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Summary of
research:
Our research interests are directed to four separate, but complementary
areas of investigation: (1) understanding the molecular mechanisms
regulating liver-specific gene expression and these regulatory networks
provide metabolic homeostasis; (2) developing methods to implement
novel gene therapies centering on liver targeting; and, (3)
Identification of bacterial signals (“quorum sensing molecules”) and
defining how they mediate host immune responses; (4) Elucidating the functional role of the
regulatory protein Txnip in regulating redox balance and metabolic control.
1) Liver-specific gene expression and metabolic
regulation
The liver is the major tissue site responsible for regulating
energy, carbohydrate and lipid metabolism. For this reason, many of the chemicals (drugs)
developed for ameliorating diseases associated with abnormal energy
(obesity), carbohydrate (diabetes) and lipid (obesity, diabetes and
heart disease) are targeted to liver-specific processes. A major goal of our research is
to understand how the transcription of the genes responsible for controlling
these liver-specific processes are regulated. We have concentrated on three individual genes:
cholesterol-7a-hydroxylase (CYP7A1) which controls the conversion of
cholesterol to bile acids, the major route to destroy excess
cholesterol, paraoxonase1 (PON1) which is carried in blood as a
component of HDL and whose concentrations accurately predict
susceptibility to atherosclerosis and microsomal triglyceride transfer
protein (MTP) which controls the production of lipoprotein particles
necessary to transport fat in blood.
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Figure 1. MTP and L-FABP demonstrate similar cell-type
specific differences in both mRNA and promoter activity levels. A, proximal regions of both MTP and L-FABP promoters
(rat) are aligned. The conserved direct repeat (DR1) is underlined. B, sybr green real time PCR analysis of MTP and
L-FABP mRNA levels in L35 and FAO cells. All values normalized to levels of 36B4 mRNA. C, Luciferase constructs
driven by either the MTP (-135/+66) or L-FABP (-141/+66) promoters
were transiently transfected into L35 and FAO cells. Constructs
containing mutant DR1 elements consist of base pair changes in the 5'
hexameric half sites of each promoter from AC to TG. Luciferase activities are
represented by filled bars (FAO cells) and empty bars (L35 cells).
All luciferase values were normalized to a Renilla control. Error
bars indicate S.D. of triplicate samples.
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Recent papers:
Kang, S., Spann, N.J., Hui,
T.Y., and Davis, R.A. 2003. ARP-1/COUP-TF II determines hepatoma
phenotype by acting as both a transcriptional repressor of microsomal
triglyceride transfer protein and an inducer of CYP7A1. J Biol Chem
278:30478-30486.
Gutierrez,
A., Ratliff, E.P., Andres, A.M., Huang, X., McKeehan, W.L., and Davis,
R.A. 2005. Bile Acids Decrease Hepatic Paraoxonase 1 Expression and
Plasma High-Density Lipoprotein Levels via FXR-Mediated Signaling of FGFR4.
Arterioscler Thromb Vasc Biol
26:301-306.
Spann, N.J., Kang, S., Li, A., and
Davis, R.A. 2006. Conserved DR1 Elements in the L-FABP and MTP
Promoters Coordinately Induce Transcription via PPARα-PGC-1β Occupancy.
(in review).
2) Gene therapy
Owing to its large size and unrestricted
contact with blood, the liver is a key tissue target for gene
therapy. The liver,
composed of many distinct cell types, is the largest reservoir of
macrophages. Since
macrophages are derived from hematopoietic stem cells (HSCs), we
developed a method to rapidly deliver therapeutic transgenes to the
liver using HSCs. To test this possibility we have created a new method
based on the ability of GdCl3 to induce a rapid and
short-lived apotosis in hepatic macrophages (i.e. Kupffer cells). The subsequent rapid
replacement of new Kupffer cells derived from the bone marrow HSCs,
allows transfer of theraoeutic transgenes to the liver. Using HSCs expressing a PON1
transgene, this method allowed us to increase the amount of PON1 protein
in mice that were genetically modified so that they rapidly develop
atherosclerosis. Transfer of PON1 into the livers of mice reduced
atherosclerosis formation by 50%.
This method is being adapted to transfer genes that are targets
for metabolic diseases.
Figure 2. GdCl3 treatment decreased
atherosclerotic lesions in LDL receptor-deficient mice engrafted with
HSCs expressing PON1. Atherosclerotic lesion area was quantitated
using aortic sinus frozen thin sections stained with oil red O obtained
from mice sacrificed after 12 weeks of being fed an atherogenic,
cholesterol-enriched diet. The mean lesion area ± S.D. for 12 separate mice per
group are shown. Statistical
differences (p values) among the groups are indicated. GFP-Tg
represents mice receiving bone marrow expressing the GFP transgene;
GFP-Tg/PON1-Tg represents mice receiving bone marrow expressing both
the GFP transgene and the PON1 transgene. Saline-treated represents mice receiving the designated
bone marrow and subsequently treated with saline only; GdCl3-treated
represents mice receiving the designated bone marrow and subsequently
treated with GdCl3 (25 mg/kg in saline).
Recent papers:
Bradshaw,
G., Gutierrez, A., Miyake, J.H., Davis, K.R., Li, A.C., Glass, C.K.,
Curtiss, L.K., and Davis, R.A. 2005. Facilitated replacement of Kupffer
cells expressing a paraoxonase-1 transgene is essential for
ameliorating atherosclerosis in mice. Proc Natl Acad Sci U S A 102:11029-11034.
3) Identification of bacterial signals (“quorum
sensing molecules”) and defining how they mediate host immune responses
Through a serendipitous series of experiments, we discovered
that PON1 blocks the ability of Salmonella bacteria from infecting mice. Salmonella infections are common and are the major cause
of food poisoning. In some
people, infections with Salmonella lead to typhoid fever and death. Recent studies suggest that
PON1 protects from Salmonella infections by inactivating chemical signals (i.e. quorum sensing
lactones) made by Salmonella
and are necessary to allow them to overcome immune defenses and invade
hosts. We are presently
identifying the quorum sensing lactones.
Figure 3. Absence of paraoxonase1 increases susceptibility of mice to Salmonella
infection. (A) C57BL/6/PON1 KO and C57BL/6 (wild-type) mice
were infected with 4,000 bacteria/mouse. Mice had free access chow to water. The number of mice surviving is
indicated. (B) C57BL/6/PON1
KO and C57BL/6 (wild-type) mice were infected with 4,000 bacteria/mouse and sacrificed 4 days
later. Livers and spleens
were homogenized in LB media, serial diluted and CFU’s were determined.
Values represent the mean ± SD for 6 mice in each group. (C) C57BL/6/PON1 KO and C57BL/6 (wild-type) mice
were infected with 4,000 bacteria expressing Green Fluorescent Protein
(GFP)/mouse and sacrificed 4 days later. Confocal fluorescent
microscopy images of frozen thin sections of livers from mice are shown
(100 X). Topo-3 nuclear stain is shown in blue. *Designates significant
difference between control and PON1 KO mice treated with Salmonella as determined by Student’s T test.
Recent papers:
Walls, M.Y., Gutierrez, A., Post,
N., Shih, D.M., Lusis, A.J., Maloy, S., and Davis, R.A. 2006.
Paraoxonase1 Protects Against Salmonella Infection. (in review).
4) Elucidating the functional
role of Txnip in redox balance and metabolic control
We have identified a protein (TXNIP)which is
found in every cell type and controls the ability to transfer chemical
energy into proteins. We have
used a new technique to selectively delete TXNIP from individual
tissues in mice. This has
allowed us to discover how TXNIP operates. Our results have revealed that TXNIP affects many
processes including: the way insulin is secreted in response to glucose
of energy, the ability of muscle and hearts to use fat as a source of
energy and the ability of all cell to tolerate oxidative injury. We have genetically
modified mice so that both Txnip alleles have lox sites surrounding exons 1 and 2. These floxed Txnip mice have
been used to create mice exhibiting tissue specific deletions of Txnip
in order to identify the mechanism through which Txnip helps to adapt
metabolism in response to nutritional state.
Recent papers:
Hui, T.Y., Sheth, S.S., J.M., D.,
Potter, D.W., Lusis, A.J., Attie, A.D., and Davis, R.A. 2004. Mice
Lacking Thioredoxin Interacting Protein Provide Evidence Linking
Cellular Redox State to Appropriate Response to Nutritional Signals. J.
Biol. Chem.
279:24387-24393.
Derivation
of mice carrying Txnip Floxed gene for studying the effect of
tissue-specific deletion
Three positive
ES cell clones were microinjected into C57BL/6 blastocysts, which were
then implanted into pseudopregnant female recipients to produce chimeras. Twenty-two chimera mice were
bred with C57/BL6 mice and their offspring were screened for germline
transmission of the floxed allele by PCR analysis. Of the 22 chimera mice
screened, 5 of them (from 2 independent ES cell clones) showed germline
transmission of the floxed allele. Heterozygous floxed mice from each founder line were
inter-bred to generate homozygous floxed mice (Fig. 1).
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Figure 4.
Genotyping analysis of progeny.
The genotype of each mouse floxed Mice by PCR was determined by
PCR analysis using DNA isolated from the tails. The size of the PCR products
diagnostic to wild-type and floxed alleles are 400 bp and 211 bp
respectively.
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Generation
of total body Txnip knockout mice
To generate Txnip total
knockout mice, homozygous floxed mice were bred with zp3-cre mice,
which express cre recombinase specifically in the female germline. Heterozygous knockout mice
(TxnipD/+) were interbred to produce homozygous knockout
mice (TxnipD/D) and wildtype littermates (Txnip++/+)
as control (Fig. 5).
Figure
5. Genotyping of Txnip Knockout Mice by PCR
Analysis
The genotype of each mouse was determined by PCR
analysis using DNA isolated from the tails. The wildtype Txnip allele and floxed allele yield a
699 bp and 734 bp PCR products respectively. When deleted by cre recombinase, the deleted allele
produces a 530 diagnostic PCR product.
Expression
of Txnip is completely abolished in Txnip knockout mice as evident from
the absence of RNA expression in the liver. (Fig. 6).
Figure 6. Expression
of Txnip mRNA in TKO Mice. Total RNA was isolated from
livers of wildtype (WT), heterozygous (TxnipD/+) and
homozygous (TxnipD/D) Total Txnip knockout mice. Txnip mRNA level was determined
by quantitative real-time PCR and normalized to 18S RNA. Results are represented as Mean
+ SD from 4 mice in each group.
Generation
of pancreatic beta-cell-specific Txnip knockout mice
Pancreatic
beta-cells-specific knockout (PKO) mice were generated by crossing
homozygous Txnip floxed (Txnipfl/fl) mice with Rip-cre mice,
which express cre recombinase specifically in pancreatic beta-cells
through the control of rat insulin promoter. Heterozygous PKO (Txnipfl/+, Ripcre/f)
mice were bred with Txnipfl/+ mice to produce homozygous PKO
(Txnipfl/fl, Ripcre/f) mice (Fig. 7).
Figure
7. Genotyping of Pancreatic beta-cells-specific Mice by PCR
Analysis
The
genotype of each mouse was determined by PCR analysis using DNA isolated
from the tails. PCR
analysis for the wildtype and floxed Txnip alleles were done as
described in Fig.1. The
presence of Rip-cre gene was determined by the presence of 100 bp
specific PCR product. A
representative genotyping result is presented to show the 6 possible
genotypes from the breeding between heterozygous PKO mice and
heterozygous floxed mice.
Natural Products mass spectroscopy center
The recent
acquisition of a new LC-mass spectrometer (Thermo LCQ Advantage MAX
Ultra Sensitive LC-MS/MS Ion Trap System) and a new GC-mass
spectrometer (Thermo TRACE
DSQ, TRACE GC/MS system) has allowed us to develop a natural products
chemical analysis core in order to identify the structures of factors
that alter metabolism as well as affect host immune response. This core lab will be housed in
the new BioSciences Building and will facilitate a coordinated research
approach involving natural products chemistry, microbiology and
mammalian physiology.
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