(Received for publication, January 16, 1997, and in revised form, April 21, 1997)
From the Department of Molecular Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
The expression of P-glycoproteins encoded by the
mdr gene family is associated with the emergence of
multidrug resistance phenotype in animal cells. However, the mechanisms
controlling the expression of these genes have not been well
elucidated. Here, we report that the expression of rat
mdr1b gene in cultured H-4-II-E hepatoma cells can be
induced by insulin. Transient transfection assays using reporter gene
constructs containing various 5 mdr1b sequences showed
that the sequence located between base pairs
243 and
163 is
important for insulin's induction of mdr1b promoter activity. Further analyses revealed that a NF-
B-binding site (located between base pairs
167 and
158) is required for
insulin-induced promoter activity. Gel mobility shift assay
demonstrated that insulin stimulates the binding of nuclear p50/p65
subunits to the mdr1b NF-
B sequence. Cotransfection of
plasmids expressing either the p50/p65 NF-
B subunits or Raf-1 kinase
or both resulted in increased expression of the gene containing
wild-type but not NF-
B site-mutated mdr1b promoter.
Finally, expression of either the antisense p65 subunit of NF-
B or
dominant negative Raf-1 kinase blocked insulin's induction of the
mdr1b promoter activity. Taken together, our results
suggest that the insulin-induced mdr1b expression is
mediated by transcription factor NF-
B via the Raf-1 kinase signaling
pathway.
Multidrug resistance (MDR),1 a major obstacle to the effective chemotherapy of many human malignancies, is commonly associated with overexpression of a family of membrane glycoproteins called P-glycoprotein. These proteins are believed to function as energy-dependent efflux pumps that prevent intracellular cytotoxic drug accumulation (for a review, see Ref. 1). P-glycoprotein is encoded by the mdr gene family, which contains two members (MDR1 and MDR2) in humans (2, 3) and three (mdr1a, mdr1b, and mdr2) in rodents (4, 5). However, only MDR1, mdr1a, and mdr1b are able to confer the MDR phenotype (6, 7), MDR2 and mdr2 only function as phospholipid transporters (8, 9).
mdr genes are tissue-specifically expressed in normal tissue and highly expressed in many tumors (1, 10, 11). Many extracellular stimuli including antitumor drugs (12), differentiating agents (13), hormones (14, 15), UV irradiation (16), heat shock (17), serum (18, 19), growth factors (18, 20), 12-O-tetradecanoylphorbol-13-acetate (21, 22), and carcinogens (23, 24) have been shown to regulate mdr expression. In the rat, mdr1b is highly expressed in lung but not in liver (25, 26). However, the expression of mdr1b is activated during hepatocarcinogenesis and liver regeneration (25, 27-29). In addition, expression of rat mdr1b mRNA can be induced by a number of cytotoxic agents including xenobiotics and protein synthesis inhibitors (23, 24, 30).
Previous studies with primary hepatocytes have shown the mRNA level
of the rat mdr1b gene to be spontaneously up-regulated due
to the increased transcription (30). Further studies have indicated
that the expression of the rat mdr1b in primary hepatocyte cultures can be modulated by dexamethasone (14), extracellular matrix
(31), epidermal growth factor, and insulin-like growth factor I (20),
suggesting that factors or stress in culture may contribute to the
induction of rat mdr1b expression in primary hepatocytes.
However, the underlying regulatory mechanisms involved in the induced
expression of rat mdr1b by these factors are still largely
unclear. Although the 5-flanking sequences of rat mdr1b have been isolated (26, 32) and a cis-acting element
important for its promoter function has been identified (26), until now little has been known about the cis- and
trans-acting factors responsible for the inducible
expression of the rat mdr1b gene by extracellular
stimuli.
In the present report, we show that expression of the rat
mdr1b gene in rat H-4-II-E hepatoma cells can be induced by
insulin. Using transient transfection assays, we confirmed that both an NF-B-binding site located at bp
167 to
158 (5
-GGGGAATTCC-3
) and a previously identified palindromic sequence (5
-AGACATGTCT-3
) located at bp
189 to
180 from the transcription start site (26) are
involved in basal transcriptional regulation as well as insulin induction of the promoter function of the rat mdr1b gene. In
addition, we demonstrated that Raf-1 kinase is also implicated in the
increased NF-
B-mediated expression of the rat mdr1b gene
by insulin. Because a number of agents known to induce mdr
expression are also known to modulate NF-
B activities, our present
findings have a broad implication in the regulation of mdr
expression under various conditions.
Reagents were purchased from the following
companies: [-32P]dNTPs, [
-32P]UTP,
and [14C]chloramphenicol from ICN Biomedicals (Costa
Mesa, CA); poly(dI-dC)·poly(dI-dC) and acetyl coenzyme A from
Pharmacia/LKB (Upsala, Sweden); rabbit polyclonal antibodies against
p50 and p65 subunits of NF-
B from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA); and oligonucleotides from Genosys Biotechnologies
Inc. (Houston, TX). All other reagents were purchased from Sigma.
The rat mdr1b-CAT reporter constructs
(1288 RMICAT,
1288 RMICAT-Fm1,
243 RMICAT,
163 RMICAT) were
constructed as described previously (26). The site-directed mutant
constructs were generated by a two-step PCR strategy (33) using
243
RMICAT as the template; the T7 promoter sequence and
5
-TCCATTTTAGCTTCCTTAG-3
as the exterior primers; and
5
-ACCTGAACATGTAGCTGAATGTCTGTGTTAATGTCTG-3
, 5
-TCTGTGTTAATGTCTGCTCAATTCCAGCTCCCTT-3
, and their
complementary sequences as the interior primers, respectively (mutated
sequences are underlined). Then, after digestion with XbaI,
the PCR products were used to replace the XbaI insert of
243 RMICAT. This generated
243 RMICAT-Fm1 and
243 RMICAT-
m,
respectively. A similar approach was used to generate
1288
RMICAT-
m except that two interior primers
(5
-TCTGTGTTAATGTCTGCTCAATTCCAGCTCCCTT-3
and its
complementary sequence) were used for PCR amplification. The sequences
of all the mutant versions were confirmed by sequencing.
243 RMICAT,
243 RMICAT-Fm1, and
243 RMICAT-
m were used as
templates for PCR amplification to construct 3x-243 RMICAT, 3x-243
RMICAT-Fm1, and 3x-243 RMICAT-
m, respectively. To do this, the
following paired primer sets were used: pair 1, 5
-AGGGATGGTACCGTGCACTAT-3
(forward primer, with
Asp718 site created underlined) and
5
-TGGGATCCAGGCTTCTCTGA-3
(backward primer, with
BamHI site created underlined); pair 2, 5
-CAGGGATCCAACCGTGCAC-3
(forward primer, with
BamHI site created underlined) and
5
-TGGGTACCAGGCTTCTCTGA-3
(backward primer, with the
Asp718 site created underlined). The PCR products of pair 1 and 2 primers were then cleaved with BamHI and ligated.
After being purified from 3% agarose gel, the ligated products were cleaved at their Asp718 site and inserted into the
Asp718 site of
243 RMICAT,
243 RMICAT-Fm1, and
243
RMICAT-
m, respectively, to generate the 3x-243 RMICAT, 3x-243
RMICAT-Fm1, 3x-243 RMICAT-
m. The orientation and sequence of each
insert was then confirmed by sequencing.
The expression vectors RSV-p65 and RSV-p50 were provided by Gary J. Nabel (University of Michigan Medical Center, Ann Arbor, MI) (34).
Plasmid pSVK3 was purchased from Pharmacia. Recombinant antisense p65
(p65) (35) was a gift of Tom Maniatis (Harvard University, Boston,
MA). Plasmid pcDNA1 was purchased from Invitrogen. Plasmids
expressing an activated Raf-1 kinase domain (Raf BXB), its dominant
negative mutant (Raf BXB-301), and the control expression vector
pKRSPA (36) were generously provided by U. R. Rapp (Institute of Medical Radiobiology and Cell Biology, University of Wurzburg, Wurzburg, Germany).
The mouse hepatoma cell line
Hepa1c1c-BprC1 (a generous gift from James Whitlock, Stanford
University Medical School, Palo Alto, CA) (37) and the rat hepatoma
cell line H-4-II-E (American Type Culture Collection number 1548) were
maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented
with 10% fetal calf serum (FCS) (Life Technologies, Inc.), 1 mM glutamine, and 50 µg of neomycin/ml in a humidified
incubator containing 5% CO2. Prior to stimulation of
insulin, cells at 70-80% confluence were grown in 0.3%
FCS-containing medium for 48 h. Cells were then treated with
insulin (107 M) for various periods and then
harvested for use in preparing nuclear extracts and RNA.
The calcium phosphate precipitation method (38) was used to transfect
cells with DNA. Two hours before transfection, cells in exponential
growth phase (approximately 70-80% confluence) were plated in Corning
six-well plates. DNA-CaPO4 precipitate was added to the
medium with 10% serum and incubated for 5-6 h. After being shocked
with 15% glycerol for 30 s and washed with phosphate-buffered
saline, cells were grown in Dulbecco's modified Eagle's medium
containing 0.3% FCS (low serum medium). When expression vectors of
NF-B p50/p65 subunits, antisense p65, Raf-1 kinase, and its dominant
negative mutant were used to transfect cells, cells were harvested
approximately 60 h after culture in the low serum medium. For
insulin stimulation, 10
7 M insulin was added
directly to the spent low serum medium 48 h after incubation.
After 12 h of insulin treatment, cells were harvested. activities
in the cell extracts were measured by a method previously described
(39) using total extract protein (as measured by the Bio-Rad protein
assay kit) as reference. Relative CAT activity levels were calculated
from the conversion of [14C]chloramphenicol into
acetylated chloramphenicol by PhosphorImager (model 400S,
Molecular Dynamics).
Nuclear extracts
were prepared from BprC1 and H-4-II-E cells by the method of Digman
et al. (40) with modifications. Briefly, cells were washed
with ice-cold PBS. After centrifugation, the cell pellet was
resuspended in 5 packed cell volumes of buffer A (10 mM
HEPES/KOH, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 0.74 mM spermidine, 0.15 mM
spermine, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin) and then incubated on ice for 10 min. Supernatants were
removed after centrifugation, and 2 packed cell volumes of buffer A was added. Cells were then lysed by 10-15 strokes in a tight fitting Dounce homogenizer. Then, volume of 68% RNase-free sucrose
was mixed with the cell lysates and centrifuged at 16,000 × g for 30 s at 4 °C. The resulting pellet was washed
with buffer A and resuspended in
packed cell volumes of
buffer B (20 mM HEPES/KOH, pH 7.9, 0.42 M NaCl,
1.5 mM MgCl2, 25% glycerol, 0.2 mM
EDTA, 0.2 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin). After constant agitation for 30 min at 4 °C, nuclear debris was centrifuged at 15,000 × g for 6 min, and
supernatants (nuclear extracts) were stored at
80 °C until
analysis by EMSA.
GMSAs were performed with approximately 5 µg of nuclear proteins in a
total volume of 20 µl of binding mixture (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 10%
glycerol, 0.2% Nonidet P-40, 3 µg of poly(dI-dC)·poly(dI-dC)) and
radiolabeled DNA probe at room temperature for 20 min. If antibodies
were added, the mixtures were preincubated for 10 min before the
addition of the radiolabeled probe. Protein concentration was
determined using the Bio-Rad kit according to the manufacturer's
instruction. DNA probe was radiolabeled with
[-32P]dCTP using the Klenow fragment of DNA polymerase
I (Boehringer Mannheim). For competition assays, a 100-fold excess of
unlabeled oligonucleotide was added to the reaction. The consensus
sequences of AP-1 (41), HNF-1 (41), HNF-3 (41), HNF-4 (41), C/EBP (41),
Ig
B (42), PRDII (43), and Ig
B-m (44) were synthesized, annealed
to double strands, and used in competition experiments. The final
reaction mixture was analyzed on a 4% nondenaturing polyacrylamide gel
with 0.25 × Tris borate-EDTA electrophoresis buffer.
A 162-nucleotide (37 to +125)
antisense RNA probe was synthesized using T7 RNA polymerase as
described previously (26). Either 20 µg (for the mdr1b
probe) or 1 µg (for the 18 S rRNA probe) of total RNA from
serum-treated, insulin-treated, or untreated H-4-II-E cells was
hybridized with 32P-labeled antisense RNA probes (2 × 105 cpm) and subjected to the RNase protection assays as
described previously (26, 45). The protected RNA products were analyzed on a 7% denaturing polyacrylamide gel and quantitated using a Personal
Densitometer SI (Molecular Dynamics).
To
investigate whether the expression of the rat mdr1b gene
expression is regulated by insulin, we treated serum-starved rat hepatoma H-4-II-E cells (0.3% serum, 48 h) with insulin. At
various time intervals, cells were harvested and mdr1b
mRNA levels were measured by RNase protection assay. As shown in
Fig. 1A, the steady-state rat
mdr1b mRNA levels elevated rapidly in the first hour
after the addition of insulin, reaching to about 3.5-fold by 12 h
(Fig. 1B). Similarly, when serum-starved H-4-II-E cells were
treated with serum (20%) for 12 h, a 3-4-fold increase of the
mdr1b mRNA level was also observed (data not shown).
Consistent with previous observations (23, 30), treating these cells with the protein synthesis inhibitor cycloheximide (10 µg/ml) also induced rat mdr1b expression (Fig. 1A, lane 7). This suggests that rat mdr1b expression may be regulated by an inhibitor that can be repressed by protein synthesis inhibition (see below).
Rat mdr1b Promoter Responds to Insulin StimulationTo
investigate the possible involvement of transcriptional regulation in
the induction of the rat mdr1b gene expression by insulin
and to identify DNA sequences involved in the insulin induction of
mdr1b expression, we generated a set of 5 deletion mutant
CAT constructs (Fig. 2C) and transfected them
into H-4-II-E cells in the presence or absence of insulin stimulation
(Fig. 2A). When
1288 RMICAT and
243 RMICAT containing
1288 and 243 bp of the rat mdr1b upstream sequences,
respectively, plus 125 bp downstream from the transcription start site,
were transfected into H-4-II-E cells followed by insulin treatment, CAT
activities increased an approximately 2-fold in comparison with the
untreated controls (p < 0.05) (compare Fig.
2A, lanes 2 and 1 and lanes 4 and 3). However, when
163RMICAT, which contains
additional deletion to
163 bp was transfected, basal transcriptional
activities were reduced more than 80% (compare lanes 5 and
3), consistent with our previous findings that sequences
between
243 and
163 are important for the basal transcription of
mdr1b (26). More importantly, the deletion also resulted in
the loss of insulin inducibility (lane 6). Together, these
results indicated that the rat mdr1b promoter can respond to
insulin stimulation and that the sequence from
243 to
163 bp is
essential for the promoter's insulin responsiveness.
Identification of an NF-
In examining the 243/
163 sequence of the rat
mdr1b promoter, we found that a sequence between bp
167
and
158 (5
-GGGGAATTCC-3
) (Fig. 3A) was
strikingly similar to the transcription factor NF-
B-binding consensus sequence (5
-GGGRNNYYCC-3
) (46). A comparison of the
putative mdr1b NF-
B-binding site with several known
NF-
B-binding sequences, including the interferon-
gene's PRDII
site (INF
PRDII) (43) and the immunoglobulin gene's
B site
(Ig
B) (42) is shown in Fig. 3A. Since insulin can
reportedly activate NF-
B activity in CHO-R cells that overexpress
insulin receptor (47), we speculated that this putative NF-
B-binding
site in the rat mdr1b promoter may be involved in the
observed insulin's induction of the promoter function. We therefore
designed experiments to test this possibility.
First, we determined whether the sequence located between bp 167 and
158 is indeed an NF-
B-binding site. To this end, we carried out
GMSA using a double-stranded oligonucleotide spanning bp
169 to
153
as the probe (designated the mdr-
B fragment in Fig. 3A)
and various unlabeled oligonucleotides as competitors. As shown in Fig.
3B, a DNA-protein complex was formed in nuclear extracts
prepared from mouse hepatoma BprC1 cells (lane 1). More importantly, formation of this complex was efficiently competed only by
the unlabeled probe (lane 2) and NF-
B sequences derived from Ig
B (lane 8) and PRDII (lane 9), and not
by the mutated sequence of Ig
B (Ig
Bm, lane 10) or
other unrelated consensus sequences, i.e. HNF-1, HNF-3,
HNF-4, AP-1, or C/EBP (lanes 3-7).
NF-B is an inducible transcription factor, and its nuclear DNA
binding activity could be induced by mitogens such as the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (48). To further
substantiate the aforementioned results, we treated BprC1 cells with
12-O-tetradecanoylphorbol-13-acetate (50 ng/ml) and prepared
nuclear extracts from the treated cells. GMSA using the mdr-
B probe
showed that the protein binding activity was rapidly induced in nuclear
extracts from cells treated for 30 min and that the increase lasted at
least 1 h (Fig. 3C, lanes 1-3).
Furthermore, the formation of protein-DNA complex could be blocked by
antiserum raised against the p65 subunit of NF-
B (Fig.
3C, lanes 5, 7, and 9),
whereas the preimmune serum did not inhibit the formation of
DNA-protein complexes (lanes 4, 6, and
8). Thus, taken together, these results strongly suggest
that the rat mdr1b promoter sequence located between bp
167 and
158 (5
-GGGGAATTCC-3
) is an authentic NF-
B-binding
site.
To
determine whether the mdr-B site is functionally involved in
mdr1b promoter activity, we first generated site-directed mutations in the NF-
B sequence of the wild-type
1288 RMICAT recombinant (designated
1288 RMICAT-
m, Fig.
4B) and then transiently transfected them
into rat H-4-II-E cells. In the absence of insulin, basal expression of
CAT activity was reduced about 65% in cells transfected with mutant
1288 RMICAT-
m compared with that in cells transfected with the
wild-type construct (
1288 RMICAT) (Fig. 4A). More
importantly, the induction of CAT expression by insulin was only seen
in cells transfected with the wild-type construct (
1288 RMICAT) and
not with the mutated version (
1288 RMICAT-
m). Together, these
results suggest that the NF-
B binding site is involved in both basal
and insulin-inducible mdr1b promoter function.
We previously identified in the rat mdr1b promoter region a
palindromic sequence (5-AGACATGTCT-3
) located between bp
189 and
180 (designated the F site in Fig. 4B). This site is
important for basal promoter function in many different cell lines
(26). Since the F site also falls within the bp
243 to
163 region, which is essential for the induction of the promoter activity as
described above, we tested whether the palindromic sequence is also
involved in the insulin-inducible promoter activity. Thus, a mutant
designated
1288 RMICAT-Fm1 was generated (Fig. 4B) and subjected to transient transfection assays. The assays demonstrated that, like the NF-
B site, the palindromic sequence is also required for insulin induction. The disruption of this palindromic sequence concomitantly abolished basal and insulin-induced mdr1b
promoter activity in rat H-4-II-E cells (Fig. 4A).
To further substantiate these findings, we generated two additional
sets of constructs. The first set contained mutations in either the
NF-B site or F site of
243 RMICAT. The constructs were designated
243 RMICAT-
m and
243 RMICAT-Fm1, respectively (Fig.
5). These mutants were then transfected into rat
H-4-II-E cells and subjected to insulin inducibility analysis.
Consistent with the results shown in Fig. 4A, mutations in
either site not only reduced basal expression levels of CAT activity
but also abolished the insulin inducibility of the reporter gene's
expression (Fig. 5). The second set of plasmids consisted of three
constructs, each containing three copies of a
243 to
127
mdr1b sequence linked in tandem to the 5
end of a promoter
sequence beginning at bp
127. Mutations in either the F site or
NF-
B site were introduced in wild-type constructs (3x-243 RMICAT),
and the resulting mutant plasmids were designated 3x-243 RMICAT-Fm1 and
3x-243 RMICAT-
m (Fig. 5). These constructs then transiently
transfected into H-4-II-E cells. In the absence of insulin, wild-type
3x-243 RMICAT showed an approximately 3-fold higher CAT activity than
243 RMICAT, which contains only one copy of the
243/
127 sequence
(Fig. 5). CAT expression was also induced by insulin in 3x-243 RMICAT,
although no additive induction was evident. However, mutations in
either the NF-
B site or F site impaired not only basal promoter
activity but also insulin inducibility. Collectively, these results
suggest that both NF-
B and the palindromic sequence are involved in
insulin-induced up-regulation of the rat mdr1b expression.
Since the transcription factor(s) recognizing the F site (palindromic
sequence) are currently unknown, the involvement of this palindromic
sequence in insulin induction was not characterized further.
Insulin Induces Nuclear NF-
To test whether treatment of rat H-4-II-E cells with
insulin could induce nuclear NF-B activity, we prepared nuclear
extracts in the presence or absence of insulin stimulation and
performed GMSAs using mdr-
B (Fig. 3A) as the probe. As
shown in Fig. 6A, a major DNA-protein complex
(arrow) was rapidly induced by insulin treatment within the
first 0.5 h (lanes 1 and 2). Binding
activity levels remained elevated throughout the 12-h induction period (lanes 3-6). Cycloheximide, already shown to be capable of
inducing rat mdr1b expression (Fig. 1A,
lane 7) (23, 30), also induced DNA binding activity (Fig.
6A, lane 7). Furthermore, the induced DNA-protein
complex was efficiently competed only by unlabeled mdr-
B and Ig
B
consensus sequences (Fig. 6B, lanes 2 and
4), and not by a nonspecific competitor AP-1 consensus
sequence (lane 3).
To further characterize the identity of insulin-induced DNA-protein
complex, antibodies directed against the p50 and p65 NF-B subunits
were used. Both antibodies, especially the p65 antibody, markedly
reduced the formation of DNA-protein complex, whereas preimmune serum
had no effect (Fig. 6B, lanes 5-7). These
results indicate that the induced complex is composed of both p50 and p65 NF-
B subunits and most likely a p50/p65 heterodimer, a major effective and inducible form of NF-
B (49).
To verify whether p50 and p65 subunits of the NF-B
family directly trans-activate the expression of the rat
mdr1b promoter, we cotransfected p50/p65 expression vectors
with reporter constructs containing either wild-type NF-
B-binding
site (
243 RMICAT or 3x-243 RMICAT) or mutated
B site (
243
RMICAT-
m or 3x-243 RMICAT-
m). As shown in Fig. 7,
a p50/p65 plasmid DNA dose-dependent increase in CAT
activities was observed in cells cotransfected with wild-type constructs but not in cells cotransfected with constructs containing mutated NF-
B site. These results further demonstrated that p50/p65 subunits of the NF-
B family are able to trans-activate
the rat mdr1b promoter activity specifically via
the NF-
B-binding site.
Raf-1 Kinase Is Involved in the NF-
The biological function of insulin is
believed to be mediated by several signal transduction pathways,
including the Ras/Raf mitogen-activated protein kinase pathway (for a
review, see Ref. 50). Coincidentally, it has been shown that NF-B
site-dependent induction of gene expression by diverse
inducers of the nuclear factor NF-
B requires Raf-1 kinase (51).
Thus, we deemed it important to investigate whether Raf-1 kinase also
regulates the rat mdr1b promoter through the NF-
B-binding
site. Plasmid expressing Raf-1 kinase (Raf BXB) (36) was cotransfected
with
243 RMICAT,
243 RMICAT-
m, or
243 RMICAT-Fm1 into H-4-II-E
cells. As shown in Fig. 8A, a Raf-1 DNA
concentration-dependent increase in CAT activity was seen
in cells cotransfected with the wild-type construct but not in those
cells transfected with constructs containing a mutant NF-
B-binding
or in the F site. These results indicate that the integrity of both the
NF-
B site and the palindromic sequences is required for the promoter
to be activated by Raf-1 kinase. Similar results were obtained using
3x-243 RMICAT and the corresponding mutants (Fig. 8B).
Furthermore, cotransfection of expression recombinants of p50/p65
NF-
B subunits and Raf-1 resulted in a synergistic effect on rat
md1b promoter activity (Fig. 8C).
Both Antisense p65 and Dominant Negative Raf-1 Mutant Inhibit Insulin-induced Promoter Function of the Rat mdr1b Gene
To obtain
more direct evidence of the involvement of NF-B and Raf-1 signaling
in insulin-induced rat mdr1b expression, we cotransfected
antisense p65 expression vector (
p65) (35) or a dominant negative
Raf-1 mutant (Raf-BXB301) (36) into H-4-II-E cells with the
243
RMICAT vector. Approximately 48 h after transfection, cells were
either mock-stimulated or insulin-stimulated for 12 h, and CAT
activity was determined. As shown in Fig. 9, insulin inducibility was abolished when cells were transfected with either
p65 or Raf-BXB301 plasmids. These results, taken together, lend additional credence to the notion that both NF-
B and Raf-1 kinase are involved in the insulin-induced expression of rat
mdr1b.
We have previously identified a palindromic sequence located at bp
189 to
180 that is essential for rat mdr1b promoter
function in many types of cells (26). Here, we report the
identification of a NF-
B-binding site located at bp
167 to
158
that is important for the basal as well as insulin-inducible promoter
activity of the rat mdr1b gene. To the best of our
knowledge, this is the first evidence that NF-
B is involved in the
transcriptional regulation of genes in the mdr family.
The transcription factor NF-B/Rel family has been shown to be
involved in immune, inflammatory, and acute phase responses; cellular
proliferation; oncogenic transformation; and programmed cell death (for
reviews, see Refs. 46 and 52-54). This family is made up of five
members (p50, p52, p65, c-Rel, and RelB) that are able to bind DNA as
homo- or heterodimers and are implicated in the expression of many
inducible genes. Indeed, NF-
B is functionally active in many
tissues, and its activity can be induced by a wide variety of stimuli,
including serum, insulin, growth factors, UV irradiation, cytotoxic
compounds, and protein synthesis inhibitors (for a review, see Ref.
49). Strikingly, many of these stimulants are also known to induce
mdr gene expression (Fig. 1A) (18, 19, 23),
suggesting that the induction of mdr gene expression by
these stimulants may be mediated through the NF-
B signaling pathway.
The pleiotropic function of insulin is mediated by several signal
transduction pathways, including the mitogen-activated protein kinase-dependent pathway (for a review, see Ref. 50). The
Raf-1 kinase works downstream of the Ras signaling pathway and upstream of the mitogen-activated protein kinase kinase and mitogen-activated protein kinase pathway (for a review, see Ref. 55). Consistent with the
previous observation that Raf-1 is involved in the signal transduction
pathway of B site-dependent induction of gene expression by many NF-
B inducers (51), our present results demonstrate that
NF-
B-dependent induction of rat mdr1b
expression by insulin also requires Raf-1. As we noted in the
Introduction, epidermal growth factor and insulin-like growth factor I
have been shown to induce expression of the rat mdr1b gene
in primary rat hepatocyte culture (20). Given that a similar signal
transduction pathway is elicited by these growth factors in activating
gene expression (56, 57), it is likely that regulation of the rat
mdr1b gene by these growth factors may follow a similar
signal transduction pathway as described in this paper.
It has been known that in unstimulated cells, NF-B is sequestered in
the cytoplasm by virtue of its association with members of a set of
inhibitor proteins belonging to the I
B family (for a review, see
Ref. 53). Through noncovalent association, the I
B proteins mask the
nuclear localization signal of NF-
B. Extracellular inducers of
NF-
B usually activate signal transduction pathways that result in
the phosphorylation and subsequent degradation or dissociation of
I
B. As a result, the released NF-
B translocates into the nucleus
where it regulates gene expression by binding directly to cognate DNA
sequences (for reviews, see Refs. 46, 52-54, and 58). The mechanism by
which Raf-1 activates NF-
B activity is not clear. Although there was
a controversial report suggesting that I
B-
can be phosphorylated
by Raf-1 (59), it is now clear that Raf-1 cannot directly phosphorylate
I
B-
(60). A recent study showed that phosphorylation of I
B-
appears to be mediated by a large, multisubunit I
B-
kinase
complex (61). However, it remains unclear that whether Raf-1, like
other MEKKs such as MEKK1, can activate NF-
B by phosphorylating
I
B-
kinase directly (62) or by an intermediate activator. It also
remains possible that Raf-1 may induce synthesis of a factor, possibly a cytokine, which in turn could have been the true activator of one or
more mitogen-activated protein kinase pathways leading to the
activation of NF-
B. A similar example was observed in HeLa cells in
which Raf-1 activates stress-activated protein kinase via an induced
cytokine (63). We also cannot rule out the possibility that Raf-1 may
modulate NF-
B activity by directly targeting p50/p65 rather than
I
B. It has recently been demonstrated that a splice variant of
adenovirus E1A product can activate NF-
B activity not only by the
I
B phosphorylation pathway but also through direct interaction with
the C-terminal 80 amino acids of the p65 component (64). Furthermore,
Perkins et al. (65) reported that regulation of NF-
B can
be achieved by cyclin-dependent kinases through association with the p300 coactivator. Additional studies are needed to determine the exact roles of Raf-1 in NF-
B regulation.
Overexpression of rat mdr1b is associated with liver
regeneration (25, 27). NF-B activity in normal liver is very low but
rapidly induced in regenerating liver (for a review, see Ref. 66).
Although other reports have suggested that the elevated RNA levels in
regenerating livers are largely attributed to increased stabilization
of the mdr1b mRNA (27), the elevated NF-
B activities during liver regeneration may also contribute to the overall increase of mdr1b mRNA levels seen during liver regeneration. In
addition, the rat mdr1b gene is consistently activated
during hepatocarcinogenesis due to its increased rate of transcription
(23, 24). Expression of human MDR1 is also elevated in many
forms of neoplasms (1, 11). Interestingly, there is increasing evidence
to suggest that NF-
B plays an important role in the proliferation
and malignant transformation of tumor cells (for reviews, see Refs. 52
and 67), implying that NF-
B may be involved in the regulation of mdr gene expression in these tumors. Furthermore,
mdr gene expression in cultured rodent cells can be acutely
induced by various cytotoxic compounds (68). Many of these compounds
exert cytotoxic stress on the cells, resulting in the concomitant
induction of stress-response NF-
B transcription factors (for a
review, see Ref. 69). Thus, it is likely that the induction of
mdr expression by many of these cytotoxic compounds may be
due to the elevated NF-
B activities.
Cycloheximide has been shown to induce rat mdr1b expression
in primary rat hepatocyte cultures and in other different cell lines
(23, 30). A previous study even suggested that such induction may be
due to the release of an unknown trans-acting transcriptional repressor (23). In the present study, we have confirmed
that cycloheximide can induce rat mdr1b expression (Fig. 1A, lane 7). More importantly, we have shown that
cycloheximide can also induce NF-B activity in rat H-4-II-E cells
(Fig. 6A, lane 7), as previously reported in
other types of cells (47, 70). The mechanism underlying the induction
of nuclear NF-
B activity by cycloheximide is still unclear. However,
because I
B has a shorter half-life than NF-
B (71), the levels of
NF-
B relative to I
B may be increased in the presence of protein
synthesis inhibitor, thereby resulting in the elevated expression of
rat mdr1b. This would be consistent with the I
B/NF-
B
scenario discussed above, implying that I
B may be the previously
unidentified trans-acting repressor. All in all, our present
findings suggest that NF-
B signaling may play an important role in
the regulation of the rat mdr1b expression by many
environmental influences that have been previously described in the
literature.
Our present study also demonstrates that the induction of rat
mdr1b promoter function by insulin requires the integrity of both the NF-B-binding site and the palindromic sequence, suggesting that these two cis-acting elements somehow cooperate in
regulating rat mdr1b expression. But because there is no
clear information on the trans-activating factors that
recognize the palindromic sequence, the activation mechanism remains
elusive. We still cannot rule out the possibility that the
palindrome-binding protein is also directly induced or regulated by
insulin and Raf-1 kinase. Therefore, the identification, through
molecular cloning, of transcription factors that recognize the
palindromic sequence will be very helpful in elucidating the activation
mechanism. It has been known that most inducible cis-acting
elements contain multiple, distinct transcription factor-binding sites
that are part of a combinatorial mechanism that relies on cooperative
binding, interaction of transcriptional activator proteins, and
transcriptional synergy (for reviews, see Refs. 72 and 73). Moreover,
NF-
B has been shown to cooperate with other transcription factors
such as ATF-2/c-Jun (35, 74, 75), IRF-1 (76), Sp1 (77), AP-1 (78), and
C/EBP (79) in modulating gene expression. Because the NF-
B-binding
site and palindromic sequence lie so close to each other, we speculate that NF-
B may interact with palindrome-binding proteins and other unidentified transcription factors in the the mdr1b promoter
to form a high order transcription enhancer complex, much like the enhanceosome organized in the interferon
enhancer region (75), and
so regulate rat mdr1b gene expression.
Finally, the observation that insulin can induce mdr1b gene
expression in rat liver cancer cells in culture may have important clinical implications. For example, breast cancer MCF-7 cells treated
with insulin or insulin-like growth factor I have reportedly high
levels of resistance to many antitumor drugs, including actinomycin D,
adriamycin, and puromycin (80). Although the levels of mdr expression in the treated cells were not reported for that study, the
drugs used are known substrates for the mdr-encoded
P-glycoproteins (for a review, see Ref. 1). It is not known, however,
if insulin would modulate such drug resistance to chemotherapeutic
agents in vivo. Nevertheless, our present communication
provides an important basis for future investigations on the potential
roles of NF-B, insulin, and other growth factors in the regulation
of drug resistance during cancer chemotherapy.
We are grateful to G. J. Nabel for providing RSV-p50 and RSV-p65 expression vectors; T. Maniatis for antisense p65 expression vector; and U. R. Rapp for Raf-BXB, Raf-BXB301, and pKRSPA vectors. We thank J. H. Huang for research support during early phases of this work. We thank M. Furuichi, R-D. Song, and J-J. Bao for excellent technical advice and encouragement during this work. We also thank other members of Dr. Kuo's laboratory for critical comments and discussions on the manuscript.