(Received for publication, August 17, 1995; and in revised form, October 19, 1995)
From the
Activation of gene expression by hypolipidemic peroxisome proliferators (e.g. native and substituted long chain fatty acids, aryloxyalkanoic fibrate drugs) is accompanied by transcriptional suppression of liver transferrin gene in treated animals or human hepatoma cell line. Transcriptional suppression of liver transferrin by hypolipidemic peroxisome proliferators results from (a) displacement of hepatic nuclear factor (HNF)-4 from the transferrin promoter by nonproductive binding of the peroxisome proliferator-activated receptor-retinoic acid X receptor heterodimer to the (-76/-52) PRI promoter element of the human transferrin gene and (b) suppression of liver HNF-4 gene expression by hypolipidemic peroxisome proliferators with a concomitant decrease in its availability for binding to the transferrin PRI promoter element. HNF-4 gene suppression and its displacement from the transferrin promoter result in eliminating HNF-4-enhanced transcription of transferrin. Liver transferrin suppression by hypolipidemic peroxisome proliferators may result in reduced iron availability as well as modulation of transferrin-induced differentiation processes. Transcriptional suppression of HNF-4-enhanced liver genes (e.g. apolipoprotein C-III, transferrin) may complement the pleiotropic biological effect exerted by hypolipidemic peroxisome proliferators.
Transferrin (Tf) ()is highly expressed in the adult
mammalian liver and is secreted by hepatocytes into the serum where it
functions as an iron transport protein and growth factor for a variety
of cells (reviewed in (1) and (2) ). Liver Tf
expression was reported to be activated by steroid hormones (3) and iron deficiency(3, 4) . Tf is
synthesized to a lower extent by Sertoli cells in the adult testis as
well as by adult brain oligodentrocytes, astrocytes, and epithelial
cells of the choroid plexus, where it is involved in the maturation of
germinal cells and in central nervous system proliferation and
differentiation processes.
In experiments to be reported elsewhere, ()serum iron, iron binding capacity and plasma Tf were found
to be 50% reduced in rats treated by xenobiotic amphipathic
carboxylates (e.g. aryloxyalkanoic acids (bezafibrate),
substituted long chain dicarboxylic acids (Medica 16)) known
collectively as hypolipidemic drugs/peroxisome proliferators (HD/PPs)
(reviewed in (5) ). HD/PPs have previously been reported to
activate the expression of a variety of discrete genes (e.g. peroxisomal
-oxidation genes(6) ,
-oxidation
P450IV genes(7) , liver genes coding for thyroid
hormone-dependent activities(8) , and others) as a result of
transcriptional activation mediated by binding of peroxisome
proliferators-activated receptors (PPARs) to sequence-specific
PPAR-activated response elements (PPREs) in the respective promoters (9, 10, 11, 12, 13) . (
)PPAR binding to PPREs requires the retinoic acid X
receptor (RXR) for forming the high affinity PPAR
RXR
heterodimer(14) . The putative binding of HD/PPs to PPARs and
the role of HD/PPs in initiating the binding of PPAR/RXR to PPREs still
remains to be investigated(9) .
Since some HD/PPs are extensively used in humans as hypolipidemic drugs (15) and since transcriptional suppression, rather than activation, mediated by the HD/PP-PPAR/RXR-PPRE transduction pathway may complement the pleiotropic biological effect exerted by HD/PPs, we became interested in elucidating the mode of action of HD/PPs as putative suppressors of liver Tf. Liver Tf gene suppression by HD/PPs will be shown here to be mediated by PPAR/RXR and to involve the HNF-4 enhancer element of the Tf gene promoter.
Figure 1:
The effect of Medica 16 on Tf mRNA
levels in rat liver and Hep G2 cells. A, rats were treated for
5 days with Medica 16 as described under ``Experimental
Procedures.'' Total RNA was prepared according to (41) ,
and 25 µg were subjected to Northern blot analysis. Rat Tf mRNA was
determined using the 1.4-kilobase PstI restriction fragment of
rTf cDNA (20) P-labeled by the random priming
method(42) . Representative experiment is shown out of three
independent experiments. B, Hep G2 cells were cultured for 72
h as described under ``Experimental Procedures'' in the
presence or absence of Medica 16. Total RNA was prepared according to (43) , and 25 µg were subjected to Northern blot analysis.
Hep G2 Tf mRNA was probed by the 1.9-kilobase BamHI-ClaI restriction fragment of hTf
cDNA(21) . Representative experiment is shown out of four
independent experiments.
Suppression of liver Tf mRNA by Medica 16 was accompanied by inhibition of Tf transcription rate as verified by run-on transcription assays in Hep G2 cell nuclei (Fig. 2). Incubation in the presence of Medica 16 for 72 h resulted in a pronounced inhibition of Tf transcription rate, indicating that reduction in Tf mRNA exerted by Medica 16 treatment may be ascribed to suppression of transcription of liver Tf gene by Medica 16.
Figure 2:
The effect of Medica 16 on the rate of
transcription of the Tf gene in Hep G2 cells. Hep G2 cells were
cultured for 72 h in the presence or absence of Medica 16, and run-on
transcription assays were determined on isolated nuclei as described
previously(17) . Newly synthesized P-RNA was
hybridized with the hTf cDNA plasmid linearized with BamHI
restriction enzyme. The extent of hybridization was normalized to the
signal obtained with poly(A) binding protein cDNA. Representative
experiment is shown out of three independent
experiments.
Essentially similar decreases in Tf mRNA and Tf transcription rates were observed using bezafibrate (not shown), thus generalizing the observed effect to other members of the HD/PP class of compounds.
Figure 3:
The effect of Medica 16 and PPAR/RXR on
the transcriptional activity of the human Tf promoter. A, Hep
G2 cells were transfected as described under ``Experimental
Procedures'' with the (-620/+39)hTf-CAT construct (5
µg) and cotransfected with the parent pSG5 vector or expression
vectors for PPAR and/or RXR (1 µg). The amount of expression
vectors was equalized by adding the parent pSG5 vector. Following
transfection, cells were incubated for 48 h with either
MeSO (
) or Medica 16 (&cjs2113;) as indicated. CAT
activity of the (-620/+39)hTf-CAT construct in cells
transfected with the pSG5 vector and in the absence of added Medica 16
was arbitrarily set to 1.0. Representative experiment is shown out of
four independent experiments. B, conditions as in A except that cells were transfected with an expression vector for
PPAR-G instead of PPAR. Representative experiment is shown out of two
independent experiments. C, for deletion analysis, Hep G2
cells were transfected with the (-620/+39)hTf-CAT,
(-125/+39)hTf-CAT, (-82/+39)hTf-CAT, or
(-52/+39)hTf-CAT (5 µg) constructs as indicated and
cotransfected with the parent pSG5 vector or expression vectors for
PPAR and RXR. The amount of expression vectors was equalized by adding
the parent pSG5 vector. Following transfection, cells were incubated
for 48 h with either Me
SO (
) or Medica 16
(&cjs2113;) as indicated. CAT activity of the
(-620/+39)hTf-CAT construct in cells transfected with the
pSG5 vector and in the absence of added Medica 16 was arbitrarily set
to 1.0. Representative experiment is shown out of three independent
experiments. D, for mutation analysis, Hep G2 cells were
transfected with the (-620/+39)hTf-CAT,
(-620/+39)hTf(PRI mut)-CAT, or (-620/+39)hTf(PRII
mut)-CAT constructs. Conditions are as in C. Representative
experiment is shown out of two independent
experiments.
The promoter element involved in transcriptional suppression of the Tf gene by PPAR/RXR and Medica 16 was further characterized by transfecting Hep G2 cells with 5`-deleted constructs of the Tf promoter linked to CAT and analyzing the effect exerted by cotransfected PPAR/RXR and added Medica 16 on expression of the concerned constructs. As shown in Fig. 3C, CAT expression promoted either by the (-620/+39)hTf or (-125/+39)hTf promoter sequences, which consist of the Tf-PRI as well as the Tf-PRII elements, was suppressed by cotransfected PPAR/RXR in the absence or presence of Medica 16, thus pointing to a PPAR/RXR responsive element within the (-125/+39)hTf promoter sequence. Deleting the Tf-PRII element resulted in a 7-fold decrease in CAT expression, reflecting the synergistic role played by Tf-PRI and -II in Tf transcription. However, transcriptional suppression by PPAR/RXR was still maintained in the (-82/+39)hTf-CAT construct. On the other hand, deleting both the PRI and PRII elements resulted in loss of suppression by PPAR/RXR, thus indicating that transcriptional suppression of the Tf gene by PPAR/RXR involves the Tf-PRI element.
The role played by the Tf-PRI element in transcriptional suppression of the Tf gene by PPAR/RXR was further verified in Hep G2 cells transfected with (-620/+39)Tf-CAT constructs mutated in either the PRI ((-620/+39)hTf(PRI mut)-CAT) or PRII ((-620/+39)hTf(PRII mut)-CAT) elements (22) and cotransfected with expression vectors for PPAR and RXR. As shown in Fig. 3D, expression from the Tf-PRII mutated construct was 50% inhibited as compared with the respective wild type construct. However, the PRII mutation did not interfere with transcriptional suppression by PPAR/RXR. Expression from the Tf-PRI mutated construct was 90% inhibited as compared with the wild type construct and could not be further suppressed by PPAR/RXR. Hence, suppression of the liver Tf gene by PPAR/RXR appears to be specifically mediated by the Tf-PRI element.
Transfection studies were complemented by studying binding
of PPAR and/or RXR to the hTf-PRI sequence using mobility shift
analysis. As shown in Fig. 4A, the PPARRXR
heterodimer transcribed and translated in vitro in rabbit
reticulocytes, but not the respective individual receptors, indeed
binds to the hTf-PRI element. Similarly, PPAR derived from transfected
COS cells (Fig. 4B) and complemented by endogenous or
transfected RXR specifically binds to the hTf-PRI element and may be
supershifted by anti-mPPAR antibody.
Figure 4:
Gel shift of the hTf-PRI element by PPAR
and RXR. Gel shift analysis was as described under ``Experimental
Procedures'' using the P-labeled hTf-PRI element
(5`-agctACGGGAGGTCAAAGATTGCGCCc-3`). PPAR and RXR were transcribed and
translated in vitro in rabbit reticulocytes (A) or in
transfected COS cells (B) as described under
``Experimental Procedures.'' Specificity of binding was
analyzed by adding unlabeled hTf-PRI (lane 7, 25-fold excess),
acyl-CoA oxidase-PPRE (lane 8, 25-fold excess), or nonrelevant
oligonucleotide (5`-GATCCGTTGCTGACTAATTGAGAG-3`) (lane 9,
25-fold excess). Where denoted, nonimmune (NI) or anti-mPPAR
antibodies (1 µl immune serum) (Ab) were added to the
incubation mixture. R
for the hTf-PRI element shifted by
PPAR/RXR is denoted by (arrow).
PPAR/RXR interference with HNF-4
binding to the Tf-PRI element was studied by comparing the Tf-PRI
binding affinities of HNF-4 and PPAR/RXR using gel shift assays. As
shown in Fig. 5, both HNF-4 and PPAR/RXR could bind to the
hTf-PRI element sequence with apparent binding affinities of 2.7
± 0.3 and 3.6 ± 0.6 nM hTf-PRI (mean ±
S.D. for three independent experiments), respectively, thus indicating
that the two receptors could compete for binding to the concerned
element. Their functional interaction with the hTf-PRI element in the
context of the heterologous thymidine kinase promoter was analyzed in
CV-1 cells transfected with a hTf-PRI-TK-CAT construct consisting of
the hTf-PRI element in front of the thymidine kinase promoter (Fig. 6). Cotransfecting these cells with either HNF-4 or
PPAR/RXR resulted in 10- and 4-fold activation of CAT
expression, indicating that PPAR/RXR binding to the hTf-PRI element in
the context of the heterologous TK promoter may result in its
transactivation.
Figure 5:
Binding of HNF-4 and PPAR/RXR to the
hTf-PRI element. A, HNF-4 (lanes 1-8) or
PPAR/RXR (lanes 9-15) were derived from transfected
COS-7 cells as described under ``Experimental Procedures.''
Gel shift analysis was as described under ``Experimental
Procedures'' using the P-labeled hTf-PRI element
(5`-agctACGGGAGGTCAAAGATTGCGCCc-3`) and competing with 4-fold (lanes 2 and 10), 8-fold (lanes 3 and 11), 16-fold (lanes 4 and 12), 32-fold (lanes 5 and 13), 64-fold (lanes 6 and 14), 76-fold (lanes 7 and 15), and 88-fold (lane 8) nonlabeled Tf-PRI. B, extent of binding of
PPAR/RXR (
) and HNF-4 (
) (densitometric units) as function
of oligonucleotide concentration (ng). Representative experiment is
shown out of three independent experiments.
Figure 6: Transactivation of hTf-PRI-TK-CAT by PPAR and RXR. CV-1 cells were transfected as described under ``Experimental Procedures'' with the hTF-PRI-TK-CAT reporter construct (5 µg) promoted by the thymidine kinase promoter and cotransfected with pSG5 (0.4 µg), pSG5-HNF-4 (0.2 µg), or pSG5-PPAR (0.2 µg) and pSG5-RXR (0.2 µg) expression vectors as indicated. CAT activity is presented by its fold induction, where the activity of the reporter construct cotransfected with pSG5 is taken as 1. Representative experiment is shown out of three experiments.
The functional relationship between PPAR/RXR and HNF-4 within the context of the homologous hTf promoter was studied in CV-1 cells transfected with the (-125/+39)hTf-CAT construct and cotransfected with expression vectors for HNF-4, PPAR, and RXR. As shown in Fig. 7, HNF-4, but not PPAR/RXR, activated CAT expression promoted by the homologous (-125/+39)hTf proximal promoter. Furthermore, cotransfecting the cells with both HNF-4 and PPAR/RXR resulted in eliminating HNF-4 activation of the Tf gene. Hence, PPAR/RXR binding to the hTf-PRI element in the context of the homologous Tf promoter is nonproductive but interferes with HNF-4 productive binding to this element, resulting in inhibition of HNF-4-enhanced transcriptional transactivation of the liver Tf gene.
Figure 7: Inhibition of the HNF-4-activated Tf promoter by PPAR/RXR in CV-1 cells. CV-1 cells were transfected as described under ``Experimental Procedures'' with (-125/+39)h-Tf-CAT construct (5 µg) and cotransfected with pSG5-HNF-4 and/or pSG5-PPAR and pSG5-RXR expression vectors (1 µg). CAT activity in cells transfected with pSG5 is taken as 1. Representative experiment is shown out of three experiments.
Figure 8:
Supershift analysis of Tf-PRI binding to
nuclear extracts and HNF-4 derived from rats treated with Medica 16.
Rat liver nuclei were prepared according to (19) . Nuclear
extracts from nontreated and rats treated for 5 days with Medica 16
were analyzed as described in Methods for binding to P-labeled hTf-PRI element
(5`-agctACGGGAGGTCAAAGATTGCGCCc-3`). R
for nuclear extract
binding in the absence of added anti HNF-4 antibodies is denoted by (arrow). HNF-4 specific binding was determined by incubating
0.2 µg (lanes 1 and 5), 0.4 µg (lanes 2 and 6), 0.6 µg (lanes 3 and 7), and
0.8 µg (lanes 4 and 8) of the respective extracts
with anti-rat HNF-4 antibodies(17) , thus producing the HNF-4
supershifted bands (arrow head). Representative experiment is
shown out of four independent experiments.
The decrease in plasma Tf observed in rats treated with hypolipidemic peroxisome proliferators was shown here to be accompanied by a decrease in liver Tf mRNA and to result from transcriptional suppression of the liver Tf gene as verified by run-on transcription assays in liver nuclei derived from Hep G2 cells. Since the level of plasma Tf is dominated by liver Tf expression and secretion, transcriptional suppression of liver Tf by HD/PP may account for the reduced plasma Tf levels in treated animals. Furthermore, since Tf transcriptional suppression was similarly observed in human Hep G2 cells incubated in the presence of added HD/PP, Tf suppression could be relevant to dyslipoproteinemic patients treated with HD/PP.
Transcriptional suppression of liver Tf gene by Medica 16 was found
here to be related to HNF-4-enhanced transcription of the Tf gene and
ascribed to displacement of HNF-4 from the Tf promoter by nonproductive
binding of PPAR/RXR to the Tf-PRI (HNF-4 enhancer) element. The
PPARRXR heterodimer behaves in this respect similarly to other
previously reported transcription factors, e.g. ARP-1, which
may compete with HNF-4 for binding to HNF-4 enhancer
elements(33, 34) . The extent of inhibition of Tf
transcription by PPAR/RXR in a specific cell type may therefore be
expected to reflect the prevailing content of concerned transcription
factors and their respective binding affinities for the Tf-PRI element.
Since the liver system is highly enriched in HNF-4 as compared with
chicken ovalbumin upstream promoter transcription factor or
ARP-1(34) , transfection with PPAR/RXR, or activating the
endogenous PPAR/RXR by HD/PP or using both intervention modes results
in Tf suppression. However, in cell types where the Tf-PRI function is
dominated by suppressive transcription factors, HD/PP may exert an
apparent transactivation of transcription mediated by displacing the
concerned suppressive transcription factors by nonproductive binding of
PPAR/RXR. The resultant effect in a specific cell type may be further
confounded by competition for RXR between some of the concerned
transcription factors in addition to competing for the same promoter
element. It should be pointed out, however, that generalizing the mode
of action of HD/PP as verified here in cells transiently transfected
with Tf-promoted CAT constructs to the endogenous Tf promoter still
remains to be complemented by studying the chromatin context of the
endogenous gene as well as the role played by additional regulatory
sequences of the Tf promoter not present in the transiently transfected
promoter constructs used here.
In addition to suppressing Tf transcription by PPAR/RXR binding to the Tf-PRI element, HD/PP may suppress Tf transcription by suppressing HNF-4 expression. Suppression of HNF-4 transcription by HD/PP has previously been verified by showing that HNF-4 transcription rates and transcript and protein levels were significantly reduced in livers of treated animals(17) . These previous results have been confirmed here in the context of the Tf gene by showing that the availability of HNF-4 for binding to the hTf-PRI element was significantly reduced in liver nuclear extracts derived from treated animals. Since HNF-4 expression is positively modulated by HNF-4 itself(35) , suppression of HNF-4 gene expression by HD/PP may perhaps result as well from PPAR/RXR binding and displacement of HNF-4 from its putative enhancer in the HNF-4 gene promoter.
The
increasing list of promoter elements reported to bind PPAR/RXR and be
involved in transcriptional modulation of various genes by PPAR/RXR may
call for updating the PPRE consensus sequence. As shown in Table 1, PPRE sequences consist of a direct repeat separated by
one-nucleotide spacer (DR-1). However, only half of the nucleotides
within each repeat sequence are strictly conserved, while others vary
considerably. Hence, additional cis and trans parameters other than
those dictated by the direct repeat and/or the PPARRXR
heterodimer, respectively, are presumably involved in binding and
transactivation mediated by the PPAR/RXR-PPRE transduction pathway.
Indeed, in the enoyl-CoA hydratase (37) and the malic
enzyme
genes, half repeats adjacent to the PPRE direct
repeat were shown to modulate PPAR/RXR binding to PPRE or
transactivation of PPRE-promoted transcription. In two other cases (e.g. P450IV (12) and malic enzyme
),
additional distant upstream promoter sequences were shown to be
involved in modulating the PPAR effect. Furthermore, putative PPAR/RXR
interacting proteins similar to those recently reported for the thyroid
hormone nuclear receptor (39) may modulate transcriptional
activity driven by the PPAR-PPRE basal unit.
Transcriptional suppression of Tf by HD/PP is essentially similar to that recently reported for the liver apolipoprotein C-III gene(17) . In both, the suppressive effect appears to be related to HNF-4-enhanced transcription of the concerned gene and to result from displacement of HNF-4 by PPAR/RXR from the HNF-4 element of the concerned gene together with HNF-4 suppression by HD/PP, resulting in its reduced liver availability. Transcriptional suppression by HD/PP therefore complements transcriptional transactivation induced by HD/PP, thus extending the scope of effects of HD/PP as pleiotropic gene modulators. Other HNF-4-activated genes should similarly be considered as candidates for transcriptional suppression by HD/PP.
The functional
significance of Tf suppression by xenobiotic HD/PP still remains to be
investigated. Tf suppression could result in a decrease in iron
availability and, if not compensated by increase in iron saturation or
in Tf receptors, could lead to anemia. Slight anemia has indeed been
observed in rats under conditions of subchromic treatment with HD/PP. ()Since HNF-4 has recently been shown to mediate
transcriptional activation of the erythropoietin gene by
hypoxia(40) , Tf suppression by HD/PP could be complemented by
erythropoietin suppression similarly mediated by the PPAR/RXR
transduction pathway. Moreover, Tf ferric reduction catalyzed by the
plasma membrane NADH reductase has recently been reported to initiate
intracellular alkalinization and mitogenic growth (reviewed in (1) ). Tf suppression by endogenous activators of the PPAR-PPRE
transduction pathway could therefore be biologically significant in
differentiation processes modulated by Tf availability.