H. Franklin Bunn, MD
Research
Director
Hematology
Division
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
Email:
bunn@calvin.bwh.harvard.edu
H. Franklin Bunn, MD
March, 2007
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.
The
Lab
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)