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J Cell Biol. Dec 1, 1990; 111(6): 2385–2395.
PMCID: PMC2116405

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25- dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors

Abstract

Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.

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Selected References

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  • Antakly T, Thompson EB, O'Donnell D. Demonstration of the intracellular localization and up-regulation of glucocorticoid receptor by in situ hybridization and immunocytochemistry. Cancer Res. 1989 Apr 15;49(8 Suppl):2230s–2234s. [PubMed]
  • Baran DT, Sorensen AM, Honeyman TW, Ray R, Holick MF. Rapid actions of 1 alpha,25-dihydroxyvitamin D3 on Ca2+ and phospholipids in isolated rat liver nuclei. FEBS Lett. 1989 Dec 18;259(1):205–208. [PubMed]
  • Barrack ER. Steroid hormone receptor localization in the nuclear matrix: interaction with acceptor sites. J Steroid Biochem. 1987;27(1-3):115–121. [PubMed]
  • Barsony J, Marx SJ. Receptor-mediated rapid action of 1 alpha,25-dihydroxycholecalciferol: increase of intracellular cGMP in human skin fibroblasts. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1223–1226. [PMC free article] [PubMed]
  • Barsony J, McKoy W, DeGrange DA, Liberman UA, Marx SJ. Selective expression of a normal action of the 1,25-dihydroxyvitamin D3 receptor in human skin fibroblasts with hereditary severe defects in multiple actions of that receptor. J Clin Invest. 1989 Jun;83(6):2093–2101. [PMC free article] [PubMed]
  • Beato M, Chalepakis G, Schauer M, Slater EP. DNA regulatory elements for steroid hormones. J Steroid Biochem. 1989 May;32(5):737–747. [PubMed]
  • Ben-Or S, Chrambach A. Transformation of the glucocorticoid receptor in the cell-free cytosol of the neural retina of the chick embryo: changes in the size and charge of the receptor complex during transformation suggest a multistage process. J Steroid Biochem. 1988 Jan;29(1):47–56. [PubMed]
  • Berger U, Wilson P, McClelland RA, Colston K, Haussler MR, Pike JW, Coombes RC. Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissues. J Clin Endocrinol Metab. 1988 Sep;67(3):607–613. [PubMed]
  • Berger U, Wilson P, Thethi S, McClelland RA, Greene GL, Coombes RC. Comparison of an immunocytochemical assay for progesterone receptor with a biochemical method of measurement and immunocytochemical examination of the relationship between progesterone and estrogen receptors. Cancer Res. 1989 Sep 15;49(18):5176–5179. [PubMed]
  • Bindal RD, Carlson KE, Katzenellenbogen BS, Katzenellenbogen JA. Lipophilic impurities, not phenolsulfonphthalein, account for the estrogenic activity in commercial preparations of phenol red. J Steroid Biochem. 1988 Sep;31(3):287–293. [PubMed]
  • Boivin G, Mesguich P, Pike JW, Bouillon R, Meunier PJ, Haussler MR, Dubois PM, Morel G. Ultrastructural immunocytochemical localization of endogenous 1,25-dihydroxyvitamin D3 and its receptors in osteoblasts and osteocytes from neonatal mouse and rat calvaria. Bone Miner. 1987 Nov;3(2):125–136. [PubMed]
  • Brown TA, DeLuca HF. Phosphorylation of the 1,25-dihydroxyvitamin D3 receptor. A primary event in 1,25-dihydroxyvitamin D3 action. J Biol Chem. 1990 Jun 15;265(17):10025–10029. [PubMed]
  • Clemens TL, Garrett KP, Zhou XY, Pike JW, Haussler MR, Dempster DW. Immunocytochemical localization of the 1,25-dihydroxyvitamin D3 receptor in target cells. Endocrinology. 1988 Apr;122(4):1224–1230. [PubMed]
  • Dame MC, Pierce EA, Prahl JM, Hayes CE, DeLuca HF. Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains. Biochemistry. 1986 Aug 12;25(16):4523–4534. [PubMed]
  • Duval D, Durant S, Homo-Delarche F. Non-genomic effects of steroids. Interactions of steroid molecules with membrane structures and functions. Biochim Biophys Acta. 1983 Aug 11;737(3-4):409–442. [PubMed]
  • Edelman A, Garabedian M, Anagnostopoulos T. Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: role of Ca2+-dependent K+ channels. J Membr Biol. 1986;90(2):137–143. [PubMed]
  • Eil C, Liberman UA, Rosen JF, Marx SJ. A cellular defect in hereditary vitamin-D-dependent rickets type II: defective nuclear uptake of 1,25-dihydroxyvitamin D in cultured skin fibroblasts. N Engl J Med. 1981 Jun 25;304(26):1588–1591. [PubMed]
  • Elashry-Stowers D, Zava DT, Speers WC, Edwards DP. Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies. Cancer Res. 1988 Nov 15;48(22):6462–6474. [PubMed]
  • Gasc JM, Delahaye F, Baulieu EE. Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study. Exp Cell Res. 1989 Apr;181(2):492–504. [PubMed]
  • Glass CK, Lipkin SM, Devary OV, Rosenfeld MG. Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone receptor heterodimer. Cell. 1989 Nov 17;59(4):697–708. [PubMed]
  • Gorski J, Welshons WV, Sakai D, Hansen J, Walent J, Kassis J, Shull J, Stack G, Campen C. Evolution of a model of estrogen action. Recent Prog Horm Res. 1986;42:297–329. [PubMed]
  • Govindan MV. Immunofluorescence microscopy of the intracellular translocation of glucocorticoid-receptor complexes in rat hepatoma (HTC) cells. Exp Cell Res. 1980 Jun;127(2):293–297. [PubMed]
  • Guiochon-Mantel A, Loosfelt H, Lescop P, Sar S, Atger M, Perrot-Applanat M, Milgrom E. Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers. Cell. 1989 Jun 30;57(7):1147–1154. [PubMed]
  • Haussler MR, Mangelsdorf DJ, Komm BS, Terpening CM, Yamaoka K, Allegretto EA, Baker AR, Shine J, McDonnell DP, Hughes M, et al. Molecular biology of the vitamin D hormone. Recent Prog Horm Res. 1988;44:263–305. [PubMed]
  • Hirst M, Feldman D. Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form. J Biol Chem. 1987 May 25;262(15):7072–7075. [PubMed]
  • Hodin RA, Lazar MA, Chin WW. Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J Clin Invest. 1990 Jan;85(1):101–105. [PMC free article] [PubMed]
  • Hopwood D, Coghill G, Ramsay J, Milne G, Kerr M. Microwave fixation: its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy. Histochem J. 1984 Nov;16(11):1171–1191. [PubMed]
  • Kayser K, Stute H, Lübcke J, Wazinski U. Rapid microwave fixation--a comparative morphometric study. Histochem J. 1988 Jun-Jul;20(6-7):347–352. [PubMed]
  • King WJ, Greene GL. Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature. 1984 Feb 23;307(5953):745–747. [PubMed]
  • Koenig H, Fan CC, Goldstone AD, Lu CY, Trout JJ. Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes. Circ Res. 1989 Mar;64(3):415–426. [PubMed]
  • Kost SL, Smith DF, Sullivan WP, Welch WJ, Toft DO. Binding of heat shock proteins to the avian progesterone receptor. Mol Cell Biol. 1989 Sep;9(9):3829–3838. [PMC free article] [PubMed]
  • Kumar V, Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988 Oct 7;55(1):145–156. [PubMed]
  • Leong AS. Microwave irradiation in histopathology. Pathol Annu. 1988;23(Pt 2):213–234. [PubMed]
  • Leong AS, Milios J, Duncis CG. Antigen preservation in microwave-irradiated tissues: a comparison with formaldehyde fixation. J Pathol. 1988 Dec;156(4):275–282. [PubMed]
  • Liberman UA, Eil C, Marx SJ. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts. J Clin Invest. 1983 Feb;71(2):192–200. [PMC free article] [PubMed]
  • Liberman UA, Eil C, Marx SJ. Receptor-positive hereditary resistance to 1,25-dihydroxyvitamin D: chromatography of hormone-receptor complexes on deoxyribonucleic acid-cellulose shows two classes of mutation. J Clin Endocrinol Metab. 1986 Jan;62(1):122–126. [PubMed]
  • Lieberherr M, Grosse B, Duchambon P, Drüeke T. A functional cell surface type receptor is required for the early action of 1,25-dihydroxyvitamin D3 on the phosphoinositide metabolism in rat enterocytes. J Biol Chem. 1989 Dec 5;264(34):20403–20406. [PubMed]
  • Login GR, Dvorak AM. Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy. Histochem J. 1988 Jun-Jul;20(6-7):373–387. [PubMed]
  • Login GR, Galli SJ, Morgan E, Arizono N, Schwartz LB, Dvorak AM. Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique. Lab Invest. 1987 Nov;57(5):592–599. [PubMed]
  • Martin PM, Sheridan PJ. Intracellular localization of steroid hormone receptors. A challenging problem. Autoradiographic and biochemical study. Ann Pathol. 1986;6(2):115–129. [PubMed]
  • McDonnell DP, Scott RA, Kerner SA, O'Malley BW, Pike JW. Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression. Mol Endocrinol. 1989 Apr;3(4):635–644. [PubMed]
  • Migliaccio A, Di Domenico M, Green S, de Falco A, Kajtaniak EL, Blasi F, Chambon P, Auricchio F. Phosphorylation on tyrosine of in vitro synthesized human estrogen receptor activates its hormone binding. Mol Endocrinol. 1989 Jul;3(7):1061–1069. [PubMed]
  • Milarski KL, Morimoto RI. Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding. J Cell Biol. 1989 Nov;109(5):1947–1962. [PMC free article] [PubMed]
  • Milde P, Merke J, Ritz E, Haussler MR, Rauterberg EW. Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods. J Histochem Cytochem. 1989 Nov;37(11):1609–1617. [PubMed]
  • Nakada M, Simpson RU, DeLuca HF. Subcellular distribution of DNA-binding and non-DNA-binding 1,25-dihydroxyvitamin D receptors in chicken intestine. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6711–6713. [PMC free article] [PubMed]
  • Newmeyer DD, Forbes DJ. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. [PubMed]
  • Ortí E, Mendel DB, Smith LI, Munck A. Agonist-dependent phosphorylation and nuclear dephosphorylation of glucocorticoid receptors in intact cells. J Biol Chem. 1989 Jun 15;264(17):9728–9731. [PubMed]
  • Parikh I, Rajendran KG, Su JL, Lopez T, Sar M. Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies. J Steroid Biochem. 1987;27(1-3):185–192. [PubMed]
  • Picard D, Yamamoto KR. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. [PMC free article] [PubMed]
  • Picard D, Kumar V, Chambon P, Yamamoto KR. Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul. 1990 Feb;1(3):291–299. [PMC free article] [PubMed]
  • Pierce EA, Dame MC, DeLuca HF. Size and charge of the functional 1,25-dihydroxyvitamin D receptor in porcine intestine. J Biol Chem. 1987 Dec 15;262(35):17092–17099. [PubMed]
  • Pike JW, Sleator NM. Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts. Biochem Biophys Res Commun. 1985 Aug 30;131(1):378–385. [PubMed]
  • Pike JW, Dokoh S, Haussler MR, Liberman UA, Marx SJ, Eil C. Vitamin D3--resistant fibroblasts have immunoassayable 1,25-dihydroxyvitamin D3 receptors. Science. 1984 May 25;224(4651):879–881. [PubMed]
  • Pike JW, Marion SL, Donaldson CA, Haussler MR. Serum and monoclonal antibodies against the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Generation by a preparation enriched in a 64,000-dalton protein. J Biol Chem. 1983 Jan 25;258(2):1289–1296. [PubMed]
  • Pike JW, Sleator NM, Haussler MR. Chicken intestinal receptor for 1,25-dihydroxyvitamin D3. Immunologic characterization and homogeneous isolation of a 60,000-dalton protein. J Biol Chem. 1987 Jan 25;262(3):1305–1311. [PubMed]
  • Pratt WB, Sanchez ER, Bresnick EH, Meshinchi S, Scherrer LC, Dalman FC, Welsh MJ. Interaction of the glucocorticoid receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated receptor transformation and translocation. Cancer Res. 1989 Apr 15;49(8 Suppl):2222s–2229s. [PubMed]
  • Raam S, Richardson GS, Bradley F, MacLaughlin D, Sun L, Frankel F, Cohen JL. Translocation of cytoplasmic estrogen receptors to the nucleus: immunohistochemical demonstration utilizing rabbit antibodies to estrogen receptors of mammary carcinomas. Breast Cancer Res Treat. 1983;3(2):179–199. [PubMed]
  • Ritchie HH, Hughes MR, Thompson ET, Malloy PJ, Hochberg Z, Feldman D, Pike JW, O'Malley BW. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D3-resistant rickets in three families. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9783–9787. [PMC free article] [PubMed]
  • Robertson NM, Kusmik WF, Grove BF, Miller-Diener A, Webb ML, Litwack G. Characterization of a monoclonal antibody that probes the functional domains of the glucocorticoid receptor. Biochem J. 1987 Aug 15;246(1):55–65. [PMC free article] [PubMed]
  • Sanchez ER, Redmond T, Scherrer LC, Bresnick EH, Welsh MJ, Pratt WB. Evidence that the 90-kilodalton heat shock protein is associated with tubulin-containing complexes in L cell cytosol and in intact PtK cells. Mol Endocrinol. 1988 Aug;2(8):756–760. [PubMed]
  • Sandgren ME, Deluca HF. An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor. Anal Biochem. 1989 Nov 15;183(1):57–63. [PubMed]
  • Schwartz Z, Schlader DL, Swain LD, Boyan BD. Direct effects of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting zone chondrocyte membrane alkaline phosphatase and phospholipase-A2 specific activities. Endocrinology. 1988 Dec;123(6):2878–2884. [PubMed]
  • Smith LD. The induction of oocyte maturation: transmembrane signaling events and regulation of the cell cycle. Development. 1989 Dec;107(4):685–699. [PubMed]
  • Sone T, Marx SJ, Liberman UA, Pike JW. A unique point mutation in the human vitamin D receptor chromosomal gene confers hereditary resistance to 1,25-dihydroxyvitamin D3. Mol Endocrinol. 1990 Apr;4(4):623–631. [PubMed]
  • Szego CM. Mechanisms of hormone action: parallels in receptor-mediated signal propagation for steroid and peptide effectors. Life Sci. 1984 Dec 10;35(24):2383–2396. [PubMed]
  • Usuda N, Ma HJ, Hanai T, Yokota S, Hashimoto T, Nagata T. Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes. J Histochem Cytochem. 1990 May;38(5):617–623. [PubMed]
  • Walters MR. Steroid hormone receptors and the nucleus. Endocr Rev. 1985 Fall;6(4):512–543. [PubMed]
  • Weisman Y, Bab I, Gazit D, Spirer Z, Jaffe M, Hochberg Z. Long-term intracaval calcium infusion therapy in end-organ resistance to 1,25-dihydroxyvitamin D. Am J Med. 1987 Nov;83(5):984–990. [PubMed]
  • Wikström AC, Bakke O, Okret S, Brönnegård M, Gustafsson JA. Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology. 1987 Apr;120(4):1232–1242. [PubMed]
  • Williams D, Gorski J. Kinetic and equilibrium analysis of estradiol in uterus: a model of binding-site distribution in uterine cells. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3464–3468. [PMC free article] [PubMed]
  • Wolff B, Willingham MC, Hanover JA. Nuclear protein import: specificity for transport across the nuclear pore. Exp Cell Res. 1988 Oct;178(2):318–334. [PubMed]

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