(Received for publication, December 11, 1995)
From the
Human apolipoprotein E is a plasma lipoprotein that appears to play an important protective role in the development of atherosclerosis. While little is known about the regulation of apoE, recent studies have shown that cytokines repress apoE synthesis both in vivo and in vitro. Furthermore, we have recently shown that the endogenous apoE gene is negatively regulated by the nuclear trans-repressor BEF-1 in the human HepG2 cell line. In this study we demonstrate that treatment of HepG2 cells with the cytokine interleukin-1 and interleukin-6 resulted in the induction of an isoform of BEF-1, designated B1. The induction of the B1 isoform could be blocked by the protein kinase inhibitor staurosporine, suggesting that B1 is a phosphorylated form of BEF-1. As further support, the B1 isoform could also be induced by phorbol ester, and subsequently inhibited by staurosporine, implicating a role for protein kinase C-mediated phosphorylation. Quantitation of the levels of the BEF-1 isoforms, and studies in the presence of cyclohexamide, provided evidence for the phosphorylation of an existing intracellular pool of BEF-1, with no change in the total intracellular level. Under conditions that generated increased levels of the B1 isoform, there was a concomitant and proportional decrease in the level of apoE mRNA. The effect did not appear to be the result of improved binding to the apoE regulatory region as the DNA binding affinity of B1 was identical to native BEF-1. Our data suggest that the regulation of apoE by BEF-1 is modulated by differential phosphorylation, possibly through the protein kinase C pathway.
Apolipoprotein E (apoE), ()a primary constituent of
several classes of mammalian lipoproteins, functions in transport and
redistribution of cholesterol and other lipids among various cells in
the body(1, 2, 3) . There is mounting
evidence that apoE plays a major protective role in the development of
arteriosclerosis (4, 5, 6, 7, 8, 9) .
In addition, apoE has proposed roles in immunoregulation and in the
genesis of Alzheimer's disease (reviewed in (10) and (11) ).
The factors that regulate apoE expression from hepatic tissue, the primary source of this circulating lipoprotein, are not well understood. Using the human hepatoma cell line HepG2 as a model system, several groups have identified regulatory elements required for efficient expression of apoE(12, 13) , and we previously determined that the nuclear repressor factor BEF-1 negatively regulated apoE gene expression in these cells(14) . BEF-1 is a member of the NF-1 family of nuclear factors(15, 16) , and studies have demonstrated that both its nuclear level and DNA binding activity are regulated via intracellular signaling, as demonstrated by effects mediated through the viral oncogene E1a and through a tyrosine phosphorylation that is required for its DNA binding activity(15, 17) . In different cells, BEF-1 exists in two isoforms designated B1 and B2. While the functional role of each of these isoforms is unknown, phosphatase studies have provided evidence suggesting that they represent a difference in serine/threonine phosphorylation(17) . In confluent cultures of HepG2 cells, where apoE is under BEF-1-mediated repression(14) , only the B2 isoform is expressed.
In a recent study, hamster hepatic apoE mRNA expression has been demonstrated to be repressed by the cytokines IL-1 and tumor necrosis factor(18) . This result is consistent with previous studies demonstrating a similar repressive effect of IL-1 on macrophage apoE mRNA synthesis in culture(19) . Because cytokines, including IL-1, are known to induce phosphorylation of many proteins (including transcription factors) (reviewed in (20) and (21) ), we have sought to investigate phosphorylation of BEF-1 as a potential mechanism for cytokine action in the repression of the apoE gene in HepG2 cells.
In this paper, we
show that treatment of HepG2 cells with IL-1, as well as IL-6,
induces an isoform of BEF-1 (B1) that binds to the apoE regulatory
region with equal affinity of the uninduced B2 isoform. This induction
appeared to be due to increased phosphorylation, as B1 could also be
induced by phorbol ester, and the induction could be blocked by the
protein kinase inhibitor staurosporine. Furthermore we show that both
IL-1 and IL-6 suppress the synthesis of apoE, and with increasing
phosphorylation of BEF-1 we observed a proportional repression of apoE
mRNA. Our data suggest that differential phosphorylation of
trans-repressor BEF-1, possibly through the protein kinase C (PKC)
pathway, plays a role in cytokine induced apoE gene repression.
Figure 1:
BEF-1 B1 induction in IL-1-treated
HepG2 cells. A, HepG2 cells were plated the day before
treatment at a density of 10 cells/100-mm culture dish.
Cells were then treated with 10 ng/ml IL-1, incubated for 16-20
h, and nuclear extracts were prepared. An EMSA was performed using 1.0
µg of the untreated or IL-1-treated extracts and 0.32 pmol of the
5` end-labeled ABEF-1 oligonucleotide which contains the human apoE
regulatory sequences from -104 to -74. The BEF-1 isoforms
leading to the two distinct protein-DNA complexes in EMSAs were labeled
as B1 and B2. B, a gel mobility retardation assay was
performed with 3 µg of a cytokine-treated HepG2 cell nuclear
extract and 0.32 pmol of a 5` end-labeled ABEF-1 oligonucleotide as the
probe in a 10-µl binding reaction. The amount of B1 and B2 factor
bound by the
P-labeled ABEF-1 probe was determined in the
presence of increasing concentrations of unlabeled ABEF-1. For the
competition, 5
, 10
, 50
, and 100
molar
excesses of unlabeled ABEF-1 oligonucleotide was added to the binding
reactions. The counts per minute (cpm) bound to the 98-kDa protein was
determined using a Betascope 603 blot analyzer and plotted as cpm bound
in B1 and B2 versus molar excess of competitor
oligomer.
Figure 2:
IL-1 induction of BEF-1 B1 is due to
phosphorylation of BEF-1 B2. A, HepG2 cells were plated the
day before treatment, treated with 10 ng/ml IL-1 or 10 ng/ml IL-1 plus
100 nM STS for 16-20 h, and nuclear extracts were
prepared. An EMSA was performed using 0.5 and 1.0 µg of the
untreated or IL-1-treated extracts and 0.32 pmol (approximately
10 counts) of the 5` end-labeled ABEF-1 oligonucleotide. Lane 1, HeLa nuclear control extract; lanes
2-3, untreated; lanes 4-5, IL-1 treated; lanes 6 and 7, IL-1 plus staurosporine-treated. B, comparison of the effect of IL-1 and PMA on B1 induction.
Cells were treated with either IL-1 alone or IL-1 plus STS as above and
by PMA alone or PMA plus STS (100 nM). The amount of B1
present in nuclear extracts 16 h later was determined as
above.
In additional experiments, we quantitated the induction of B1 by IL-1 as well as by the PKC activator, phorbol 12-myristate 13-acetate (PMA). As shown in Fig. 2B, we consistently observed an induction of the B1 isoform by IL-1 that could be completely inhibited by STS. The treatment of HepG2 cells with PMA also resulted in an induction of the B1 isoform, although to a lesser extent than with IL-1 (Fig. 2B). Furthermore, the PMA-induced phosphorylation of BEF-1 was also inhibited by STS. While we cannot rule out the possibility of some other post-translational modification leading to induction of B1, the above data suggest that the cytokine induction of B1 in HepG2 cells is due to increased phosphorylation of B2, possibly acting through the PKC pathway. Furthermore, the relative lack of effect of STS on the low basal level of B1, compared with its block of induced phosphorylation, suggests that prephosphorylated BEF-1 is relatively long-lived
Figure 3:
IL-1
induction of B1 is due to phosphorylation of an existing pool. The
total amount of BEF-1 (A) and the relative amount of B1 and B2 (B) in IL-1-treated HepG2 cells were quantitated using a
Molecular Devices PhosphorImager and the Signal Analytics Corp. IPLab
gel software following EMSAs using 0.5-1.5 µg of untreated or
IL-1-treated HepG2 cell derived nuclear extracts and a P-labeled ABEF-1 probe. The data are the mean ±
S.E. of three independent experiments. C, cells were also
treated for 16 h with or without 1 µg/ml CHX and the level of B1
induction determined.
Further analysis was performed to compare the degree of BEF-1 phosphorylation and mRNA levels in cells treated with or without IL-1 or IL-6. As shown in Fig. 4A, we observed the expected increase in IL-1-induced phosphorylation; however, we observed an even greater degree of BEF-1 phosphorylation with IL-6 in this and repeated experiments. As indicated for IL-1 above, there was no change in the total level of BEF-1 following treatment of the cells with IL-6; only a shift in the degree of phosphorylation. As shown in Fig. 4B, we observed approximately a 50% decrease in apoE mRNA following IL-1 treatment but an 81% decrease following IL-6 treatment. The proportionally greater decrease in mRNA levels with increasing degree of phosphorylation, and no change in total BEF-1 binding activity, suggests that the B1 isoform is a more potent repressor than B2 at the BEF-1 repressor binding site in the apoE upstream regulatory region.
Figure 4: Increased phosphorylation of BEF-1 correlates with increased repression of apoE mRNA. HepG2 cells were treated with or without IL-1 or IL-6, incubated for 16-20 h, and the level of nuclear BEF-1 B1 and B2 isoforms (A) was determined as above, and apoE mRNA levels (B) were quantitated with a RPA using an antisense probe synthesized to the 3` end of apoE as described under ``Experimental Procedures.'' ApoE mRNA levels were normalized to human actin or human glyceraldehyde phosphate dehydrogenase mRNA levels. The data are presented as a percent of the control and are the result of the average ± the S.E. of three independent experiments. For each experiment, RPA analysis was performed in triplicate.
Figure 5:
Binding of BEF-1 B1 and BEF-1 B2 to apoE
regulatory region. Varying concentrations of the P-labeled
ABEF-1 probe were incubated with a nuclear extract derived from
IL-1-treated HepG2 cells, and EMSAs were performed as described in the
legend to Fig. 1. Dissociation rate constants were determined by
Enzfitter (Elsevier Biosoft), a weighted nonlinear, least square
regression analysis program.
Changes in cellular gene transcription patterns induced by
extracellular signals are an important part of many biological
processes (reviewed in (27) ), and protein phosphorylation
clearly has evolved as the most versatile post-translational
modification for situations where rapid modulation of transcription
factor activity is required in response to signals from receptors on
the cell surface (reviewed in (28) and (29) ).
Extracellular signaling molecules such as cytokines have been well
described as affecting gene expression by modulating the
phosphorylation of proteins directly involved in transcriptional
control (reviewed in (20) ). These effects have been well
described for the induction of expression mediated by NF B, c-Jun,
c-Fos, and NF-IL-6(29, 30) . In this study, we have
provided evidence that the nuclear repressor factor BEF-1 becomes
phosphorylated in response to cytokine activation of HepG2 cells, with
a resulting increase in its functional repressor activity.
We were
able to induce the phosphorylation of BEF-1 with IL-1, IL-6, and PMA.
In preliminary studies, we also have shown that transforming growth
factor- similarly induced the phosphorylation of BEF-1 generating
the B1 isoform. Thus, extracellular signals from several agents appear
to generate converging intracellular pathways that ultimately result in
phosphorylation of nuclear repressor BEF-1. The PMA induction, and
staurosporine inhibition of phosphorylation of B1, suggests PKC
involvement in the differential phosphorylation of BEF-1. Researchers
initially had attempted to implicate PKC in IL-1 signaling, since PMA,
which directly activates PKC, mimics many of the actions of
IL-1(21) . However, evidence has been presented questioning the
importance of PKC in IL-1 action. Many studies have shown that
inhibitors of protein kinase C such as staurosporine fail to block IL-1
responses in different cell types; and it has been suggested that
staurosporine may even up-regulate IL-1 receptors and thereby increase
the effect of IL-1 on some cells (reviewed in (21) ). In the
case of BEF-1, however, we clearly show that STS blocked the induction
by both cytokines and PMA. While our data would suggest that the
mechanism for phosphorylation of B1 by these different agents is
through a common pathway, it is possible that the induced
phosphorylation by each agent is mediated through different kinases
each inhibited by STS.
It is not clear how increased phosphorylation might facilitate the repressive activity of BEF-1 against apoE. Until we understand what factors bind to previously mapped elements in the apoE upstream regulatory region(12, 13, 31) , and how they interrelate with one another and BEF-1, we can only speculate on the molecular mechanism of apoE repression. As reviewed recently by Hill and Treisman(32) , a number of transcription factors contain signal-regulated transcription activation domains, and it is presumed that regulated phosphorylation facilitates their interaction with the basal transcriptional machinery or co-activator proteins. We have shown that the phosphorylation of BEF-1 does not alter its binding affinity for the DNA, suggesting that the effect on activity is likely a post-DNA binding mechanism. The increased repressor activity of phosphorylated BEF-1 on apoE gene expression possibly is the result of enhanced interactions with auxiliary transcription factors at the apoE promoter region. Overall, the balance between and interaction of various positively and negatively acting factors plays a critical role in controlling the expression of genes, and with regard to apoE, the differential phosphorylation of the nuclear repressor BEF-1 appears at least in part to play a role in determining its expression and response to cell signals. Understanding how factors such as BEF-1 control apoE gene regulation may aid in the development of agents to modulate its expression and ultimately in controlling implicated disease processes.