Abstract
Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb -2.8 to -1.0 of the marine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb -2.5 to -2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor gamma (PPARgamma) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR gamma antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR-gamma mRNA levels increase correspondingly. Although forced expression of PPAR gamma and retinoid X receptor alpha activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR gamma is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.
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- Ailhaud G., Grimaldi P., Négrel R. Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr. 1992;12:207–233. doi: 10.1146/annurev.nu.12.070192.001231. [DOI] [PubMed] [Google Scholar]
- Alvarez R., de Andrés J., Yubero P., Viñas O., Mampel T., Iglesias R., Giralt M., Villarroya F. A novel regulatory pathway of brown fat thermogenesis. Retinoic acid is a transcriptional activator of the mitochondrial uncoupling protein gene. J Biol Chem. 1995 Mar 10;270(10):5666–5673. doi: 10.1074/jbc.270.10.5666. [DOI] [PubMed] [Google Scholar]
- Boyer B. B., Kozak L. P. The mitochondrial uncoupling protein gene in brown fat: correlation between DNase I hypersensitivity and expression in transgenic mice. Mol Cell Biol. 1991 Aug;11(8):4147–4156. doi: 10.1128/mcb.11.8.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cassard-Doulcier A. M., Gelly C., Fox N., Schrementi J., Raimbault S., Klaus S., Forest C., Bouillaud F., Ricquier D. Tissue-specific and beta-adrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5'-flanking region. Mol Endocrinol. 1993 Apr;7(4):497–506. doi: 10.1210/mend.7.4.8388995. [DOI] [PubMed] [Google Scholar]
- Cassard-Doulcier A. M., Larose M., Matamala J. C., Champigny O., Bouillaud F., Ricquier D. In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein. J Biol Chem. 1994 Sep 30;269(39):24335–24342. [PubMed] [Google Scholar]
- Chawla A., Lazar M. A. Peroxisome proliferator and retinoid signaling pathways co-regulate preadipocyte phenotype and survival. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1786–1790. doi: 10.1073/pnas.91.5.1786. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cornelius P., MacDougald O. A., Lane M. D. Regulation of adipocyte development. Annu Rev Nutr. 1994;14:99–129. doi: 10.1146/annurev.nu.14.070194.000531. [DOI] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
- Forman B. M., Tontonoz P., Chen J., Brun R. P., Spiegelman B. M., Evans R. M. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell. 1995 Dec 1;83(5):803–812. doi: 10.1016/0092-8674(95)90193-0. [DOI] [PubMed] [Google Scholar]
- Freytag S. O., Paielli D. L., Gilbert J. D. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 1994 Jul 15;8(14):1654–1663. doi: 10.1101/gad.8.14.1654. [DOI] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Graves R. A., Tontonoz P., Ross S. R., Spiegelman B. M. Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor. Genes Dev. 1991 Mar;5(3):428–437. doi: 10.1101/gad.5.3.428. [DOI] [PubMed] [Google Scholar]
- Graves R. A., Tontonoz P., Spiegelman B. M. Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression. Mol Cell Biol. 1992 Mar;12(3):1202–1208. doi: 10.1128/mcb.12.3.1202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green H., Kehinde O. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975 May;5(1):19–27. doi: 10.1016/0092-8674(75)90087-2. [DOI] [PubMed] [Google Scholar]
- Himms-Hagen J. Brown adipose tissue thermogenesis and obesity. Prog Lipid Res. 1989;28(2):67–115. doi: 10.1016/0163-7827(89)90009-x. [DOI] [PubMed] [Google Scholar]
- Himms-Hagen J. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB J. 1990 Aug;4(11):2890–2898. [PubMed] [Google Scholar]
- Hynes R. O., Bye J. M. Density and cell cycle dependence of cell surface proteins in hamster fibroblasts. Cell. 1974 Oct;3(2):113–120. doi: 10.1016/0092-8674(74)90114-7. [DOI] [PubMed] [Google Scholar]
- Keller H., Mahfoudi A., Dreyer C., Hihi A. K., Medin J., Ozato K., Wahli W. Peroxisome proliferator-activated receptors and lipid metabolism. Ann N Y Acad Sci. 1993 Jun 11;684:157–173. doi: 10.1111/j.1749-6632.1993.tb32279.x. [DOI] [PubMed] [Google Scholar]
- Klaus S., Choy L., Champigny O., Cassard-Doulcier A. M., Ross S., Spiegelman B., Ricquier D. Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression. J Cell Sci. 1994 Jan;107(Pt 1):313–319. doi: 10.1242/jcs.107.1.313. [DOI] [PubMed] [Google Scholar]
- Kletzien R. F., Clarke S. D., Ulrich R. G. Enhancement of adipocyte differentiation by an insulin-sensitizing agent. Mol Pharmacol. 1992 Feb;41(2):393–398. [PubMed] [Google Scholar]
- Kliewer S. A., Lenhard J. M., Willson T. M., Patel I., Morris D. C., Lehmann J. M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell. 1995 Dec 1;83(5):813–819. doi: 10.1016/0092-8674(95)90194-9. [DOI] [PubMed] [Google Scholar]
- Kliewer S. A., Umesono K., Mangelsdorf D. J., Evans R. M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature. 1992 Jan 30;355(6359):446–449. doi: 10.1038/355446a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kliewer S. A., Umesono K., Noonan D. J., Heyman R. A., Evans R. M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 1992 Aug 27;358(6389):771–774. doi: 10.1038/358771a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kopecký J., Baudysová M., Zanotti F., Janíková D., Pavelka S., Houstek J. Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture. J Biol Chem. 1990 Dec 25;265(36):22204–22209. [PubMed] [Google Scholar]
- Kozak U. C., Kopecky J., Teisinger J., Enerbäck S., Boyer B., Kozak L. P. An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene. Mol Cell Biol. 1994 Jan;14(1):59–67. doi: 10.1128/mcb.14.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laborda J. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucleic Acids Res. 1991 Jul 25;19(14):3998–3998. doi: 10.1093/nar/19.14.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lehmann J. M., Moore L. B., Smith-Oliver T. A., Wilkison W. O., Willson T. M., Kliewer S. A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem. 1995 Jun 2;270(22):12953–12956. doi: 10.1074/jbc.270.22.12953. [DOI] [PubMed] [Google Scholar]
- Lowell B. B., S-Susulic V., Hamann A., Lawitts J. A., Himms-Hagen J., Boyer B. B., Kozak L. P., Flier J. S. Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature. 1993 Dec 23;366(6457):740–742. doi: 10.1038/366740a0. [DOI] [PubMed] [Google Scholar]
- McGrane M. M., Yun J. S., Moorman A. F., Lamers W. H., Hendrick G. K., Arafah B. M., Park E. A., Wagner T. E., Hanson R. W. Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice. J Biol Chem. 1990 Dec 25;265(36):22371–22379. [PubMed] [Google Scholar]
- Mercer S. W., Trayhurn P. Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice. FEBS Lett. 1986 Jan 20;195(1-2):12–16. doi: 10.1016/0014-5793(86)80120-x. [DOI] [PubMed] [Google Scholar]
- Nicholls D. G., Locke R. M. Thermogenic mechanisms in brown fat. Physiol Rev. 1984 Jan;64(1):1–64. doi: 10.1152/physrev.1984.64.1.1. [DOI] [PubMed] [Google Scholar]
- Rabelo R., Schifman A., Rubio A., Sheng X., Silva J. E. Delineation of thyroid hormone-responsive sequences within a critical enhancer in the rat uncoupling protein gene. Endocrinology. 1995 Mar;136(3):1003–1013. doi: 10.1210/endo.136.3.7867554. [DOI] [PubMed] [Google Scholar]
- Rehnmark S., Kopecký J., Jacobsson A., Néchad M., Herron D., Nelson B. D., Obregon M. J., Nedergaard J., Cannon B. Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine. Exp Cell Res. 1989 May;182(1):75–83. doi: 10.1016/0014-4827(89)90280-2. [DOI] [PubMed] [Google Scholar]
- Rehnmark S., Néchad M., Herron D., Cannon B., Nedergaard J. Alpha- and beta-adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture. J Biol Chem. 1990 Sep 25;265(27):16464–16471. [PubMed] [Google Scholar]
- Ricquier D., Bouillaud F., Toumelin P., Mory G., Bazin R., Arch J., Pénicaud L. Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue. Evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis. J Biol Chem. 1986 Oct 25;261(30):13905–13910. [PubMed] [Google Scholar]
- Ross S. R., Choy L., Graves R. A., Fox N., Solevjeva V., Klaus S., Ricquier D., Spiegelman B. M. Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7561–7565. doi: 10.1073/pnas.89.16.7561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ross S. R., Graves R. A., Greenstein A., Platt K. A., Shyu H. L., Mellovitz B., Spiegelman B. M. A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9590–9594. doi: 10.1073/pnas.87.24.9590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothwell N. J., Stock M. J., Tedstone A. E. Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat. Mol Cell Endocrinol. 1987 Jun;51(3):253–257. doi: 10.1016/0303-7207(87)90035-9. [DOI] [PubMed] [Google Scholar]
- Shapiro D. J., Sharp P. A., Wahli W. W., Keller M. J. A high-efficiency HeLa cell nuclear transcription extract. DNA. 1988 Jan-Feb;7(1):47–55. doi: 10.1089/dna.1988.7.47. [DOI] [PubMed] [Google Scholar]
- Short M. K., Clouthier D. E., Schaefer I. M., Hammer R. E., Magnuson M. A., Beale E. G. Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice. Mol Cell Biol. 1992 Mar;12(3):1007–1020. doi: 10.1128/mcb.12.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spiegelman B. M., Green H. Control of specific protein biosynthesis during the adipose conversion of 3T3 cells. J Biol Chem. 1980 Sep 25;255(18):8811–8818. [PubMed] [Google Scholar]
- Tontonoz P., Graves R. A., Budavari A. I., Erdjument-Bromage H., Lui M., Hu E., Tempst P., Spiegelman B. M. Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha. Nucleic Acids Res. 1994 Dec 25;22(25):5628–5634. doi: 10.1093/nar/22.25.5628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tontonoz P., Hu E., Devine J., Beale E. G., Spiegelman B. M. PPAR gamma 2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1995 Jan;15(1):351–357. doi: 10.1128/mcb.15.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tontonoz P., Hu E., Graves R. A., Budavari A. I., Spiegelman B. M. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994 May 15;8(10):1224–1234. doi: 10.1101/gad.8.10.1224. [DOI] [PubMed] [Google Scholar]
- Tontonoz P., Hu E., Spiegelman B. M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994 Dec 30;79(7):1147–1156. doi: 10.1016/0092-8674(94)90006-x. [DOI] [PubMed] [Google Scholar]
- 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]
- Yubero P., Manchado C., Cassard-Doulcier A. M., Mampel T., Viñas O., Iglesias R., Giralt M., Villarroya F. CCAAT/enhancer binding proteins alpha and beta are transcriptional activators of the brown fat uncoupling protein gene promoter. Biochem Biophys Res Commun. 1994 Jan 28;198(2):653–659. doi: 10.1006/bbrc.1994.1095. [DOI] [PubMed] [Google Scholar]
- Yubero P., Viñas O., Iglesias R., Mampel T., Villarroya F., Giralt M. Identification of tissue-specific protein binding domains in the 5'-proximal regulatory region of the rat mitochondrial brown fat uncoupling protein gene. Biochem Biophys Res Commun. 1994 Oct 28;204(2):867–873. doi: 10.1006/bbrc.1994.2540. [DOI] [PubMed] [Google Scholar]