclear retinoic acid receptors (RAR) on the basis of the retinoid specificity, the sensitivity of repression (half-maximal repression at 0.2 nM), and the additional repression caused by cotransfection of an alpha-RAR expression vector. The sequences required for retinoic acid repression were localized to an 18-bp element containing cell-specific enhancer activity. The enhancer binds helix-loop-helix (HLH) and octamer transcription factors that act synergistically to activate transcription. Retinoic acid repression requires both these factors since mutations in either motif resulted in the loss of repression. Furthermore, repression was observed only in cell lines containing enhancer activity. We have used electrophoretic mobility shift assays to show that repression does not involve direct DNA binding of RAR or RAR-retinoid X receptor heterodimers. Instead, repression appears to involve interactions with the stimulatory enhancer factors. Following retinoic acid treatment, there was a specific decrease in an enhancer complex containing both HLH and octamer proteins. Formation of the HLH-octamer complex was also specifically blocked by the addition of exogenous RAR-retinoid X receptor protein. These results demonstrate that RAR can repress CT/CGRP gene transcription by interfering with combinatorial activation by cell-specific HLH and octamer proteins.

Regulation of the calcitonin/calcitonin gene-related peptide gene by cell-specific synergy between helix-loop-helix and octamer-binding transcription factors.

The calcitonin/calcitonin gene-related peptide (CGRP) gene is transcribed in thyroid C-cells and a subset of neurons. We have localized sequences required for cell-specific enhancement of calcitonin/CGRP transcription in rat thyroid C-cell lines. An 18-base pair element approximately 1 kilobase pair upstream of the transcriptional start site stimulated expression of a luciferase reporter gene 50-fold in 44-2C C-cells. There was less than 2-fold stimulation in HeLa and Rat-1 cells, which do not express the endogenous calcitonin/CGRP gene. The enhancer contains potential binding sites for helix-loop-helix (HLH) and octamer transcription factors based on sequence homologies. The functional significance of these sites was shown by point mutations in the HLH and octamer motifs and by separation of the two motifs, all of which decreased enhancer activity greater than 10-fold. The involvement of HLH proteins was further shown by co-expression of the mammalian achaete-scute homologue-1 HLH protein, which activated the enhancer severalfold in HeLa cells. Electrophoretic mobility shift analyses revealed several DNA-protein complexes containing HLH and octamer-binding proteins. One octamer-binding complex (OB1) most likely contains the ubiquitous Oct-1 protein, whereas a second complex (OB2) was cell-specific. In contrast to OB1, OB2 had lower affinity for a consensus octamer motif, and its DNA binding was not affected by addition of antiserum that recognizes Oct-1 and Oct-2 proteins. In addition, we observed a large complex that appears to contain both an HLH protein and OB2. These results demonstrate that calcitonin/CGRP enhancer activity is controlled by a cell-specific synergistic activation involving HLH and octamer-binding factors.

Cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide transcription. Localization to an 18-base pair basal enhancer element.

We have investigated the mechanisms underlying cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide (CGRP) gene expression. Treatment with the synthetic glucocorticoid dexamethasone has been shown to decrease mRNA levels in the 44-2C thyroid C cell line. Nuclear run-on assays showed that dexamethasone repressed transcription 2-3-fold in 44-2C cells. In contrast, dexamethasone stimulated calcitonin/CGRP transcription 4-6-fold in the CA77 thyroid C cell line. Transient transfection assays were used to map repression of reporter gene activity in 44-2C cells to a neuroendocrine cell-specific enhancer located between -920 and -1125 base pairs (bp). Within this region, an 18-bp element was found that conferred both full basal enhancer activity and dexamethasone-dependent repression in 44-2C cells. The 18-bp region contains possible binding sites for AP-1 and helix-loop-helix transcription factors as well as a glucocorticoid receptor half-site. Colocalization of repression and enhancer activity was then investigated in other cell lines. In CA77 cells, while the 920-1125 region strongly enhanced transcription, the 18-bp region conferred only partial activation and dexamethasone had little effect on reporter gene activity. Dexamethasone did not repress the calcitonin/CGRP activity in the heterologous HeLa and Rat1 fibroblast cell lines. These results suggest that glucocorticoids repress transcription of the calcitonin/CGRP gene by inhibiting cell-specific transcription factor activity.