Corepressor/coactivator paradox: potential constitutive coactivation by corepressor splice variants

The functional consequences of the interaction of transcriptional coregulators with the human thyroid hormone receptor (TR) in mammalian cells are complex. We have used the yeast, Saccharomyces cerevisiae, which lack endogenous nuclear receptors (NRs) and NR coregulators, as a model to decipher mechanisms regulating transcriptional activation by TR. In effect, this system allows the reconstitution of TR mediated transcription complexes by the expression of specific combinations of mammalian proteins in yeast. In this yeast system, human adenovirus 5 early region 1A (E1A), a natural N-CoR splice variant (N-CoR(I)) or an artificial N-CoR truncation (N-CoR(C)) coactivate unliganded TRs and these effects are inhibited by thyroid hormone (TH). E1A contains a short peptide sequence that resembles known corepressor-NR interaction motifs (CoRNR box motif, CBM), and this motif is required for TR binding and coactivation. N-CoR(I) and N-CoR(C) contain three CBMs, but only the C-terminal CBM1 is critical for coactivation. These observations in a yeast model system suggest that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical corepressor mode. These findings also raise the possibility that alternative splicing events which form corepressor proteins containing only C-terminal CBM motifs could represent a novel mechanism in mammalian cells for regulating constitutive transcriptional activation by TRs

Nuclear and Cytoplasmic Accumulation of Ep-ICD Is Frequently Detected in Human Epithelial Cancers

BACKGROUND: We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. METHODOLOGY AND RESULTS: Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. CONCLUSIONS: Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers

Thyroid hormone response element organization dictates the composition of active receptor

Thyroid hormone (triiodothyronine, T3) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T3 response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TRβ activates transcription more efficiently than TRα at an inverted palindrome (F2), and this correlates with preferential TRβ homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TRβ homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TRβ mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TRβ homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TRβ requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR·TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo

Randomized comparison of the effects of the vitamin D(3 )adequate intake versus 100 mcg (4000 IU) per day on biochemical responses and the wellbeing of patients

BACKGROUND: For adults, vitamin D intake of 100 mcg (4000 IU)/day is physiologic and safe. The adequate intake (AI) for older adults is 15 mcg (600 IU)/day, but there has been no report focusing on use of this dose. METHODS: We compared effects of these doses on biochemical responses and sense of wellbeing in a blinded, randomized trial. In Study 1, 64 outpatients (recruited if summer 2001 25(OH)D <61 nmol/L) were given 15 or 100 mcg/day vitamin D in December 2001. Biochemical responses were followed at subsequent visits that were part of clinical care; 37 patients completed a wellbeing questionnaire in December 2001 and February 2002. Subjects for Study 2 were recruited if their 25(OH)D was <51 nmol/L in summer 2001. 66 outpatients were given vitamin D; 51 completed a wellbeing questionnaire in both December 2002 and February 2003. RESULTS: In Study 1, basal summer 25-hydroxyvitamin D [25(OH)D] averaged 48 ± 9 (SD) nmol/L. Supplementation for more than 6 months produced mean 25(OH)D levels of 79 ± 30 nmol/L for the 15 mcg/day group, and 112 ± 41 nmol/L for the 100 mcg/day group. Both doses lowered plasma parathyroid hormone with no effect on plasma calcium. Between December and February, wellbeing score improved more for the 100-mcg/day group than for the lower-dosed group (1-tail Mann-Whitney p = 0.036). In Study 2, 25(OH)D averaged 39 ± 9 nmol/L, and winter wellbeing scores improved with both doses of vitamin D (two-tail p < 0.001). CONCLUSION: The highest AI for vitamin D brought summertime 25(OH)D to >40 nmol/L, lowered PTH, and its use was associated with improved wellbeing. The 100 mcg/day dose produced greater responses. Since it was ethically necessary to provide a meaningful dose of vitamin D to these insufficient patients, we cannot rule out a placebo wellbeing response, particularly for those on the lower dose. This work confirms the safety and efficacy of both 15 and 100 mcg/day vitamin D(3 )in patients who needed additional vitamin D

Randomized comparison of the effects of the vitamin D3adequate intake versus 100 mcg (4000 IU) per day on biochemical responses and the wellbeing of patients

Abstract Background For adults, vitamin D intake of 100 mcg (4000 IU)/day is physiologic and safe. The adequate intake (AI) for older adults is 15 mcg (600 IU)/day, but there has been no report focusing on use of this dose. Methods We compared effects of these doses on biochemical responses and sense of wellbeing in a blinded, randomized trial. In Study 1, 64 outpatients (recruited if summer 2001 25(OH)D <61 nmol/L) were given 15 or 100 mcg/day vitamin D in December 2001. Biochemical responses were followed at subsequent visits that were part of clinical care; 37 patients completed a wellbeing questionnaire in December 2001 and February 2002. Subjects for Study 2 were recruited if their 25(OH)D was <51 nmol/L in summer 2001. 66 outpatients were given vitamin D; 51 completed a wellbeing questionnaire in both December 2002 and February 2003. Results In Study 1, basal summer 25-hydroxyvitamin D [25(OH)D] averaged 48 ± 9 (SD) nmol/L. Supplementation for more than 6 months produced mean 25(OH)D levels of 79 ± 30 nmol/L for the 15 mcg/day group, and 112 ± 41 nmol/L for the 100 mcg/day group. Both doses lowered plasma parathyroid hormone with no effect on plasma calcium. Between December and February, wellbeing score improved more for the 100-mcg/day group than for the lower-dosed group (1-tail Mann-Whitney p = 0.036). In Study 2, 25(OH)D averaged 39 ± 9 nmol/L, and winter wellbeing scores improved with both doses of vitamin D (two-tail p < 0.001). Conclusion The highest AI for vitamin D brought summertime 25(OH)D to >40 nmol/L, lowered PTH, and its use was associated with improved wellbeing. The 100 mcg/day dose produced greater responses. Since it was ethically necessary to provide a meaningful dose of vitamin D to these insufficient patients, we cannot rule out a placebo wellbeing response, particularly for those on the lower dose. This work confirms the safety and efficacy of both 15 and 100 mcg/day vitamin D3 in patients who needed additional vitamin D