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. 1996 Jul;16(7):3393–3400. doi: 10.1128/mcb.16.7.3393

Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

K Ebihara 1, Y Masuhiro 1, T Kitamoto 1, M Suzawa 1, Y Uematsu 1, T Yoshizawa 1, T Ono 1, H Harada 1, K Matsuda 1, T Hasegawa 1, S Masushige 1, S Kato 1
PMCID: PMC231333  PMID: 8668154

Abstract

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

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Selected References

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  1. Baker A. R., McDonnell D. P., Hughes M., Crisp T. M., Mangelsdorf D. J., Haussler M. R., Pike J. W., Shine J., O'Malley B. W. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci U S A. 1988 May;85(10):3294–3298. doi: 10.1073/pnas.85.10.3294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  3. Brandi M. L., Bianchi M. L., Eisman J. A., Glorieux F., Adami S., Fiore C. E., Nuti R., Ortolani S. Genetics of osteoporosis. Calcif Tissue Int. 1994 Sep;55(3):161–163. doi: 10.1007/BF00425868. [DOI] [PubMed] [Google Scholar]
  4. Burmester J. K., Maeda N., DeLuca H. F. Isolation and expression of rat 1,25-dihydroxyvitamin D3 receptor cDNA. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1005–1009. doi: 10.1073/pnas.85.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlberg C., Bendik I., Wyss A., Meier E., Sturzenbecker L. J., Grippo J. F., Hunziker W. Two nuclear signalling pathways for vitamin D. Nature. 1993 Feb 18;361(6413):657–660. doi: 10.1038/361657a0. [DOI] [PubMed] [Google Scholar]
  6. Chen Z. P., Shemshedini L., Durand B., Noy N., Chambon P., Gronemeyer H. Pure and functionally homogeneous recombinant retinoid X receptor. J Biol Chem. 1994 Oct 14;269(41):25770–25776. [PubMed] [Google Scholar]
  7. Cheskis B., Freedman L. P. Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers. Mol Cell Biol. 1994 May;14(5):3329–3338. doi: 10.1128/mcb.14.5.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Darwish H., DeLuca H. F. Vitamin D-regulated gene expression. Crit Rev Eukaryot Gene Expr. 1993;3(2):89–116. [PubMed] [Google Scholar]
  9. Dempster D. W., Lindsay R. Pathogenesis of osteoporosis. Lancet. 1993 Mar 27;341(8848):797–801. doi: 10.1016/0140-6736(93)90570-7. [DOI] [PubMed] [Google Scholar]
  10. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freedman L. P., Arce V., Perez Fernandez R. DNA sequences that act as high affinity targets for the vitamin D3 receptor in the absence of the retinoid X receptor. Mol Endocrinol. 1994 Mar;8(3):265–273. doi: 10.1210/mend.8.3.8015545. [DOI] [PubMed] [Google Scholar]
  12. Giguère V. Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev. 1994 Feb;15(1):61–79. doi: 10.1210/edrv-15-1-61. [DOI] [PubMed] [Google Scholar]
  13. Green S., Chambon P. Nuclear receptors enhance our understanding of transcription regulation. Trends Genet. 1988 Nov;4(11):309–314. doi: 10.1016/0168-9525(88)90108-4. [DOI] [PubMed] [Google Scholar]
  14. Gronemeyer H. Transcription activation by estrogen and progesterone receptors. Annu Rev Genet. 1991;25:89–123. doi: 10.1146/annurev.ge.25.120191.000513. [DOI] [PubMed] [Google Scholar]
  15. Hughes M. R., Malloy P. J., Kieback D. G., Kesterson R. A., Pike J. W., Feldman D., O'Malley B. W. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 1988 Dec 23;242(4886):1702–1705. doi: 10.1126/science.2849209. [DOI] [PubMed] [Google Scholar]
  16. Kastner P., Grondona J. M., Mark M., Gansmuller A., LeMeur M., Decimo D., Vonesch J. L., Dollé P., Chambon P. Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell. 1994 Sep 23;78(6):987–1003. doi: 10.1016/0092-8674(94)90274-7. [DOI] [PubMed] [Google Scholar]
  17. Kastner P., Krust A., Mendelsohn C., Garnier J. M., Zelent A., Leroy P., Staub A., Chambon P. Murine isoforms of retinoic acid receptor gamma with specific patterns of expression. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2700–2704. doi: 10.1073/pnas.87.7.2700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kato S., Sasaki H., Suzawa M., Masushige S., Tora L., Chambon P., Gronemeyer H. Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors. Mol Cell Biol. 1995 Nov;15(11):5858–5867. doi: 10.1128/mcb.15.11.5858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kato S., Tora L., Yamauchi J., Masushige S., Bellard M., Chambon P. A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically. Cell. 1992 Feb 21;68(4):731–742. doi: 10.1016/0092-8674(92)90148-6. [DOI] [PubMed] [Google Scholar]
  20. Koszewski N. J., Reinhardt T. A., Beitz D. C., Horst R. L. Developmental changes in rat kidney 1,25-dihydroxyvitamin D receptor. Biochem Biophys Res Commun. 1990 Jul 16;170(1):65–72. doi: 10.1016/0006-291x(90)91241-j. [DOI] [PubMed] [Google Scholar]
  21. Kristjansson K., Rut A. R., Hewison M., O'Riordan J. L., Hughes M. R. Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3. J Clin Invest. 1993 Jul;92(1):12–16. doi: 10.1172/JCI116539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leid M., Kastner P., Chambon P. Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem Sci. 1992 Oct;17(10):427–433. doi: 10.1016/0968-0004(92)90014-z. [DOI] [PubMed] [Google Scholar]
  23. Leid M., Kastner P., Lyons R., Nakshatri H., Saunders M., Zacharewski T., Chen J. Y., Staub A., Garnier J. M., Mader S. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 1992 Jan 24;68(2):377–395. doi: 10.1016/0092-8674(92)90478-u. [DOI] [PubMed] [Google Scholar]
  24. Leroy P., Krust A., Zelent A., Mendelsohn C., Garnier J. M., Kastner P., Dierich A., Chambon P. Multiple isoforms of the mouse retinoic acid receptor alpha are generated by alternative splicing and differential induction by retinoic acid. EMBO J. 1991 Jan;10(1):59–69. doi: 10.1002/j.1460-2075.1991.tb07921.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lohnes D., Kastner P., Dierich A., Mark M., LeMeur M., Chambon P. Function of retinoic acid receptor gamma in the mouse. Cell. 1993 May 21;73(4):643–658. doi: 10.1016/0092-8674(93)90246-m. [DOI] [PubMed] [Google Scholar]
  26. Mader S., Chen J. Y., Chen Z., White J., Chambon P., Gronemeyer H. The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains. EMBO J. 1993 Dec 15;12(13):5029–5041. doi: 10.1002/j.1460-2075.1993.tb06196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Malloy P. J., Hochberg Z., Tiosano D., Pike J. W., Hughes M. R., Feldman D. The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families. J Clin Invest. 1990 Dec;86(6):2071–2079. doi: 10.1172/JCI114944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mano H., Mori R., Ozawa T., Takeyama K., Yoshizawa Y., Kojima R., Arao Y., Masushige S., Kato S. Positive and negative regulation of retinoid X receptor gene expression by thyroid hormone in the rat. Transcriptional and post-transcriptional controls by thyroid hormone. J Biol Chem. 1994 Jan 21;269(3):1591–1594. [PubMed] [Google Scholar]
  29. Manolagas S. C., Jilka R. L. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med. 1995 Feb 2;332(5):305–311. doi: 10.1056/NEJM199502023320506. [DOI] [PubMed] [Google Scholar]
  30. McDonnell D. P., Scott R. A., Kerner S. A., O'Malley B. W., Pike J. W. Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression. Mol Endocrinol. 1989 Apr;3(4):635–644. doi: 10.1210/mend-3-4-635. [DOI] [PubMed] [Google Scholar]
  31. Meyer M. E., Gronemeyer H., Turcotte B., Bocquel M. T., Tasset D., Chambon P. Steroid hormone receptors compete for factors that mediate their enhancer function. Cell. 1989 May 5;57(3):433–442. doi: 10.1016/0092-8674(89)90918-5. [DOI] [PubMed] [Google Scholar]
  32. Morrison N. A., Qi J. C., Tokita A., Kelly P. J., Crofts L., Nguyen T. V., Sambrook P. N., Eisman J. A. Prediction of bone density from vitamin D receptor alleles. Nature. 1994 Jan 20;367(6460):284–287. doi: 10.1038/367284a0. [DOI] [PubMed] [Google Scholar]
  33. Morrison N. A., Yeoman R., Kelly P. J., Eisman J. A. Contribution of trans-acting factor alleles to normal physiological variability: vitamin D receptor gene polymorphism and circulating osteocalcin. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6665–6669. doi: 10.1073/pnas.89.15.6665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nagpal S., Saunders M., Kastner P., Durand B., Nakshatri H., Chambon P. Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors. Cell. 1992 Sep 18;70(6):1007–1019. doi: 10.1016/0092-8674(92)90250-g. [DOI] [PubMed] [Google Scholar]
  35. Nakajima S., Hsieh J. C., MacDonald P. N., Galligan M. A., Haussler C. A., Whitfield G. K., Haussler M. R. The C-terminal region of the vitamin D receptor is essential to form a complex with a receptor auxiliary factor required for high affinity binding to the vitamin D-responsive element. Mol Endocrinol. 1994 Feb;8(2):159–172. doi: 10.1210/mend.8.2.8170472. [DOI] [PubMed] [Google Scholar]
  36. Nakamura Y., Komatsu N., Nakauchi H. A truncated erythropoietin receptor that fails to prevent programmed cell death of erythroid cells. Science. 1992 Aug 21;257(5073):1138–1141. doi: 10.1126/science.257.5073.1138. [DOI] [PubMed] [Google Scholar]
  37. Ninomiya Y., Mochii M., Eguchi G., Hasegawa T., Masushige S., Kato S. Tissue-specific response of estrogen receptor gene expression to estrogen in chick. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1374–1380. doi: 10.1016/0006-291x(92)90454-s. [DOI] [PubMed] [Google Scholar]
  38. Orkin S. H., Kazazian H. H., Jr The mutation and polymorphism of the human beta-globin gene and its surrounding DNA. Annu Rev Genet. 1984;18:131–171. doi: 10.1146/annurev.ge.18.120184.001023. [DOI] [PubMed] [Google Scholar]
  39. Parker M. G. Steroid and related receptors. Curr Opin Cell Biol. 1993 Jun;5(3):499–504. doi: 10.1016/0955-0674(93)90016-j. [DOI] [PubMed] [Google Scholar]
  40. Perlmann T., Rangarajan P. N., Umesono K., Evans R. M. Determinants for selective RAR and TR recognition of direct repeat HREs. Genes Dev. 1993 Jul;7(7B):1411–1422. doi: 10.1101/gad.7.7b.1411. [DOI] [PubMed] [Google Scholar]
  41. Rastinejad F., Perlmann T., Evans R. M., Sigler P. B. Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature. 1995 May 18;375(6528):203–211. doi: 10.1038/375203a0. [DOI] [PubMed] [Google Scholar]
  42. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  43. Sasaki H., Harada H., Handa Y., Morino H., Suzawa M., Shimpo E., Katsumata T., Masuhiro Y., Matsuda K., Ebihara K. Transcriptional activity of a fluorinated vitamin D analog on VDR-RXR-mediated gene expression. Biochemistry. 1995 Jan 10;34(1):370–377. doi: 10.1021/bi00001a045. [DOI] [PubMed] [Google Scholar]
  44. Sicinski P., Geng Y., Ryder-Cook A. S., Barnard E. A., Darlison M. G., Barnard P. J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989 Jun 30;244(4912):1578–1580. doi: 10.1126/science.2662404. [DOI] [PubMed] [Google Scholar]
  45. Spotila L. D., Constantinou C. D., Sereda L., Ganguly A., Riggs B. L., Prockop D. J. Mutation in a gene for type I procollagen (COL1A2) in a woman with postmenopausal osteoporosis: evidence for phenotypic and genotypic overlap with mild osteogenesis imperfecta. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5423–5427. doi: 10.1073/pnas.88.12.5423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sucov H. M., Dyson E., Gumeringer C. L., Price J., Chien K. R., Evans R. M. RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev. 1994 May 1;8(9):1007–1018. doi: 10.1101/gad.8.9.1007. [DOI] [PubMed] [Google Scholar]
  47. Tasset D., Tora L., Fromental C., Scheer E., Chambon P. Distinct classes of transcriptional activating domains function by different mechanisms. Cell. 1990 Sep 21;62(6):1177–1187. doi: 10.1016/0092-8674(90)90394-t. [DOI] [PubMed] [Google Scholar]
  48. Towers T. L., Luisi B. F., Asianov A., Freedman L. P. DNA target selectivity by the vitamin D3 receptor: mechanism of dimer binding to an asymmetric repeat element. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6310–6314. doi: 10.1073/pnas.90.13.6310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Umesono K., Evans R. M. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell. 1989 Jun 30;57(7):1139–1146. doi: 10.1016/0092-8674(89)90051-2. [DOI] [PubMed] [Google Scholar]
  50. Umesono K., Murakami K. K., Thompson C. C., Evans R. M. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991 Jun 28;65(7):1255–1266. doi: 10.1016/0092-8674(91)90020-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yoshimoto Y., Norman A. W. Biological activity of vitamin D metabolites and analogs: dose-response study of 45Ca transport in an isolated chick duodenum perfusion system. J Steroid Biochem. 1986 Dec;25(6):905–909. doi: 10.1016/0022-4731(86)90322-5. [DOI] [PubMed] [Google Scholar]
  52. Zechel C., Shen X. Q., Chen J. Y., Chen Z. P., Chambon P., Gronemeyer H. The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats. EMBO J. 1994 Mar 15;13(6):1425–1433. doi: 10.1002/j.1460-2075.1994.tb06396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zelent A., Mendelsohn C., Kastner P., Krust A., Garnier J. M., Ruffenach F., Leroy P., Chambon P. Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing. EMBO J. 1991 Jan;10(1):71–81. doi: 10.1002/j.1460-2075.1991.tb07922.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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