From the Endocrine Research Group, Departments of
Medicine and § Biochemistry & Molecular Biology and
Pharmacology & Therapeutics, the Faculty of Medicine, University
of Calgary, Calgary, Alberta, Canada T2N 4N1
Received for publication, December 7, 2000, and in revised form, January 10, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Insulin induces apolipoprotein A-I,
apoA-I gene transcription via a membrane receptor
with intrinsic tyrosine kinase activity. This finding prompted us to
ask whether the gene is stimulated by epidermal growth factor (EGF),
EGF a peptide hormone that binds to another member of the receptor
superfamily with tyrosine kinase activity. Our data showed that like
insulin, EGF increased abundance of apoA-I protein and transcription of
the gene in human hepatoma, Hep G2 cells. The effects of both hormones
appeared direct because their induction of apoA-I gene
transcription was not affected by the protein synthesis inhibitor,
cycloheximide. Although both insulin and EGF stimulate apoA-I
expression, each hormone binds to a distinct membrane receptor thus
suggesting differential intracellular signaling. Therefore, we used a
panel of inhibitors to define the pathway(s) that mediate the actions
of these hormones. Whereas, the actions of EGF required only the
Ras-mitogen-activated protein, MAP kinase, those of insulin were
mediated by equal participation of both the Ras-MAP kinase and protein
kinase C, PKC cascades. Despite differences in signaling pathways
triggered by each hormone receptor, the activation of
apoA-I transcription required the participation of a single
transcription factor, Sp1. Furthermore, EGF induction of transcription
was attenuated by mutating the MAP kinase site at amino acid,
Thr266 rendering Sp1 phosphorylation deficient. In summary,
EGF stimulation of apoA-I expression is mediated solely by
the Ras-MAP kinase cascade and enhanced activity of this pathway
requires Sp1 with an intact phosphorylation site at Thr266.
However, insulin induction of this gene is different and requires both
Ras-MAP kinase and PKC pathways but their actions are also mediated by Sp1.
Apolipoprotein A-I
(apoA-I)1 is a major protein
component of the serum high-density lipoprotein (HDL) particles (1, 2). The anti-atherogenic properties of apoA-I alone or as part of HDL
underlie their inverse correlation with the incidence of ischemic cardiovascular disease, the number 1 cause of premature death in modern
societies (3, 4). The cardioprotective actions of apoA-I or HDL arises
from their participation in a normal physiologic process, so called
"reverse cholesterol transport" (5, 6). ApoA-I acts as a
cofactor to facilitate an interaction between HDL particles and the
cell membrane. This interaction enables the efflux of intracellular
cholesterol to HDL particles, which in turn shuttles the sterol to the
liver for further metabolism and excretion (5, 7). Enhanced reverse
cholesterol transport lowers total body cholesterol, as demonstrated
clearly following the infusion of apoA-I protein into humans (8). The
reduction in cholesterol lowers the risk of arteriosclerosis (9), a
major cause of ischemic cardiovascular disease. In support of this
idea, transgenic mice that overexpress human apoA-I protein had
significant reductions of atherosclerotic lesions in vessel walls (10). Therefore, understanding the mechanisms that enhance apoA-I
expression will lead us to better ways to enhance its expression and
thus lower the risk of ischemic cardiovascular disease (11).
We showed recently that insulin induces rat apoA-I gene
transcription and this induction is mediated by an insulin responsive core element (IRCE) a motif recognized by Sp1 (12, 13). Insulin action
is initiated by its binding to a membrane receptor with intrinsic
tyrosine kinase activity, prompting us to wonder whether this mechanism
is unique to the peptide hormone. The actions of insulin can be
categorized into immediate responses: such as glucose transport or
delayed responses; including cell differentiation and proliferation
(14, 15). Several intracellular pathways are activated by insulin
action and may include the Ras-MAP kinase, PI 3-kinase, and
phospholipase C Plasmid Constructs--
Construction of the reporter,
pAI.474-CAT was described previously (17). The deletion constructs;
pAI.425-, pAI.375-, pAI.325-, and pAI.235-CAT containing rat
apoA-I DNA spanning Transient and Stable Transfection--
Human hepatoma Hep G2
cells were transiently transfected with plasmid DNA of interest using
LipofectAMINE (Life Technologies, Inc.) as per the instructions
recommended by the manufacturer. The efficiency of DNA uptake was
monitored by co-transfecting 1 µg of the plasmid,
RSV- Cell Culture and CAT Activity Assay--
Hep G2 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% bovine calf serum (Life Technologies,
Inc.) and penicillin/streptomycin at 37 °C. Hep G2 cells serve as an
accepted model for the studying apoA-I (12, 13, 22). Cells
were cultured overnight in serum-free medium prior to the addition of
the agent(s) of interest, followed by measuring CAT activity (12,
23).
SDS-Polyacrylamide Gel Electrophoresis and Western
Immunoblotting--
Whole cell extract from control or cells treated
with various factors were harvested and lysed in buffer containing
orthovanadate 2 mM, Triton X-100 1%, SDS 0.1%, leupeptin
and apoprotin 5 µg/ml each, benzamidine and bacitracin 1 mg/ml each,
dithiothreitrol 600 mM, Tris 20 mM (pH 7.4),
NaCl 300 mM, EDTA 5 mM, NaF 50 mM, sodium pyrophosphate 40 mM, KH2PO4
50 mM, and Na molybdate 10 mM. An aliquot of
equal numbers of control or treated hepatoma cells or culture medium
(10 µg of total protein) containing secreted apoA-I protein was
separated by electrophoresis in a 10% SDS-polyacrylamide gel and
transferred to nitrocellulose membrane. The blot was probed using a
monoclonal antibody (Calbiochem) as described (13). To investigate the
activation state of p42/44 MAP kinase in cell lysates of Hep G2 cells
in response to various treatments, we employed a phospho-specific
antibody probe directed against the activated form of ERK-1/2 (New
England Biolabs, Inc.) (24). Protein samples of total cell lysates were
analyzed for activated p42/44 MAP kinase using Western blot techniques
(21).
RNA Preparation and RT-PCR--
Total RNA from cells was
extracted from cells using TRITM-reagent (Molecular
Research Center, Cincinnati, OH) (25). The RNA was reverse-transcribed
with a first strand cDNA synthesis kit using pd(N)6
primer (Amersham Pharmacia Biotech) according to manufacturer's
protocol. 3 µl of this solution was amplify using PCR primed with a
forward primer 5'-CCTGATGAATGCTCATCCG-3' and reverse primer
5'-AAGCATTCTGCCGACATGG-3' homologous to the CAT gene. The
RT-PCR signal from CAT mRNA transcripts was normalized with the
signal obtained from Retroviral Infection of HepG2 Cell--
The pBabe-Puro
retroviral expression vector (27) with and without
RasAsn-17 insert was transfected into
AmphoPckTM-293 cell (CLONTECH), using
LipofectAMINE as per the manufacturer's instructions. The supernatants
containing the virus were used to infect stable Hep G2 cells that
harbored the pAI.474-CAT reporter gene in the presence of 8 µg/ml
Polybrene. After elimination of uninfected cells by adding 2.5 µg/ml
puromycin to culture medium, the cells were treated with 17 nM EGF, 100 microunits/ml insulin or 5 µM
bpV(phen), prior to assaying for CAT activity.
Site-directed Mutagenesis of Sp1--
Sp1 mutants were created
using the QuickChangTM site-directed mutagenesis kit
(Stratagene, Heidelberg, Germany) according to manufacturer's
instruction (28, 29). In brief, CMV-Sp1 plasmid DNA was denatured and
annealed with oligonucleotide primers containing the desired mutation.
Using the nonstrand-displacing action of PfuTurbo DNA polymerase, the
enzyme extended and incorporated the mutant primers resulting in nicked
circular DNA. The methylated, nonmutated parental DNA templates were
subsequently digested with DpnI. The circular, nicked
double-stranded DNA was transformed into XL1-Blue supercompetent cells.
After transformation, the XL1-Blue supercompetent cells repaired the
nicks in the mutated plasmid. The mutations were verified using
nucleotide sequencing before use in transfection of Hep G2 cells.
EGF and Insulin Increase ApoA-I Gene Expression--
We recently
described the creation of a stable Hep G2 cell line harboring the
reporter, pAI.474-CAT. This plasmid is comprised of the
In search of a potential mechanism underlying EGF induction of
apoA-I promoter activity, the cells were pretreated for 30 min with 10 µM cycloheximide, a protein synthesis
inhibitor, or 1 µM actinomycin D, a transcription
inhibitor, prior to 24 h of exposure to EGF or insulin. Total RNA
was extracted from these cells and assayed for abundance of CAT
mRNA using RT-PCR. Results showed that whereas, cycloheximide did
not inhibit EGF or insulin induction of CAT mRNA expression (Fig.
1B), as expected actinomycin D blocked transcription of the
CAT gene. These findings suggest that both hormones had a
direct effect on apoA-I promoter activity and did not
require de novo synthesis of other proteins.
Whether EGF or insulin induction of apoA-I promoter
correlated with increases in apoA-I protein is not known. Therefore,
Western blot analysis was used to measure the abundance of the protein in both whole cell lysate and culture medium from cells treated with
either EGF or insulin or 5 µM bpV(phen), a insulin
mimetic, for 24 h. All three agents increased apoA-I protein in
both cell lysate and culture medium (Fig. 1C). Together the
above results show that EGF and insulin share similar activities in the
direct induction of apoA-I promoter activity leading to
increased abundance of the protein.
Activation of EGF Receptor Kinase Induces ApoA-I
Expression--
The action(s) of EGF is initiated by its binding to a
specific membrane receptor with intrinsic tyrosine kinase activity. To
determine whether EGF induction of apoA-I is mediated by its receptor, stably transfected Hep G2 cells were exposed to 1 µM PD153035, a specific inhibitor for the EGF receptor
(30), prior to the addition of hormone. The results (Fig. 2) showed
that treatment with PD153035 completely blocked EGF induction of
apoA-I activity. In contrast, neither insulin nor bpV(phen)
induction of CAT activity in the same cells was affected by PD153035
(Fig. 2). These findings show that the activation of EGF receptor
tyrosine kinase is required for apoA-I induction by the
hormone. Furthermore, blockade of EGF receptor activity had no effect
on the actions of insulin or bpV(phen).
Intracellular Signaling Pathways Underlying EGF and Insulin
Action--
Ligand binding to a receptor with intrinsic tyrosine
kinase activity triggers this function leading to receptor
autophosphorylation. These events initiate signal transduction and
cellular responses. Activated tyrosine kinase receptor can initiate
several intracellular pathways including; Ras-MAP kinase cascade, PI
3-kinase, and PLC
To identify the signaling pathways underlying the actions of EGF and
insulin, we used a panel of specific inhibitors known to block selected
pathways. The results show that inhibitors of PI 3-kinase, 100 nM wortmannin or 10 µM LY294002 (32), did not affect the actions of EGF, insulin, or bpV(phen) (Fig.
3). Whereas, the MEK1 inhibitor PD98059
(1 µM) completely blocked EGF induction of
apoA-I expression (Fig. 3), it only inhibited 50% of
insulin action on apoA-I activity (Fig. 3). Although the PKC
inhibitor GF109203X (2 µM) (33, 34) did not affect EGF
response, it did inhibit insulin induction of apoA-I gene by
50% (Fig. 3). More importantly, the combination of PD98059 and GFX
together completely blocked insulin induction of apoA-I
(Fig. 3). Like insulin, apoA-I induction by bpV(phen) was
also blocked in the presence of both inhibitors (data not shown).
That MAP kinase was activated during EGF, insulin, or bpV(phen)
induction of the gene was assessed by treating the cells with or
without the MEK1 inhibitor, PD98059. Cell lysate was assayed for p42/44
kinase using Western blot analysis (Fig. 3B, inset). The
treatment of cells with PD98059 inhibited the phosphorylation of p42/44
MAP kinase (Fig. 3B, inset). The addition of these data to
that above show the following: (i) the PI 3-kinase pathway does not
participate in either EGF or insulin induction of apoA-I expression; (ii) the MAP kinase cascade is the sole mediator of EGF
stimulation of apoA-I; and (iii) insulin or bpV(phen) action requires two independent pathways mediated by PKC and MAP kinase to
stimulate the apoA-I gene.
PKC Activation Induces ApoA-I Expression--
Since insulin or
bpV(phen) induction of apoA-I is blocked by the PKC
inhibitor GFX, this implies that the converse where PKC is activated
should enhance apoA-I gene activity. Therefore, we tested
whether the PKC activator PDBu stimulated apoA-I expression (Fig. 4) and if this induction is
sensitive to the MEK inhibitor PD98059 (13). Results show treatment of
cells with 25 nM PDBu increased apoA-I promoter
activity and that was blocked by GFX. However, this induction was not
affected by pretreatment of cells with PD98059. These findings suggest
that PKC activation does not crossover to the MAP kinase cascade above
the level of MEK as described in other cell systems (35, 36).
Role of Ras in EGF and Insulin Induction of ApoA-I--
To
determine whether Ras participates in the EGF and insulin induction of
apoA-I, we infected the stable Hep G2 cells with RasAsn-17 retrovirus to express dominant negative
RasAsn-17. This mutant interrupts the
Ras-dependent signaling pathway (37, 38). Results (Fig.
5) showed that expression of
RasAsn-17 blocked EGF induction of the apoA-I
gene. As expected, the expression of RasAsn-17 partially
but significantly attenuated insulin induction of apoA-I promoter. Exposure of cells to GFX prior to RasAsn-17
retrovirus infection blocked both insulin (Fig. 5) or bpV(phen) (data
not shown) induction of the gene. Additionally, the insertion of the
dominant negative RasAsn-17 in cells did not affect the
stimulation of apoA-I gene induced by PDBu, a PKC activator
(data not shown). These findings show that EGF stimulates
apoA-I induction via a Ras-MAP kinase pathway and further
solidifying the finding that insulin induction of the gene is mediated
independently via both the Ras-MAP kinase and the PKC pathways.
EGF Response Requires Presence of IRCE--
Next we searched for
the cis-acting element(s) in the apoA-I promoter that
mediated the actions of EGF. Thus serial deletion constructs of
apoA-I promoter were transfected into Hep G2 cells and then
treated with EGF, insulin, or bpV(phen). Results (Fig. 6A) showed that EGF
stimulation of the promoter like insulin or bpV(phen) was abolished
following deletion of the
Our previous studies showed that insulin induction of apoA-I
gene required a motif called the IRCE Sp1 Augments EGF, Insulin, or bpV(phen) induction of
ApoA-I--
The IRCE motif is GC-rich and binds to a transcription
factor, Sp1. Therefore, we speculate that Sp1 might participate in EGF,
insulin, or bpV(phen) induction of the promoter. To investigate this
hypothesis, we used submaximal concentrations of EGF (8.5 nM), insulin (50 microunits/ml), and bpV(phen) (2.5 µM) to stimulate cells that do or do not overexpress of
Sp1. Results (Fig. 7) showed that Sp1
expression alone increased apoA-I gene expression 2-fold and
submaximal doses of EGF, insulin, and bpV(phen) caused a 2.1-, 1.8-, and 1.7-fold, respectively, in apoA-I transcription. In the
presence of both Sp1 and EGF, insulin, or bpV(phen) we observed a
marked induction of promoter activity of 7.4-, 6.5-, and 6.4-fold, respectively. The combined actions of Sp1 and hormone were not simply
additive but synergistic.
Mutation of Thr266 in Sp1 Attenuated EGF Induction of
ApoA-I--
Next we postulated that EGF stimulation of
apoA-I was mediated by the MAP kinase pathway involves
phosphorylation of Sp1. To test this idea, we inspected Sp1 protein for
potential sites of MAP kinase phosphorylation according to published
algorithms (39). Six potential sites for MAP kinase were identified
(Fig. 8) (40, 41). The prediction scores
ranged from 0 to 1.000 with values that exceeded 0.5 reflecting
potential phosphorylation sites. Three of the sites with the highest
scores were Thr266 (0.767), Thr414 (0.664), and
Thr650 (0.726) (41). Thr266 was especially
interesting because it was located toward the C-terminal region of the
second glutamine-rich domain, one of the known trans-activation domains
of Sp1 (19). Since the amino acids surrounding Thr266
appeared most homologous against a consensus MAP kinase site, we
mutated the threonine residue by replacing it with an alanine to create
Sp1-T266A.
The use of Sp1-T266A in transfection studies revealed the following
results. Although transfection of Sp1-T266A alone into stable Hep G2
cells augmented activity of pAI.474-CAT, the activity was not
significantly different from wild-type Sp1. When the transfected cells
were exposed to EGF, apoA-I induction was attenuated by 54%
in cells containing the mutant Sp1 compared with the wild-type. However, PDBu induction of apoA-I expression mediated by PKC
was the same in the presence of the mutant or wild-type Sp1. These data
suggest that amino acid Thr266 in Sp1 is required for full
EGF induction of apoA-I.
ApoA-I is an essential component of HDL. The protein alone or in
the form of HDL mediate a normal physiologic process called reverse
cholesterol transport, which lowers total body cholesterol (5, 7)
thereby reducing the risk of IHD. This function of apoA-I makes it an
important target for therapies to enhance expression of the protein. To
achieve this goal, our studies have helped define the mechanism(s) by
which hormones regulate gene activity (11, 42). This avenue of research
is attractive because simple manipulation of the hormones, mimetics, or
synthetic analogues will help us control activity of the gene.
Our initial studies showed that insulin induction of apoA-I
transcription in Hep G2 cells was mediated by a cis-acting element, the
IRCE in the promoter (12). More recently, we found that signaling
pathways involving protein kinase A, PKA, or PKC also stimulated gene
activity and this induction required the transcription factor, Sp1
(13). Sp1 binds specifically to the IRCE (12, 13). Together these
findings form the basis of an incomplete model depicting
apoA-I induction but we have yet to determine whether the
two separate sets of results are connected. To relate the findings
arising from these studies, we have defined the pathway(s) by which
insulin induce apoA-I and extended this line of thinking to
examine the potential action(s) of another peptide hormone, EGF, that
also acts via a receptor tyrosine kinase.
That EGF stimulated apoA-I expression comes from two sets of
complementary data. First, 17 nM EGF induced
apoA-I promoter 7-fold in stable Hep G2 cells that harbored
a DNA fragment ( Activation of the EGF receptor may trigger signaling pathways including
those mediated by Ras-MAP kinase, PI 3-kinase, or PLC Although the preceding observations showed the Ras-MAP kinase cascade
to mediate EGF induction of apoA-I transcription, this finding only details the cytosolic portion of the mechanism. Activation of transcription requires conversion of the cytosolic signal to nuclear
event(s). Many transcription factors are known to mediate the actions
of activated MAP kinase (56). However, the specific one(s) required for
EGF induction of apoA-I is not known. To examine this
question, we used deletion and mutational analysis to locate a motif,
the IRCE ( The finding that the Ras-MAP kinase cascade mediates EGF activity and
this pathway eventually requires Sp1 to activate transcription provides
a catalogue of the components of the induction. Further insight into
this mechanism requires the connection between the kinase cascade and
Sp1. Therefore, we postulated that Sp1 may serve as a substrate for MAP
kinase. To examine this hypothesis we searched for and found many
potential kinase sites in Sp1. However, amino acid Thr266
was targeted for mutagenesis because this motif was most homologous to
the consensus MAP kinase site. Although Thr266 is located
in the second trans-activation domain of Sp1, the mutation of this
amino acid did not affect significantly the basal activity of the
transcription factor. However, exposure of the transfected cells to EGF
showed a clear difference resulting in a 54% lower level of induction
in cells with mutant Sp1 versus the wild type (Fig. 8). This
finding is consistent with the idea that Thr266 serves as a
functional phosphorylation site for the actions of EGF.
In another model, EGF induction of gastrin gene expression in
epithelial cells was mediated by the MAP kinase cascade and required
phosphorylation of the transcription factor, Sp1 (57). Thus our finding
that MAP kinase regulates Sp1 adds to the list of kinases including:
Sp1 kinase (57) or DNA-dependent kinase (58), PKA (13, 18,
21), casein kinase II (43), and PKC (13, 48) known to modulate Sp1
activity, which in turn, leads to changes in gene expression. Although
phosphorylation of Sp1 by various kinases is possible and in some cases
demonstrated, the specific sites that are modified remain undefined.
Therefore, the observation that mutation of Thr266
attenuated EGF induction of apoA-I is consistent with the
idea that MAP kinase modulates Sp1 activity by phosphorylating
Thr266. Since EGF induction was not completely abrogated,
this suggests that additional phosphorylation sites are likely involved.
The identification of the mechanism underlying EGF induction of
apoA-I provides an opportunity to better understand the
regulation of the gene by comparing or contrasting the actions of EGF
with that of insulin in the Hep G2 cells. For these studies, the first step is to define the mechanism(s) mediating insulin induction of
apoA-I. Since insulin induction is weak, we also examined an insulin mimetic, bpV(phen) and tested its action on apoA-I
gene expression. BpV(phen) acts by inhibiting the specific
protein-tyrosine phosphatase for the insulin receptor and thus mimics
many of the actions of insulin, but intracellular signaling mechanisms
may be different from those of insulin. For example, PI 3-kinase in cultured hepatocytes is required for insulin but not for bpV(phen) to
regulate expression of the insulin-like growth factor-binding protein
(49). Our first studies showed that like insulin, bpV(phen) induced
apoA-I transcription, except that the induction was
4-5-fold (Fig. 2) as compared with 3-3.5-fold for insulin. Both
insulin and bpV(phen) induction of apoA-I was not affected
by the EGF receptor kinase inhibitor, PD153035, suggesting that
although exposure of cells to these agents increased apoA-I
gene activity, the pathways leading to induction were likely different.
Next, the approach that we used to define the signaling pathways for
EGF induction of apoA-I was applied to the actions of insulin and bpV(phen). Exposure of cells to PD98059 following infection
with RasAsn-17 retrovirus blocked both the actions of
insulin or bpV(phen) induction of apoA-I by 50%. The
remaining induction was sensitive to the PKC inhibitor, GFX. As
expected the combination of both GFX plus PD98059 or
RasAsn-17 retrovirus completely abrogated insulin or
bpV(phen) induction of apoA-I. Consistent with previous
studies, activation of PKC by exposure to PDBu enhanced
apoA-I activity, and this was blocked by prior treatment
with GFX (13, 33, 34). Our previous studies, showed that PDBu induction
of apoA-I was mediated by PKC activation and that this
pathway required the interaction of Sp1 with an intact IRCE (13). These
data suggest that the PKC and Ras-MAP kinase pathways act independently
through Sp1, because there does not appear to be cross-talk between two
pathways. For example, the inhibition of MEK1 with PD98059 or
RasAsn-17 expression does not affect the actions of PDBu.
More importantly, the Sp1-Thr266 mutant did not affect PKC
induction of this gene (Fig. 8), suggesting a distinct regulatory site
for PKC phosphorylation within Sp1.
Although the pathway by which EGF activates MAP kinase leading to Sp1
phosphorylation and apoA-I induction is clear, the role of
PKC in mediating actions of insulin or bpV(phen) is not. Insulin or
bpV(phen) induction of PKC activity may be achieved by stimulation of
PLC In summary, our studies show that EGF up-regulates apoA-I
gene transcription. This process requires a single signaling pathway mediated by Ras-MAP kinase. The activation of this kinase may potentially lead to phosphorylation of the transcription factor, Sp1 at
Thr266. The modified Sp1 interacts with the IRCE to the
enhance transcription of the gene. Insulin and bpV(phen) also induce
apoA-I transcription but rather than a single cascade, their
actions are mediated by two parallel independent pathways that involve
the activation of PKC and MAP kinase. ApoA-I induction by
these two agents also require the participation of Sp1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cascades (14). These pathways are not exclusive to
the insulin receptor and may be used by other tyrosine kinase
receptors, including that for EGF (16). Therefore, we tested the
ability of another peptide hormone, epidermal growth factor, EGF on
apoA-I expression. If apoA-I is inducible by EGF, it offers a new avenue to augment expression of the gene. But equally
important is that this model provides an opportunity to compare or
contrast the signaling mechanism(s) activated by EGF and insulin. The
results summarized here show that like insulin, EGF also enhances
apoA-I expression. However, whereas the actions of EGF are mediated
solely by the actions of a single pathway, that of insulin requires the
participation of at least two cascades.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
425,
375,
325, and
235 to
7
were synthesized using the parent pAI.474-CAT as a template in separate
PCR (17). Transverse mutation of the IRCE (
411 to
404) from
GAGGCGGG to TCTTATTT was accomplished using a mutant primer in a PCR
(12). The RasAsn-17-retroviral vector and Sp1
expression plasmid were gifts from Drs. J. Stone (University of
Alberta, Edmonton, Alberta, Canada) and Dr. R. Tjian (University
of California, Berkeley, CA), respectively (19).
-galactosidase (20). Stably transfected Hep G2 cells were
created by co-transfecting pAI.474-CAT (17) and the plasmid, pRc/CMV2
(Invitrogen) that carried neomycin resistance as described
(13).
-actin using the primer pair (forward: 5'-CGTGGGCCGCCCTAGGCACCA-3'; reverse: 5'-TTGGCCTTAGGGTTCAGGGGG-3') as described previously (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
474 to
7
fragment of the rat apoA-I promoter fused to the reporter
gene, CAT (23). In these cells, CAT activity (Fig. 1A) increased following
treatment with 17 nM EGF. Induction appeared to be rapid
with detectable increases at 3 h following exposure to the
hormone. The extrapolation of the data points to time 0 suggested an
almost instantaneous induction by EGF. Similarly, 100 milliunits/ml
insulin also stimulated CAT activity with comparable kinetics. The only
difference was that following 48 h of exposure to EGF, CAT
activity in the cells increased 7-fold versus a 3-3.5-fold caused by insulin. Like insulin, 5 µM mimetic, bpV(phen)
also enhanced apoA-I transcription 4-5-fold (Fig.
2).
View larger version (34K):
[in a new window]
Fig. 1.
EGF and insulin induction of
apoA-I. Panel A, shows the time
course detailing induction of the apoA-I promoter in stable
Hep G2 cells that harbor pAI.474-CAT following exposure to 17 nM EGF or 100 microunits/ml insulin. The inset
shows a typical autoradiograph of CAT activity in the presence of EGF.
Panel B shows an ethidium bromide-stained gel of RT-PCR
signals reflecting CAT mRNA in the same cells treated with 1 µM actinomycin D (ACTD) or 10 µM
cycloheximide (CHX) following exposure to EGF or insulin
(upper). RT-PCR signals from -actin mRNA served as
control (lower). The various treatments are denoted
below each lane. Panel C shows a Western blot
analysis of apoA-I protein in the lysates and corresponding
spent medium from Hep G2 cells exposed to EGF, insulin, or bpV(phen),
as indicated above each lane.
View larger version (44K):
[in a new window]
Fig. 2.
Inhibiting EGF receptor kinase blocks
induction of apoA-I. Panel A, shows an
autoradiograph of CAT activity in stable cells treated with either 17 nM EGF, 100 microunits/ml insulin, or 5 µM
bpV(phen) for 24 h in the presence (lanes 4, 6, and
8) or absence (lanes 3, 5, and 7) of 1 µM PD153035. Panel B, shows a graph of the
relative CAT activities in cells treated with conditions noted at the
bottom of each bar (mean ± S.E.,
n = 4). Asterisk (*) denotes a significant
difference with p < 0.01 between the groups with and
without PD153035 treatment as determined by ANOVA.
pathways (16, 31). However, the cellular responses
and their signaling mechanisms are largely dependent on the cell type
examined. Furthermore, the actions of a single hormone may vary from
one cell type to another. Therefore, we wanted to examine the
intracellular signaling pathway by which EGF and insulin induced
apoA-I gene expression in Hep G2 cells.
View larger version (56K):
[in a new window]
Fig. 3.
Effects of PD98059, a MEK inhibitor and GFX,
a PKC inhibitor, on apoA-I. Panel A shows an
autoradiograph of CAT activity in stable Hep G2 cells treated either
with EGF (lanes 2-7) or insulin (lanes 9-14)
for 24 h in the presence (lanes 3 and 10) or
absence (lanes 2 and 9) of 2 µM
PD98059 or in the presence (lanes 4 and 11) or
absence (lanes 2 and 9) of 2 µM
GFX. Lanes 5 and 12 indicate the pretreatment of
cells with both 2 µM PD98059 and 2 µM GFX
(PD98059/GFX). Other inhibitors wortmannin (lanes
6 and 13) and LY294002 (lanes 7 and
14) did not affect the actions of EGF or insulin.
Panel B shows a graph of the relative CAT activities in
cells treated with conditions noted at the bottom of each
bar (mean ± S.E., n = 4. and S.E.,
asterisk (*) denotes a significant difference with
p < 0.01 between the groups with and without the
presence of any inhibitor as determined by ANOVA). The insert of
Panel B (inset), shows a typical Western blot
that reflects the phosphorylation of p42/44 MAP kinase induced by EGF,
insulin or bpV(phen) in the presence and absence of PD98059 as
indicated for each lane.
View larger version (29K):
[in a new window]
Fig. 4.
Effect of PD98059 on PDBu induction of
apoA-I. Panel A (left) shows an
autoradiograph of CAT activity in stable cells treated with a PKC
activator, 25 nM PDBu for 24 h in the presence or
absence of the PKC inhibitor 2 µM GFX or 2 µM PD98059. Panel B shows a graph of relative
CAT activities in cells treated with the conditions noted at the
bottom of each bar (mean ± S.E.,
n = 5). Asterisk (*) denotes a significant
difference with p < 0.01 between the groups treated
with and without inhibitors as determined by ANOVA.
View larger version (21K):
[in a new window]
Fig. 5.
Effect of dominant negative
RasAsn-17 on EGF and insulin induction of
apoA-I. This graph represents the relative CAT
activities in stable Hep G2 cells infected with either pBabe-Puro
retrovirus (CV, control virus) or the virus expressing
RasAsn-17 as indicated. Puromycin-selected Hep G2 cells
were treated with the conditions as noted at the bottom of
lane (mean ± S.E., n = 4).
Asterisk (*) denotes a significant difference with
p < 0.01 between the groups with and without the
expression of RasAsn-17 in response to each agent as
determined by ANOVA.
425 to
376 fragment of the promoter.
View larger version (34K):
[in a new window]
Fig. 6.
Actions of EGF and insulin/bpV(phen) require
the IRCE. Panel A, shows the relative CAT
activities in Hep G2 cells that were transiently transfected with
various deletion constructs (2 µg each) of CAT-reporter containing
474,
425,
375, or
325 to
7 of the apoA-I promoter
(map). Cells were treated with 17 nM EGF, 100 milliunits/ml
insulin, or 5 µM bpV(phen) for 24 h, followed by CAT
activity assay as indicated at the bottom of each
bar group (asterisk (*) denotes a significant
difference with p < 0.01 between
474-CAT and other
deletion constructs in response to various treatment as determined by
ANOVA). Panel B shows the relative CAT activities in Hep G2
cells transiently transfected with 2 µg of pAI.474-CAT
(Wild-type IRCE) or an identical construct containing a
mutant of the IRCE (Mutant IRCE) in response to various
treatment as indicated (mean ± S.E., n = 4).
Asterisk (*) denotes a significant difference with
p < 0.01 between wild-type IRCE and mutant IRCE as
determined by ANOVA.
411 to
404. Deletion analysis
suggested that the same motif may also be required for response to EGF.
Therefore, we tested the activity of a reporter construct containing a
mutant of the IRCE. Results (Fig. 6B) showed that the
reporter containing the mutant IRCE was not inducible by EGF, insulin,
or bpV(phen). These finding show that the actions of all three agents
require the presence of an intact IRCE.
View larger version (36K):
[in a new window]
Fig. 7.
Expression of Sp1 potentiates EGF and
insulin/bpV(phen) induction of apoA-I. The graph shows
the relative CAT activities in Hep G2 cells with co-transfection of
apoA-I-474-CAT with either 1 µg of wild-type Sp1 (Sp1) or
empty vector (EV) for 24 h, followed by treatment with
8.5 nM EGF, 50 microunits/ml insulin (ins), and
2.5 µM bpV(phen) followed by assaying for CAT activity
(mean ± S.E., n = 4). Asterisk (*)
denotes a significant difference with p < 0.01 between
the groups with and without Sp1 expression in response to each agent as
determined by ANOVA.
View larger version (53K):
[in a new window]
Fig. 8.
Site-directed mutagenesis of
Thr266 in Sp1 attenuates EGF induction of apoA-I.
Panel A shows the amino acid sequence of Sp1. Six motifs
(XXTP, bold) were identified as potential phosphorylation
sites for MAP kinase. The underlined sequences (not bold)
show the two glutamine-rich activation domains. The bold and
underlined sequences indicate three zinc fingers. The
table shows the positions of potential MAP kinase
phosphorylation sites and the prediction scores. T indicates
the potential phosphorylation site with a score above 0.5. Panel
B demonstrates the effect of Thr266 mutant Sp1
(Thr266-Sp1) on EGF induction of apoA-I compared
with wild-type Sp1 (Sp1). The apoA-I-474-CAT plasmid DNA were
co-transfected with 1 µg each of either wild-type Sp1 or
Thr266 mutant Sp1 or empty vector (EV). After
24 h, cells were treated with and without 8 nM EGF or
25 nM PDBu, followed by assaying for CAT activity
(mean ± S.E., n = 4). Asterisk (*)
denotes a significant difference with p < 0.01 between
the groups with wild-type and Thr266 mutant Sp1 in response
to each agent as determined by ANOVA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
474 to
7) in the pAI.474-CAT reporter (Fig. 1).
Second, this induction of apoA-I transcription underlies the
increase in abundance of the protein in both cell lysate and culture
medium (Fig. 1C). The mechanism by which EGF induces
apoA-I expression appeared to be transcriptionally mediated
because hormone action was not affected by cycloheximide inhibition of
protein synthesis. This finding implies that EGF trans-activation of
this gene is direct and did not require de novo protein
synthesis. The direct effect of EGF is further supported by the rapid
response of promoter activity, noted in the kinetics of induction (Fig.
1A). Although EGF is a known hepatocyte mitogen (44, 45) and
its activity may induce gene expression in the liver, this is the first
demonstration that the hormone up-regulates apoA-I. The
preceding studies add EGF to the list of hormones that modulate
apoA-I including, thyroid hormone, steroid hormones, and
insulin (42, 46, 47). EGF is the second and insulin the first
peptide hormone shown to regulate apoA-I expression. Thus it
is of interest to compare and contrast the actions of these hormones
that act via comparable intracellular signaling pathways.
(16, 31).
Despite the variety of choices, EGF activation of apoA-I
appears to be channeled solely through the Ras-MAP kinase cascade, a
pathway commonly used by the hormone to regulate intracellular events
(40, 50). This explanation was supported by finding that the
specific EGF receptor inhibitor PD153035 blocked hormonal induction of
the gene. In addition, EGF activation of the Ras-MAP kinase cascade
appeared distinct from that of insulin or bpV(phen) because the same
inhibitor had no effect on the actions of the latter two agents.
Additional support that EGF induction of apoA-I was mediated
by a single pathway arose from the finding that hormone action was
inhibited completely by the MEK1 inhibitor PD98059 at a low
concentration of 2 µM (51). This inhibitor also abolished phosphorylation of p42/44 MAP kinase induced by EGF (Fig. 3B, inset). Infection of the cells with a retrovirus expressing a dominant negative Ras, RasAsn-17 (38), blocked EGF
induction of apoA-I and confirmed the participation of Ras
in this process. Additional evidence for the single pathway mechanism
came from data showing that the EGF action was not affected by GFX or
wortmannin, inhibitors for PKC and PI 3-kinase, respectively. These
data show that EGF induction of apoA-I is mediated by the Ras-dependent MAP kinase, a pathway known to be present and
active in Hep G2 cells (52-55). For example, recent data show that
hepatopoietin stimulates MAP kinase through EGF receptor activation in
these cells (55).
411 to
404) within the promoter that mediated the actions
of EGF. Our recent studies showed that the IRCE was bound specifically
by the transcription factor, Sp1 but not Sp2 nor Sp3 and this binding
enhanced apoA-I gene transcription (13). Overexpression of Sp1 in cells augmented EGF induction of
apoA-I, thus showing the participation of Sp1 in this
process (Fig. 7).
, which triggers the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol leading to PKC
activation (14, 15). This speculation is based on our finding that PI 3-kinase inhibitors, wortmannin and LY294002, did not affect the induction of the gene by either insulin or bpV(phen). This finding suggests that insulin or bpV(phen) activation of PKC is unlikely via
the PI-3 kinase pathway. It remains undetermined whether PKC directly
phosphorylates to stimulate Sp1 because there are 14 potential PKC
recognition sites in Sp1 (39). The mutagenesis approach may not be the
most efficient way to tackle this problem in the search for functional
Sp1 site(s) targeted by PKC.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a AstraZeneca/CIHR/PMAC Heart and Stroke Foundation of Canada fellowship.
** Recipient of scientist awards from the Canadian Institute of Health Research and Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Depts. of Medicine and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Health Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-5212; Fax: 403-270-0979; E-mail: ncwwong@ucalgary.ca.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M011031200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; IRCE, insulin responsive core element; MAP, mitogen-activated protein; PI 3-kinase, phosphatidylinositol 3-kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; EGF, epidermal growth factor; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Andersson, L. O. (1997) Curr. Opin. Lipidol. 8, 225-228[Medline] [Order article via Infotrieve] |
2. | Brouillette, C. G., and Anantharamaiah, G. M. (1995) Biochim. Biophys. Acta 1256, 103-129[Medline] [Order article via Infotrieve] |
3. | Barter, P. J., and Rye, K. A. (1996) Atherosclerosis 121, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
4. | De Backer, G., De Bacquer, D., and Kornitzer, M. (1998) Atherosclerosis 137 (suppl.), S1-S6[CrossRef][Medline] [Order article via Infotrieve] |
5. | Barter, P. J., and Rye, K. A. (1996) Curr. Opin. Lipidol. 7, 82-87[Medline] [Order article via Infotrieve] |
6. | Luoma, P. V. (1997) Pharmacol. Toxicol. 81, 57-64[Medline] [Order article via Infotrieve] |
7. | Miller, N. E., La Ville, A., and Crook, D. (1985) Nature 314, 109-111[Medline] [Order article via Infotrieve] |
8. |
Nanjee, M. N.,
Crouse, J. R.,
King, J. M.,
Hovorka, R.,
Rees, S. E.,
Carson, E. R.,
Morgenthaler, J. J.,
Lerch, P.,
and Miller, N. E.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
1203-1214 |
9. | Eisenberg, D. A. (1998) Am. J. Med. 104, 2S-5S[CrossRef][Medline] [Order article via Infotrieve] |
10. | Lawn, R. M., Wade, D. P., Hammer, R. E., Chiesa, G., Verstuyft, J. G., and Rubin, E. M. (1992) Nature 360, 670-672[CrossRef][Medline] [Order article via Infotrieve] |
11. | Taylor, A. H., Nakamura, T., and Wong, N. C. (1997) Proc. West Pharmacol. Soc. 40, 127-130[Medline] [Order article via Infotrieve] |
12. |
Murao, K.,
Wada, Y.,
Nakamura, T.,
Taylor, A. H.,
Mooradian, A. D.,
and Wong, N. C.
(1998)
J. Biol. Chem.
273,
18959-18965 |
13. |
Zheng, X. L.,
Matsubara, S.,
Diao, C.,
Hollenberg, M. D.,
and Wong, N. C.
(2000)
J. Biol. Chem.
275,
31747-31754 |
14. | Kahn, C. R. (1994) Diabetes 43, 1066-1084[Medline] [Order article via Infotrieve] |
15. |
Saltiel, A. R.
(1996)
Am. J. Physiol.
270,
E375-E385 |
16. | Carpenter, G. (2000) Bioessays 22, 697-707[CrossRef][Medline] [Order article via Infotrieve] |
17. | Romney, J. S., Chan, J., Carr, F. E., Mooradian, A. D., and Wong, N. C. (1992) Mol. Endocrinol. 6, 943-950[Abstract] |
18. |
Ahlgren, R.,
Suske, G.,
Waterman, M. R.,
and Lund, J.
(1999)
J. Biol. Chem.
274,
19422-19428 |
19. | Courey, A. J., and Tjian, R. (1988) Cell 55, 887-898[Medline] [Order article via Infotrieve] |
20. | Herbomel, P., Bourachot, B., and Yaniv, M. (1984) Cell 39, 653-662[Medline] [Order article via Infotrieve] |
21. |
Zhong, Z. D.,
Hammani, K.,
Bae, W. S.,
and DeClerck, Y. A.
(2000)
J. Biol. Chem.
275,
18602-18610 |
22. | Sakai, T., Jin, F., Kamanna, V. S., and Kashyap, M. L. (2000) Atherosclerosis 149, 43-49[CrossRef][Medline] [Order article via Infotrieve] |
23. | Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051[Medline] [Order article via Infotrieve] |
24. | Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J. H., Shabanowitz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. (1991) EMBO J. 10, 885-892[Abstract] |
25. |
Wu, J. Y.,
Wu, Y.,
Reaves, S. K.,
Wang, Y. R.,
Lei, P. P.,
and Lei, K. Y.
(1999)
Am. J. Physiol.
277,
C537-C544 |
26. |
Zheng, X. L.,
Gui, Y.,
Sharkey, K. A.,
and Hollenberg, M. D.
(1999)
J. Pharmacol. Exp. Ther.
289,
632-640 |
27. | Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract] |
28. | Nelson, M., Zhang, Y., and Van Etten, J. L. (1993) EXS 64, 186-211[Medline] [Order article via Infotrieve] |
29. | Vandeyar, M. A., Weiner, M. P., Hutton, C. J., and Batt, C. A. (1988) Gene (Amst.) 65, 129-133[CrossRef][Medline] [Order article via Infotrieve] |
30. | Fry, D. W., Kraker, A. J., McMichael, A., Ambroso, L. A., Nelson, J. M., Leopold, W. R., Connors, R. W., and Bridges, A. J. (1994) Science 265, 1093-1095[Medline] [Order article via Infotrieve] |
31. | Zwick, E., Hackel, P. O., Prenzel, N., and Ullrich, A. (1999) Trends Pharmacol. Sci. 20, 408-412[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Vlahos, C. J.,
Matter, W. F.,
Hui, K. Y.,
and Brown, R. F.
(1994)
J. Biol. Chem.
269,
5241-5248 |
33. |
Eichholtz, T.,
de Bont, D. B.,
de Widt, J.,
Liskamp, R. M.,
and Ploegh, H. L.
(1993)
J. Biol. Chem.
268,
1982-1986 |
34. |
Toullec, D.,
Pianetti, P.,
Coste, H.,
Bellevergue, P.,
Grand-Perret, T.,
Ajakane, M.,
Baudet, V.,
Boissin, P.,
Boursier, E.,
and Loriolle, F.
(1991)
J. Biol. Chem.
266,
15771-15781 |
35. | Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Marais, R.,
Light, Y.,
Mason, C.,
Paterson, H.,
Olson, M. F.,
and Marshall, C. J.
(1998)
Science
280,
109-112 |
37. | Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-654[CrossRef][Medline] [Order article via Infotrieve] |
38. | Herskowitz, I. (1987) Nature 329, 219-222[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Kreegipuu, A.,
Blom, N.,
and Brunak, S.
(1999)
Nucleic Acids Res.
27,
237-239 |
40. |
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556 |
41. | Merchant, J. L., Du, M., and Todisco, A. (1999) Biochem. Biophys. Res. Commun. 254, 454-461[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Hargrove, G. M.,
Junco, A.,
and Wong, N. C.
(1999)
J. Mol. Endocrinol.
22,
103-111 |
43. |
Armstrong, S. A.,
Barry, D. A.,
Leggett, R. W.,
and Mueller, C. R.
(1997)
J. Biol. Chem.
272,
13489-13495 |
44. | Marino, M., Spagnuolo, S., Cavallini, M., Terenzi, F., Mangiantini, M. T., and Leoni, S. (1996) Cell Signal. 8, 555-559[CrossRef][Medline] [Order article via Infotrieve] |
45. | Thoresen, G. H., Guren, T. K., Sandnes, D., Peak, M., Agius, L., and Christoffersen, T. (1998) J. Cell. Physiol. 175, 10-18[CrossRef][Medline] [Order article via Infotrieve] |
46. | Taylor, A. H., Wishart, P., Lawless, D. E., Raymond, J., and Wong, N. C. (1996) Biochemistry 35, 8281-8288[CrossRef][Medline] [Order article via Infotrieve] |
47. | Ness, G. C., Lopez, D., Chambers, C. M., Newsome, W. P., Cornelius, P., Long, C. A., and Harwood, H. J., Jr. (1998) Biochem. Pharmacol. 56, 121-129[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Pal, S.,
Claffey, K. P.,
Cohen, H. T.,
and Mukhopadhyay, D.
(1998)
J. Biol. Chem.
273,
26277-26280 |
49. |
Band, C. J.,
and Posner, B. I.
(1997)
J. Biol. Chem.
272,
138-145 |
50. | Treisman, R. (1996) Curr. Opin. Cell Biol. 8, 205-215[CrossRef][Medline] [Order article via Infotrieve] |
51. | Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract] |
52. |
Sergeant, N.,
Lyon, M.,
Rudland, P. S.,
Fernig, D. G.,
and Delehedde, M.
(2000)
J. Biol. Chem.
275,
17094-17099 |
53. | Nagase, T., Kawata, S., Tamura, S., Matsuda, Y., Inui, Y., Yamasaki, E., Ishiguro, H., Ito, T., and Matsuzawa, Y. (1996) Int. J. Cancer 65, 620-626[CrossRef][Medline] [Order article via Infotrieve] |
54. | Richards, C. A., Short, S. A., Thorgeirsson, S. S., and Huber, B. E. (1990) Cancer Res. 50, 1521-1527[Abstract] |
55. | Li, Y., Li, M., Xing, G., Hu, Z., Wang, Q., Dong, C., Wei, H., Fan, G., Chen, J., Yang, X., Zhao, S., Chen, H., Guan, K., Wu, C., Zhang, C., and He, F. (2000) J. Biol. Chem |
56. | Davis, R. J. (1995) Mol. Reprod. Dev. 42, 459-467[Medline] [Order article via Infotrieve] |
57. |
Chupreta, S.,
Du, M.,
Todisco, A.,
and Merchant, J. L.
(2000)
Am. J. Physiol. Cell Physiol.
278,
C697-C708 |
58. | Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 63, 155-165[Medline] [Order article via Infotrieve] |