1Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles 900899141; and 2Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073
Submitted 18 September 2003 ; accepted in final form 1 December 2003
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ABSTRACT |
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perisinusoidal pericytes; peroxisome proliferator-activated receptor response element; extracellular signal-regulated kinase 1/2
PPAR is a nuclear receptor that heterodimerizes with retinoid X receptor (7, 22, 34). PPAR
binds to PPAR response elements (PPRE) of target gene promoters and transactivates them on binding by its ligands such as 15-deoxy-
12,14-prostaglandin J2 and synthetic thiazolidinediones (7, 34). Lately, PPAR
ligands have been reported to render various biological effects including increased insulin sensitivity, anti-inflammation, and macrophage differentiation (7, 22, 26). A critical role of enhanced PPAR
function by its ligands or its overexpression has been best demonstrated in fat cell differentiation (26, 34). Previously, we and others (24, 25) have reported that PPAR
mRNA expression was significantly decreased in HSC isolated from various animal models of liver fibrosis (i.e., toxins and bile duct ligation). During HSC activation in culture, a well-established in vitro model of HSC activation, PPAR
mRNA expression, also decreased (24, 25). More recently, forced expression of PPAR
in culture-activated HSC was shown to revert its morphology to a more quiescent phenotype (17). These reports suggest an important role of PPAR
in maintenance of the quiescent HSC phenotype.
In response to liver injury, platelets and liver macrophages (i.e., Kupffer cells) produce and secrete increased amounts of cytokines and growth factors (8, 9, 23). TNF- is one such cytokine that is known to play pleiotropic functions in cells. Intracellular signaling pathways for TNF-
are extremely complex and can lead to multiple cell responses including cell proliferation, inflammation, or cell death (3, 14). Currently, TNF-
, on its interaction with its receptor, is known to use two distinct intracellular signaling pathways including the NF-
B pathway and MAP kinase pathway (JNK and p38 kinase) (14, 33). Here, we investigated molecular mechanisms underlying TNF-
-mediated regulation of PPAR
activity employing an HSC cell line BSC. We report that TNF-
inhibits PPAR
binding to PPRE (DNA).
In BSC, TNF- stimulates ERK1/2 and JNK and inhibits PPAR
transactivity in a manner dependent on Ser82 of PPAR
.
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MATERIALS AND METHODS |
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pCDNA3 empty and pCDNA3-human PPAR1 expression vectors were gifts from V. K. K. Chatterjee (Univ. of Cambridge, Cambridge, UK). pStud-mouse PPAR
1 and pStud-mouse Ser82Ala-PPAR
1 expression vectors were gifts from S. R. Tafuri (Warner-Lambert C., Ann Arbor, MI). NF-
B luciferase construct containing five repeats of the consensus NF-
B binding sequence was purchased from Strategene (La Jolla, CA). Other chemicals were from Sigma (St. Louis, MO) unless specified otherwise.
Cell culture. BSC cells were originated from rat HSC isolated from experimental biliary liver fibrosis rendered by 18-day bile duct obstruction (35). The spontaneously immortalized HSCs were maintained in DMEM plus 10% FBS and passed at a dilution of 1:3 every week until characterization was performed. The colonization activity and cell viability of BSC were evaluated, and subclones were established by the limiting dilution method on the 63rd passage. Trypsinized BSC were diluted and replated onto 96-well microtiter plates (Costar, Corning, NY) with one cell per well in 200 µl DMEM supplemented with 10% FBS, antibiotics, and 10% cell-free conditioned medium from original BSC culture. After 3 wk of culture, the number of colonies from single cells was counted and growing clones were trypsinized with 0.05% trypsin-EDTA (GIBCO, Carksbad, CA) and subcultured in DMEM containing 10% FBS and antibiotics in 24-well plates and were subsequently transferred to six-well plates (Costar, Corning, NY) for expansion. A total of 74 clonal cell lines were obtained in this manner. One of the clones, designated BSC-C10, was characterized by immunofluorescent microscopy and Northern blot analysis or RT-PCR for expression of HSC markers at protein and mRNA levels. The BSC-C10 clone expresses markers for HSC such as glial acidic fibrillary protein, desmin, -smooth muscle actin, synaptophysin, vascular cell adhesion molecule, neural cell adhesion molecule. BSC cells were maintained in low glucose DMEM supplemented with 10% FBS, 100 mg/ml streptomycin, 10,000 U/ml penicillin, and 25 µg/ml amphotericin B.
Isolation of HSC. HSC were isolated from normal male Wister rats as previously described (24) by the Non-Parenchymal Liver Cell Core of the Univ. of Southern California (USC)-Univ. of California Los Angeles Research Center for Alcoholic Liver and Pancreatic Diseases. The purity of isolated HSC was examined by phase-contrast microscopy and ultraviolet-excited fluorescence microscopy and viability based on trypan blue exclusion. Freshly isolated rat HSC were cultured on 100-mm culture dishes in low-glucose DMEM supplemented with 10% FBS.
Real Time RT-PCR. Total RNA was extracted from BSC treated for 18 h with either TNF- (20 ng/ml) or GW1929 (1 µM) or both using Trizol reagent. Total RNA (50 ng each for PPAR
and 5 ng each for GAPDH) was reverse-transcribed at 48°C for 30 min followed by 40 cycles of PCR using TaqMan Gold RT-PCR kit (Applied Biosystems, Foster City, CA). Probes used were 5,6-carboxyfluorescein amidite labeled at the 5' end and black hole quencher-1 labeled at the 3' end (Biosearch Technologies, Novato, CA). ABI PRISM 7900 HT machine (Molecular Biology Core, USC Liver Center) was used to detect the threshold cycle (Ct). Each Ct value for PPAR
was first normalized to the respective Ct value for GAPDH and subsequently to a control (i.e., reference) sample.
Electrophoretic mobility shift assay. BSC were treated in serum-free DMEM containing 0.1% BSA for 18 h with either TNF- (20 ng/ml) or GW1929 (1 µM) or both. Nuclear extracts were then prepared as previously described by Schreiber et al. (29). Nuclear extracts (15 µg each) were preincubated on ice for 15 min in a reaction mixture containing (in mM) 20 HEPES (pH 7.6), 100 KCl, 0.2 EDTA, and 2 DTT with 20% glycerol and 200 µg/ml poly(dI-dC) and incubated for 30 min with 12ngof
32P-labeled double-stranded ARE-7 (the PPRE from AP2 gene). For a supershift assay, antibodies to PPAR
were added to reaction mixture and incubated for an additional 20 min. The reaction mixture was then resolved by a 6% PAGE followed by autoradiography.
Transient transfection and luciferase reporter assay. For PPRE promoter activity assays, BSC in six-well plates (100,000 cells/well) were transfected with PPRE-luciferase construct (tk-PPRE X 3-luciferase; 1 µg/well) and expression vectors (1 µg/well), either empty vector or PPAR1-containing vector. For NF-
B promoter activity assays, BSC were transfected with an NF-
B-luciferase construct (0.5 µg), pCDNA3-PPAR
1 expression vector (0.5 µg), and pCMX expression vector, either empty vector (1 µg) or Ser32Ala/Ser36Ala mutant of I
B
-containing vector (0.51 µg). For the assessment of transfection efficiency, renilla phRL-TK vector (2 ng/well) was used (Promega, Madison, WI). A nonlipid cationic Targefect F2 reagent (Targeting Systems) was used at 1:1 molar ratio of total DNA (µg) and volume of F2 (µl). After 24 h of transfection, the cells were treated in serum-free DMEM containing 0.1% BSA for 1618 h with or without TNF-
(20 ng/ml) in the presence or absence of a selective PPAR
ligand GW1929 (1 µM). Next, the cells were washed in PBS three times and solubilized in 1x passive lysis buffer (Promega, Madison, WI), and cell lysates were assayed for both firefly (PPRE-luciferase or NF-
B-luciferase) and renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Ratios of firefly luciferase activity and renilla luciferase activity were calculated.
Preparation of cell lysates and Western blot analysis. To assess PPAR protein levels, BSC in 100-mm dishes were treated for 18 h with TNF-
(20 ng/ml) or GW1929 (1 µM) or both in serum-free DMEM containing 0.1% BSA, washed in PBS three times, and solubilized for 30 min in modified RIPA buffer (50 mM Tris·HCl, pH 8, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, 2 mM Na3VO4, 10 mM NaF) containing a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). To assess phosphorylation and protein levels of ERK and JNK, BSC in 100-mm dishes were treated for 510 min with or without TNF-
(20 ng/ml). After centrifugation in a microcentrifuge (15,000 g, 15 min), soluble supernatants (i.e., cell lysates) were transferred into new tubes and resolved by Western blot analysis. Briefly, cell lysates (25 µg) were resolved by SDS-PAGE followed by electrophoretic transfer of proteins onto nitocellulose membranes. The membranes were then probed with appropriate antibodies, and signals were detected by the hydrogen peroxide-enhanced chemiluminescence (ECL) method using the Pierce ECL kit (Amersham, Arlington Heights, IL) as previously described (18).
Statistical analysis. Data are presented as means ± SE of several experiments. Statistical analyses were performed by two-tailed Student's t-test.
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RESULTS |
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To ascertain this finding in BSC, an HSC-derived cell line, we treated BSC for 18 h with TNF- in the presence and absence of a selective PPAR
ligand GW1929 and measured PPAR
mRNA levels by real-time RT-PCR. Treatment of BSC with GW1929 increased PPAR
mRNA level about twofold (Fig. 1B). TNF-
decreased both basal and GW1929-induced PPAR
mRNA levels by 9095%. These data suggest that BSC responds to TNF-
in a similar manner to primary HSC.
To determine whether the decreased PPAR mRNA level by TNF-
is accompanied by decreased PPAR
protein expression, we examined PPAR
protein levels by Western blot analysis (Fig. 1C). Interestingly, TNF-
treatment for 18 h did not affect PPAR
protein levels despite diminished PPAR
mRNA levels (Fig. 1C).
TNF- decreases basal and GW1929-induced PPAR
-PPRE (DNA) binding in BSC. PPAR
is a transcription factor that binds to PPRE of target gene promoters in nucleus, leading to regulation of gene expression (7, 22, 34). To determine whether TNF-
alters PPAR
-DNA binding in BSC, we prepared nuclear extracts from BSC treated for 18 h with TNF-
in the presence and absence of GW1929 and performed the electrophoretic mobility shift assay using a labeled double-stranded oligonucleotides containing the (ARE7) PPRE as described in MATERIALS AND METHODS. The (ARE7) PPRE preferentially binds PPAR
over PPAR
(21). Treatment of BSC with GW1929 enhanced PPAR
-PPRE binding, and TNF-
decreased both basal and GW1929-induced PPAR
-PPRE binding (Fig. 2). Specificity of PPAR
in the protein-PPRE (DNA) complex was confirmed by disappearance of the band when the reaction mixture was incubated with antibodies to PPAR
(Fig. 2). Control rabbit IgG failed to affect PPAR
-PPRE complex formation.
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TNF- decreases transcriptional activity of PPAR
in BSC. To determine whether TNF-
affects PPAR
-mediated transactivation of PPRE in BSC, we transiently transfected the cells with a PPRE-luciferase reporter construct and a PPAR
1 expression vector. After 24 h of transfection, the cells were treated for 18 h with TNF-
in the presence and absence of GW1929. In BSC cotransfected with an empty expression vector, TNF-
significantly decreased GW1929-induced transactivity of endogenous PPAR
(Fig. 3). Overexpression of PPAR
in BSC increased the PPRE promoter activity fivefold, and GW1929 further increased this activity threefold. In these cells, TNF-
treatment significantly decreased both basal (GW1929-independent) and GW1929-induced PPRE promoter activity by 32% and 42%, respectively (Fig. 3). These data suggest that TNF-
inhibits PPRE transactivation by both endogenous and exogenous PPAR
in the presence and absence of the PPAR
ligand GW1929. These inhibitory effects of TNF-
are in agreement with those on PPAR
-PPRE (DNA) binding as shown in Fig. 2.
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TNF- increases activation-specific phosphorylation of ERK1/2 and JNK in BSC. The A/B domain of PPAR
contains a single consensus MAP kinase phosphorylation site that becomes phosphorylated by ERK1/2 and JNK, resulting in decreased transcriptional activity of PPAR
(1, 4, 20). To assess this possibility, we first examined whether these MAP kinases are phosphorylated by TNF-
as the evidence of their activation in BSC. We treated the cells with TNF-
for 510 min, and cell lysates were resolved by Western blot analysis. TNF-
increased activation-specific phosphorylation of both ERK1/2 (Thr202/Tyr204) and JNK (Thr183/Tyr185) in BSC (Fig. 4). TNF-
, however, failed to affect phosphorylation of p38 MAP kinase in these cells (data not shown). These data suggest that TNF-
potentially phosphorylates PPAR
in BSC via activation of ERK1/2 and JNK that may lead to inhibition of transactivation of PPAR
.
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TNF- fails to inhibit PPRE transactivation by Ser82Ala mutant form of PPAR
in BSC. To ascertain the role of PPAR
phosphorylation by TNF-
-induced MAP kinase on PPRE transactivation, we performed transient transfection experiments employing a Ser82Ala-PPAR
1 mutant expression vector together with the PPRE-luciferase reporter construct. It should be noted that HSC express predominantly PPAR
1 isoform but not the adipocyte-specific PPAR
2 isoform (25). Serine at 82 amino acids of the mouse PPAR
1 (equivalent to serine at 84 amino acids of the human PPAR
1) is the single MAP kinase consensus recognition site that is indeed phosphorylated by ERK1/2 (4) and JNK (5), resulting in inhibition of PPAR
1 activity. In BSC, overexpression of Ser82Ala-PPAR
1 mutant achieved higher PPRE promoter activity than the wild-type PPAR
1 (the first set of bars in Fig. 5). GW1929 enhanced the PPRE promoter activity mediated by both mutant and wild-type PPAR
, and the mutant still displayed greater PPRE transactivation (the second set of bars). In these cells, TNF-
, however, failed to inhibit transactivation of Ser82Ala-PPAR
1, whereas the inhibition was reproduced in the cells overexpressing wild-type PPAR
1. These data indicate that Ser82 phosphorylation of PPAR
1 is important for inhibitory action of TNF-
on ligand-induced PPAR
activity in BSC.
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Inhibitory action of TNF- on PPAR
transactivation is independent of the NF-
B-pathway in BSC. In addition to the MAP kinase cascade, the NF-
B pathway has been reported to be another major TNF-
signaling pathway in cells (33). To determine whether the NF-
B pathway plays a role in inhibitory action of TNF-
in PPAR
activity in BSC, we employed Ser32Ala/Ser36Ala mutant of NF-
B inhibitory protein
(I
B
) (4). This phosphorylation mutant form of I
B
fails to be phosphorylated by I
B kinase and thus constitutively binds to NF-
B in the cytoplasm preventing nuclear translocation of NF-
B, resulting in inhibition of the NF-
B signaling pathway (14). First, we determined the effects of Ser32Ala/Ser36Ala I
B
mutant on the NF-
B signaling pathway by assessing NF-
B promoter activity. In BSC, expression of Ser32Ala/Ser36Ala I
B
mutant inhibited NF-
B promoter activity, reaching maximal 8090% inhibition at 0.51 µg of construct (Fig. 6B). Next, we cotransfected cells with the PPRE-luciferase construct and expression vectors for PPAR
1 and Ser32Ala/Ser36Ala I
B
and measured PPRE luciferase activity following treatment of cells with TNF-
and GW1929. In control BSC transfected with pCMX empty expression vector for Ser32Ala/Ser36Ala I
B
, TNF-
inhibited both basal and GW1929-induced PPRE promoter activity as previously described (Fig. 6A). Expression of Ser32Ala/Ser36Ala I
B
(i.e., inhibition of the NF-
B pathway) in BSC moderately increased basal PPRE promoter activity. In these cells, TNF-
could still inhibit both basal and GW1929-induced PPRE promoter activity to an extent similar to that in control cells (Fig. 6A). These data suggest that inhibitory action of TNF-
on PPAR
transactivation in BSC is not altered by inhibition of the NF-
B pathway. Increased basal PPRE promoter activity by expression of Ser32Ala/Ser36Ala I
B
(i.e., inhibition of the NF-
B pathway) suggests that the NF-
B pathway may play a negative role in basal transactivity of PPAR
.
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DISCUSSION |
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Chronic injury leading to fibrosis in liver occurs in response to various insults including viral hepatitis, alcohol abuse, drugs, metabolic diseases due to overload of iron and copper, and autoimmune attacks of hepatocytes or bile duct epithelium (8, 9, 23). HSC is now well established as the key cellular element involved in the development of hepatic fibrosis (8, 9, 23). In response to various insults, normally quiescent vitamin A-storing HSC undergoes a phenotypic transformation termed "activation" or "transdifferentiation" to become actively proliferating myofibroblast-like cells. HSC activation involves the coordinated activity of several key transcriptional regulators of the HSC genome including NF-B, AP-1, Kruppel-like transcription factor 6, C/EBP, and E-box transcription factors (23). In contrast to these transcription factors associated with the activated phenotype of HSC, PPAR
has been identified as a transcription factor that is active in quiescent HSC, but its activity decreases in activated HSC (24, 25). In both primary HSC from cholestatic liver fibrosis animal model (25) and culture-activated HSC (24, 25), PPAR
mRNA expression and PPRE binding were greatly diminished when compared with control quiescent HSC. Treatment of activated HSC with PPAR
ligands restored PPAR
expression and diminished various biochemical markers associated with fibrosis (13, 25). Moreover, oral administration of synthetic PPAR
ligands, thiazolidinediones, in animal models of liver fibrosis (toxin administration or bile duct ligation) was shown to retard fibrosis in vivo (13). These studies strongly support the therapeutic potential of PPAR
ligands in liver fibrosis.
Because decreases in PPAR expression level and its activity are correlated with and seem to precede HSC activation (i.e., phenotypic changes and increases in fibrogenic activation markers), it is important to understand how PPAR
is regulated in HSC. In response to liver injury, liver macrophages are activated to release various cytokines including TNF-
. Thus we initiated this study to determine molecular mechanisms underlying TNF-
-mediated PPAR
regulation in HSC. In the present study, we demonstrated that inhibitory action of TNF-
on PPAR
activity in BSC was at the posttranslational level. Although TNF-
greatly diminished mRNA levels of PPAR
in BSC, it failed to alter PPAR
protein level. This discrepancy between mRNA and protein levels of PPAR
was rather puzzling. To this end, complex regulation of PPAR
expression by growth factors and cytokines has recently been reported. In particular, TGF-
was shown to exert a biphasic effect on PPAR
mRNA expression in human aortic smooth muscle cells (early stimulation and late repression) (11). A short-term treatment (12 h) of these cells with TNF-
was also shown rather to increase PPAR
expression (10). PDGF regulation of PPAR
was more complex in that PPAR
expression was increased at 2 h but declined to some extent at 6 h and rose again at 12 h to achieve a second peak that was sustained for 72 h (12). To make the matter even more complex, Hauser et al. (15) reported that PPAR
protein levels are significantly decreased in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones, which involves proteasome-mediated PPAR
degradation. All these reports indicate that complex regulation of PPAR
expression occurs in cells in response to various signals. It is noteworthy that discrepancy between levels of mRNA and protein has been previously reported for NADH cytochrome P-450 oxidoreductase following thyroid hormone treatment in liver (2) and for the small RING-finger protein SNURF/RNF4 in testis (28).
Counteracting potential of TNF- on PPAR
function has previously been reported. TNF-
causes insulin resistance in insulin target cells, whereas treatment of insulin-resistant animals with PPAR
ligands ameliorates insulin resistance (19, 26, 27). TNF-
has also been reported to inhibit fat cell differentiation, whereas PPAR
is a potent positive regulator of this function and PPAR
overexpression facilitates fat cell differentiation (30, 31). Our present study suggests that TNF-
may exert some of its action to counteract PPAR
activity at a posttranslational level in cells.
PPAR has two isoforms,
1 and
2, that originate from the same gene but result from differential promoter usages with a 30-amino acid difference at the NH2 terminus (26, 36). HSC express predominantly PPAR
1 but not adipocyte-specific PPAR
2 (25). The NH2-terminal A/B domain of these receptors contains a single consensus MAP kinase site (Ser82 and Ser112 of mouse PPAR
1 and
2, respectively). On their phosphorylation by growth factor-activated ERK1/2, their transcriptional activities decrease (1, 4, 20). Mutation of this phosphorylation site has been reported to greatly augment PPAR
transcriptional activity (1, 4, 20) and to enhance fat cell differentiation (20). The mutated PPAR
was also reported to be resistant to growth factor-mediated inhibition of its transcriptional function. Moreover, purified PPAR
protein was directly phosphorylated in vitro by recombinant activated ERK2 (4). Later, JNK was also shown to efficiently phosphorylate PPAR
at the same Ser82 site (5). In BSC, we found that TNF-
stimulates both ERK1/2 and JNK and inhibitory action of TNF-
on transactivation of PPAR
is abolished when the cells express the Ser82Ala-PPAR
1 mutant, suggesting the importance of PPAR
phosphorylation in the inhibitory action of TNF-
. Now, the question arises as to whether both ERK1/2 and JNK play a role in PPAR
phosphorylation and resultant inhibition of its function in BSC. Our preliminary study with the MEK/ERK inhibitor PD-98059 suggests that ERK1/2 may be responsible for PPAR
phosphorylation and subsequent inhibition of its transcriptional activity, although the role of JNK in PPAR
regulation cannot be completely ruled out (data not shown). Further analysis with dominant negative forms of these enzymes would be needed to confirm our interpretation. In BSC, TNF-
also inhibits PPAR
-PPRE (DNA) binding. However, this is most likely independent of PPAR
phosphorylation, because PPAR
phosphorylation has been reported to not alter PPAR
-DNA binding (1, 4). These results led us to propose two levels of PPAR
regulation by TNF-
in BSC: one level at PPAR
phosphorylation and the other level at decreased PPAR
-DNA binding.
Additional support for TNF--induced inhibition of PPAR
-DNA binding, being independent of PPAR
phosphorylation, comes from the recent study (32) with mesenchymal stem cell line ST2, in which PPAR
was reported to form a complex with NF-
B that, in turn, blocked PPAR
-DNA binding following TNF-
treatment. Overexpression of NF-
B-inducing kinase (i.e., increased NF-
B level in the nucleus) inhibited ligand-induced transactivation of PPAR
. This inhibitory action of NF-
B-inducing kinase on PPAR
transactivation was still observed when cells were cotransfected with Ser112Ala-PPAR
2 mutant (equivalent to Ser82Ala PPAR
1) (32). These data may be interpreted to mean that overexpression of NF-
B could inhibit PPAR
activity regardless of its phosphorylation status. To determine the contribution of the NF-
B signaling to PPAR
activity in BSC, we took a different approach to decrease endogenous NF-
B signaling by expression of the phosphorylation mutant form of I
B
(to increase I
B
-NF-
B complex formation in cytoplasm). In these cells, we observed increased basal transcriptional activity of PPAR
that is consistent with a negative role of NF-
B in PPAR
activity. In these cells, TNF-
was capable of inhibiting PPAR
activity as well as in control cells possessing a higher level of NF-
B signaling. Our data, together with results of Suzawa et al. (32), suggest that TNF-
could exert its inhibitory action on PPAR
activity by either PPAR
phosphorylation or inhibition of PPAR
-DNA binding that is independent of each other. Depending on magnitude of NF-
B signaling in cells, TNF-
may differentially use one mechanism over the other.
Taken together, we propose a working model for TNF--mediated regulation of PPAR
activity in HSC (Fig. 7). Treatment of the cells with TNF-
causes activation of ERK and JNK following the known complex formation of TNF receptor with various intracellular adaptor proteins (6). Ser82 phosphorylation of PPAR
by ERK or/and JNK results in decreased PPAR
transcriptional activity, and this molecular change underlies activation of HSC. TNF-
also diminishes PPAR
-DNA binding to further decrease PPAR
activity. In summary, TNF-
inhibits PPAR
activity at a posttranslational level in the HSC-derived cells. TNF-
inhibits PPAR
activity, at least in part, by inhibition of PPAR
-PPRE (DNA) binding and appears to also involve ERK1/2-mediated phosphorylation of Ser82-PPAR
1 but not the NF-
B pathway.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants R37AA-06603, P50AA-11199 (USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases), and R24AA-12885 (Nonparenchymal Liver Cell Core) and by the Medical Research Service of the Department of Veterans Affairs.
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FOOTNOTES |
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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.
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REFERENCES |
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