H. Franklin Bunn, MD
Brigham and Women's Hospital
Harvard Medical School
One Blackfan Circle
KARP Bldg – CHRB 05.215
Boston, MA 02115
Tel: (617) 355-9068
Fax: (617) 355-9124
H. Franklin Bunn, MD
DESCRIPTION OF RESEARCH PROGRAM
The research in our lab focuses on genes involved in regulating energy metabolism and oxygen homeostasis. The work currently in progress as well as planned projects exploit up-to-date developments in cell and molecular biology. During the last 25 years 20 PhD scientists and 15 physician-scientists received one to four years' training in our laboratory. In addition, 15 medical students have spent 6 to 12 months in our lab working on research projects. Over 30 lab assistants have spent one to three years with us. Nearly all of them have gone on either to graduate school in biomedical science or to medical school. About 40 college and medical students have joined our lab for summer research experience.
Glucose and Fat Metabolism
Our lab has cloned a gene encoding a cytochrome b/b5 reductase fusion protein (Ncb5or). This novel flavo-heme protein has NAD(P)H dependent oxidase activity and is highly conserved in all higher eukaryotes including flies, worms and mammals. It is localized in the endoplasmic reticulum and is widely expressed in most organs and tissues. We have prepared a knockout of this gene in the mouse. The Ncb5or-/- mice are fully viable at the expected 25% frequency and have the following phenotype:
1) Diabetes: Until age 4 weeks, Ncb5or-/- animals have normal blood sugar levels and yet have abnormal glucose tolerance, indicative of a prediabetic state. By the time these -/- animals are 6 weeks old they all have frank diabetes with blood glucose levels > 600 mg/dl, low plasma insulin. These animals have normal sensitivity to administration of insulin. Thus the diabetes is due to a primary defect in insulin production. Immunostaining of the pancreas reveals marked deficiency of insulin-producing beta cells in the islets. Electron miscroscopy reveals marked hypertrophy and hyperplasia in mitochondria of beta cells of Ncb5or -/- mice.
2) Lipodystrophy with markedly decreased white fat, elevated serum triglycerides and cholesterol. The lipodystrophy persists even in -/- mice in which diabetes has been corrected by islet transplantation.
3) Skeletal abnormalities consisting of thinning of bony cortex, decreased bone density, increased osteoclast activity and increased serum alkaline phosphatase indicative of increased bone turnover.
Mouse models of diabetes and lipodystrophy have provided important insights into regulation of fat metabolism and energy homeostasis. The pancreatic beta cell is unusually susceptible to oxidant stress. Our studies show that Ncb5or plays a critical role in the redox homeostasis of beta cells. A detailed understanding of the physiology of this enzyme may be relevant to the pathogenesis of both type 1 and type 2 diabetes.
We are currently carrying out enzymatic studies to identify the physiologic substrate and product of Ncb5or. Preliminary evidence suggests that the enzyme functions in electron transport required for fatty acid desaturation.
Molecular Adaptation to Hypoxia
The other central focus of our laboratory has been to elucidate the mechanism(s) by which oxygen pressure is sensed in cells and triggers a signal transduction pathway that mediates regulation of physiologically important genes. Work in the lab began 10 years ago with studies on erythropoietin (Epo), a hormone responsible for red blood cell production and markedly induced by hypoxia.
Transcriptional Regulation The marked up-regulation of Epo gene expression in response to hypoxia involves primarily transcriptional activation by HIF-1, a hypoxia inducible heterodimeric transcription factor which binds to a 3’ enhancer. Our lab showed that activation of HIF-1 involves stabilization of its a subunit during hypoxia by inhibition of ubiquitin-dependent proteolysis. This post-translational regulation is strongly dependent on the redox state of the cell, thereby providing a clue as to the mechanism by which the hypoxic signal is transduced. Regulated proteolysis depends on a 200-residue Oxygen Dependent Degradation Domain (ODD) within HIF-1a.
Daphnia Hemoglobin When the water flea Daphnia magna is exposed to hypoxic water, it turns bright red. This phenomenon is due to marked in production of Daphnia hemoglobin. Dr. Thomas Gorr completed transfection experiments that clearly demonstrate the presence of cis-acting elements in the promoter of the Daphnia magna globin-2 gene which confer marked (10-15 fold) hypoxic induction when placed upstream of a luciferase reporter gene. These elements contain consensus HIF-1 binding sequences. When these sites are mutated individually and in combination, hypoxic induction of the reporter gene is markedly suppressed. Moreover, Dr. Gorr’s gel shift experiments show that HIF is activated in hypoxic Daphnia. These experiments demonstrate that HIF response elements in the globin-2 promoter are necessary and sufficient for transcriptional activation in response to hypoxia.
Hao Zhu (Assistant Professor at KUMC)
Kevin Larade (Research Fellow)
Andre Dejam (Clinical/Research Fellow)
Yongzhao Zhang (Research Fellow)
Zhi-gang Jiang (Technician)
Relevant Publications since 1987
o Goldberg MA, Glass GA, Cunningham JM, Bunn HF. The regulated expression of erythropoietin by two human hepatoma cell lines. Proc Natl Acad Sci USA, 1987; 84:7972-76.
o Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science, 1988; 242:1412-15.
o Gilliland G, Perrin S, Blanchard K, Bunn HF: Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA, 1990; 87:2725-29.
o Goldberg MA, Gaut CC, Bunn HF. Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events. Blood, 1991; 77:271-77.
o Gilliland DG, Blanchard KL, Levy J, Perrin S, Bunn HF. Clonality in myeloproliferative disorders: Analysis by means of the polymerase chain reaction. Proc Natl Acad Sci USA 1991; 88:6848-6852.
o Rondon IJ, MacMillan LA, Beckman BS, Goldberg MA, Schneider T, Bunn HF, Malter JS. Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein. J Biol Chem 1991; 266:16594-98.
o Blanchard KL, Acquaviva AM, Galson DL, Bunn HF. Hypoxic induction of the human erythropoietin gene: Cooperation between the promoter and enhancer, each containing steroid receptor response elements. Mol Cell Biol 1992; 12:5373-85.
o Fandrey J, Bunn HF. In vivo and in vitro regulation of erythropoietin mRNA: Measurement by competitive polymerase chain reaction. Blood 1993; 81:617-23.
o Boissel J-P, Lee W-R, Presness SR, Cohen FE, Bunn HF. Erythropoietin structure-function relationships: Mutant proteins that test a model of tertiary structure. J Biol Chem 1993; 268:983-93.
o Wen D, Boissel J-PR, Tracy TE, Mulcahy LS, Czelusniak J, Goodman M, Bunn HF. Erythropoietin Structure-Function Relationships: High degree of sequence homology among mammals. Blood 1993; 82:1507-16.
o Wen D, Boissel J-P, Showers M, Ruch BC, Bunn HF. Erythropoietin Structure-Function Relationships: Identification of Functionally Important Domains. J Biol Chem 1994; 269:22839-46.
o Galson D, Tsuchiya T,Tendler DS, Ren Y, Huang E, Ogura T, Bunn HF. Nuclear orphan receptor HNF-4 functions as transcriptional activator for tissue-specific and hypoxia-inducible erythropoietin gene expression. Mol Cell Biol 1995; 15:2135-2144.
o Ho V, Acquaviva A, Duh E, Bunn HF. Use of a marked erythropoietin gene for investigation of its cis-acting elements. J Biol Chem 1995; 270:10084-10090.
o Bunn HF, Poyton RO. Oxygen sensing and molecular adaptation to hypoxia. Physiol Reviews 1996; 76:839-885.
o Ho V, Bunn HF. Effect of transition metals on the expression of the erythropoietin gene: further evidence that the oxygen sensor is a heme protein. Biochem Biophys Res Commun 1996; 223:175-180
o Arany Z, Huang LE, Eckner R, Bhattacharya S, Jiang C, Goldberg MA, Bunn HF, Livingston DM. An essential role for p300/CBP in the cellular response to hypoxia. Proc Natl Acad Sci USA 1996; 93: 12969-12973.
o Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hypoxia-inducible transcription factor depends upon redox-sensitive stabilization of its a subunit. J Biol Chem 1996; 271: 32253-32259
o Ebert BL, Bunn HF. Regulation of trasncription by hypoxia requires a multiprotein complex that includes hypoxia inducible factor 1, adjacent transcription factors and p300/CBP. Mol Cell Biol 1998;18:4089-4096
o Qiu H, Belanger A, Yoon H-WP, Bunn HF. Homodimerization restores biological activity to an inactive erythropoietin mutant. J Biol Chem 1998; 273:11173-11176
o Huang LE, Gu J, Schau M, Bunn HF. Regulation of Hypoxia-inducible factor 1a is mediated by its oxygen-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 1998; 95:7987-7992
o Huang LE, Willmore W, Gu J, Goldberg MA, Bunn HF. Inhibition of HIF-1 activation by carbon monoxide and nitric oxide: implications for oxygen sensing and signaling J. Biol. Chem. 1999; 274:9038-9044.
o Ebert BL, Bunn HF. Regulation of the erythropoietin gene. Blood 1999; 94:1864-1877.
o Zhu H, Qiu H, Yoon H-WP, Huang S, Bunn HF. Identification of a novel cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells. Proc. Natl. Acad. Sci. USA 1999, 96:14742-14747
o Horiguchi H, Bunn HF. Erythropoietin induction in Hep3B cells is not affected by inhibition of heme biosynthesis. Biochim Biophys Acta 2000; 1495:231-236
o Horiguchi H, Kayama F, Oguma E, Willmore WG, Hradecky P, Bunn HF. Cadmium and Platinum Suppression of Erythropoietin Production in Cell Culture: Clinical Implications. Blood 2000, 96:3743-3747.
o Zhu H, Bunn HF, How do cells sense oxygen? (Perspective) Science, 2001, 292:449-451.
o Huang LE, Bunn HF, O2 Sensing, HIF and Regulation of Gene Expression. J. Biol. Chem. 2003; 278, 19575-78
o Xie J, Zhu H, Larade K, Ladoux A, Seguritan A, Chu M, Ito S, Bronson RT, Leiter EH, Zhang C-Y, Rosen ED, Bunn HF. Absence of a novel oxidoreductase, NCB5OR, causes insulin-deficient diabetes. Proc Natl Acad Sci USA 2004;101:10750-5.
o Zhu H, Larade K, Jackson TA, Xie J, Ladoux A, Acker H, Berchner-Pfannschmidt U, Fandrey J, Cross AR, Lukat-Rodgers GS, Rodgers KR, Bunn HF. NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplamsic reticulum. J Biol Chem. 2004; 279:30316-25.
o Gorr TA, Cahn JD, Yamagata H, Bunn HF. Hypoxia induced synthesis of hemoglobin in the crustacean Daphnia magna is HIF dependent. J Biol Chem 2004; 279:36038-47.
o Gorr TA, Tomita T, Wappner P, Bunn HF. Regulation of Drosophila HIF activity in SL2 cells: identification of a hypoxia-induced variant isoform of the HIFa homologue gene Similar. J Biol Chem 2004; 279:36048-58.
o Andersen G, Wegner L, Rose CS, Johansen A, Ek J, Lauenborg J, Drivsholm T, Borch-Johnsen K, Damm P, Hansen T, Bunn HF, Pedersen O. Variation in NCB5OR: studies of relationships to type 2 diabetes, MODY, and gestational diabetes mellitus. Diabetes. 2004; 53(11):2992-7.
o Larade K, Bunn HF. Promoter characterization and transcriptional regulation of Ncb5or, a novel reductase necessary for pancreatic b-cell maintenance. Biochim Biophys Acta, 2006; 1759:257-62.
o Larade K, Jiang Z-G, Dejam A, Zhu H, Bunn HF. The reductase Ncb5or is responsive to the redox status in beta-cells and is not involved in the ER stress response. Biochemical Journal (In Press)