Protein kinase C α modulates liver X receptor α transactivation

in Journal of Endocrinology

Liver X receptor α (LXRα), an oxysterol-activated nuclear hormone receptor, regulates the expression of genes involved in lipid and cholesterol homeostasis and inflammation. We show here that transactivation by LXRα in monkey kidney COS-1 (Cos-1) cells is decreased by activation of the protein kinase C (PKC) signaling pathway. In transient co-transfection assays, phorbol myristate acetate (PMA) suppressed LXR-dependent transactivation of LXR-responsive reporter genes or the natural promoter of the human ATP-binding cassette (ABC), ABCA1 gene. The decrease in LXR transactivation after PMA treatment was also observed in human embryonic kidney (HEK) 293 and human hepatocellular carcinoma (HepG2) cells. Moreover, endogenous LXR target genes, ABCA1 and sterol response element-binding protein-1c, were also decreased by PMA treatment in HEK293 cells as assessed by real-time PCR. The PMA-mediated decrease of LXR activity was blocked by the PKC inhibitor bisindolylmaleimide and mimicked by constitutively active PKCα. Nuclear extracts treated with PMA show no decrease in LXRα DNA binding as assessed by mobility shift and chromatin immunoprecipitation assays. Additionally, in vitro kinase assays demonstrate that PKCα can phosphorylate LXRα. Our findings reveal a mode of regulation of LXRα that may be relevant to disease conditions where aberrant PKC signaling is observed, such as diabetes.

Abstract

Liver X receptor α (LXRα), an oxysterol-activated nuclear hormone receptor, regulates the expression of genes involved in lipid and cholesterol homeostasis and inflammation. We show here that transactivation by LXRα in monkey kidney COS-1 (Cos-1) cells is decreased by activation of the protein kinase C (PKC) signaling pathway. In transient co-transfection assays, phorbol myristate acetate (PMA) suppressed LXR-dependent transactivation of LXR-responsive reporter genes or the natural promoter of the human ATP-binding cassette (ABC), ABCA1 gene. The decrease in LXR transactivation after PMA treatment was also observed in human embryonic kidney (HEK) 293 and human hepatocellular carcinoma (HepG2) cells. Moreover, endogenous LXR target genes, ABCA1 and sterol response element-binding protein-1c, were also decreased by PMA treatment in HEK293 cells as assessed by real-time PCR. The PMA-mediated decrease of LXR activity was blocked by the PKC inhibitor bisindolylmaleimide and mimicked by constitutively active PKCα. Nuclear extracts treated with PMA show no decrease in LXRα DNA binding as assessed by mobility shift and chromatin immunoprecipitation assays. Additionally, in vitro kinase assays demonstrate that PKCα can phosphorylate LXRα. Our findings reveal a mode of regulation of LXRα that may be relevant to disease conditions where aberrant PKC signaling is observed, such as diabetes.

Introduction

The liver X receptors (LXRs; α and β subtypes) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and are activated by oxidized cholesterol metabolites known as oxysterols (Janowski et al. 1996). LXR functions as an important regulator of cholesterol homeostasis and also acts as a glucose sensor and modulator of innate immunity and inflammation (Joseph et al. 2003, 2004, Mitro et al. 2007). The LXRs function by regulating transcription of target genes by binding to LXR response elements (LXREs) as obligate heterodimers with retinoic X receptor α (RXRα; Willy et al. 1995). Since their discovery, multiple LXR target genes that are involved in cholesterol and lipid transport and homeostasis have been identified. These include the ATP-binding cassette (ABC) transporters, ABCA1, ABCG1, ABCG5 and ABCG8, lipoprotein and lipoprotein-remodeling enzymes such as apolipoprotein E, lipoprotein lipase, cholesterol ester transfer protein as well as lipogenic proteins such as sterol response element-binding protein (SREBP)-1c, and fatty acid synthase (Costet et al. 2000, Mak et al. 2002a,b, Ulven et al. 2004). Despite increasing knowledge of the physiological function and mechanisms of action of LXR, little is known about the mechanisms by which LXR itself is regulated.

The function of nuclear hormone receptors can be regulated at multiple levels, including transcriptional regulation and post-transcriptional modification such as phosphorylation. The LXR has been shown to exist as a phosphoprotein in human embryonic kidney (HEK) 293 cells; however, the functional consequence of phosphorylation is currently unclear (Chen et al. 2006). Protein kinase A (PKA) can directly phosphorylate LXRα in cultured cells and has been reported to both increase and decrease transactivation depending on cell type (Tamura et al. 2000, 2004, Yamamoto et al. 2007). In primary rat hepatocytes, PKA signaling blocks LXR activation of the SREBP1c promoter (Yamamoto et al. 2007). However, Tamura et al. (2000) have also demonstrated that PKA signaling can increase LXR transactivation on a consensus LXRE-reporter construct in renal As4.1 mouse cell lines. Therefore, LXR activity appears to be differentially modulated by phosphorylation depending on promoter context and cell type. Indeed, this has been shown to be the case for the functionally related nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). The PPARγ transactivation is repressed by MAPK signaling in adipocytes while the opposite is true in Chinese hamster ovary cells (Hu et al. 1996, Juge-Aubry et al. 1999).

The PKC family of serine/threonine kinases is composed of at least ten members (α, βI, βII, γ, δ, ε, η, θ, ζ, and λ). The multiple isoforms are classified based on the requirements for activation, which depends on the presence or absence of domains that bind to calcium or the lipid diacylglycerol (DAG; Dempsey et al. 2000). The PKC signaling pathway is activated by a broad spectrum of extracellular stimuli that promote lipid hydrolysis and plays a fundamental role in numerous biological facets such as differentiation, proliferation, apoptosis, and neuronal transmitter release (Newton 2003). PKC signaling affects the activity of multiple nuclear receptors including the androgen receptor (AR), glucocorticoid receptor, vitamin D receptor (VDR), PPAR, and the retinoic acid receptor (RAR; Darne et al. 1998, Rigas et al. 2003). Whereas PKC signaling increases the activity of some nuclear receptors such as AR and PPARα, it has also been shown to repress VDR and RARα activity (Hsieh et al. 1991, 1993, Delmotte et al. 1999).

In order to explore the potential role of PKC signaling on LXR function, we examined the activity of LXR in transient transfection assays in the presence of modulators of PKC signaling pathways. We show here that activation of PKC signaling with phorbol 12-myristate 13-acetate (PMA) repressed LXR-dependent transactivation of LXRE-reporter plasmids as determined by transient transfection assays and activation of endogenous LXR target genes as determined by real-time PCR. The effect of PMA was both dose- and time-dependent and could be mimicked by constitutively active PKCα. The inhibitory effects were abrogated upon co-incubation with the PKC inhibitor bisindolylmaleimide. Finally, PKCα was shown to phosphorylate LXRα in vitro. These findings reveal that PKC activation can regulate LXR-mediated gene expression and may have implications in diseases where PKC signaling is altered (Rask-Madsen & King 2005).

Materials and Methods

Reagents and plasmids

22R-hydroxycholesterol (22R-HC), 9-cis-retinoic acid (9-cisRA), PMA, and GW3965 were purchased from Sigma. Bisindolylmaleimide was purchased from Calbiochem (San Diego, CA, USA). Rabbit antibody to human RXRα was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and mouse antibody to human LXRα was purchased from R&D Systems (Minneapolis, MN, USA). The rabbit LXRα/β antibody (sc-1000) used for chromatin immunoprecipitation (ChIP) was purchased from Santa Cruz Biotechnology. Mammalian expression vectors expressing human LXRα, LXRβ, PPARα, and RXRα (pRC-CMV-LXRα, pRC-CMV-LXRβ, pSG5-PPARα, and pSG5-RXRα respectively) and luciferase reporter plasmids harboring response elements for LXR and PPAR have been described previously (Willy et al. 1995, Meertens et al. 1998, Miyata et al. 1998, Landis et al. 2002). PKC7, expressing constitutively active PKCα, was a generous gift from Dr J L Staudinger (University of Kansas, Lawrence, KS, USA). Human ABCA1 promoter (−928/+107) luciferase reporter plasmid was a generous gift from Dr A Tall (Columbia University, New York, NY, USA).

Cell lines

Cos-1, HEK293, and HepG2 cells were obtained from American Type Tissue Collection (ATCC, Manassas, VA, USA). Cos-1 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin/streptomycin, and 1% v/v l-glutamine. HepG2 cells were cultured in MEM-F15 supplemented with 10% v/v fetal bovine serum, 1% v/v sodium pyruvate, 1% v/v l-glutamine, and 1% penicillin/streptomycin.

Transfection and reporter gene assays

Transient transfections of cells were carried out using FuGene 6 reagent (Roche) according to manufacturer's instructions. Briefly, cells (3.5×105 cells/well in six-well plates) were transfected using 3 μl Fugene to 0.5 μg reporter plasmid, 0.25 μg pRC-CMV-LXRα, 0.25 μg pSG5-RXR, and 0.1 μg pCMV-lacZ (encoding β-galactosidase) for normalization of transfection efficiency. The total amount of DNA was kept constant using pRC-CMV and pSG5 empty vectors. Following transfection, the media was aspirated and replaced with complete DMEM supplemented with 10% charcoal-stripped FBS and appropriate ligand as indicated in the figures. LXR agonist 22(R)-hydroxycholesterol was dissolved in 95% ethanol, and 9-cisRA and GW3965 were dissolved in Me2SO (DMSO). Control cells received the equivalent amount of vehicle. Cells were harvested for 24 or 48 h post-transfection as indicated and lysed in reporter lysis buffer (Promega), and luciferase, β-galactosidase and Bradford assays were carried out as described previously (Landis et al. 2002).

Electrophoretic mobility shift assays

The Cos-1 cells were transfected with 0.5 μg LXRα and/or RXRα expression plasmids and incubated overnight, as described previously. The cells were then treated with PMA for an additional 24 h and Cos-1 nuclear extracts were prepared, as described previously (Andrews & Faller 1991). Ten micrograms of nuclear extract were used in gel retardation assays, as previously described (Landis et al. 2002). Briefly, double-stranded oligonucleotide probes (sequence 5′-GATCCCAGGTCACAGGAGTCAGAA-3′; 5′-GATCTTCTGACCTCCTGTGACCTGG-3′) were annealed and radiolabeled with [32P] ATP using Klenow enzyme. Binding reactions consisted of 5 μl Buffer C (20 mM HEPES (pH 7.9); 25% Glycerol; 0.42 M NaCl; 0.2 mM EDTA (pH 8.0); 1.5 mM MgCl2; 0.5 mM dithiothreitol), 1 μl BSA (4 mg/ml), 1 μl PolydI/dC (8 mg/ml), 1 μl salmon sperm DNA (4 mg/ml), 4 μl radiolabeled oligo (20 pmol/reaction), 10 μg nuclear extract, and H2O to 30 μl total volume. Reactions were incubated at 30 °C for 1 h and stopped by the addition of 2 μl loading dye (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol). Samples were separated on a 4% polyacrylamide gel (pre-run for 2 h) in 0.25× Tris-borate-EDTA (TBE) running buffer at 240 V at 4 °C. The gel was dried and probes were detected by autoradiography.

Chromatin immunoprecipitation assay

ChIP assays were performed in HEK293 cells using the ChIP-IT Express Enzymatic Kit according to manufacturer's instructions (Active Motif, Carlsbad, CA, USA). Briefly, HEK293 cells were grown to 85% confluency in 10 cm dishes and treated with GW3965 (2 μM) and/or PMA (80 nM) for 2 h. Cells were fixed with 1% formaldehyde for 10 min at room temperature. Nuclei were isolated and chromatin was digested enzymatically for 10 min at 37 °C resulting in chromatin fragments of ∼200–1500 bps. LXRα/β was immunoprecipitated overnight at 4 °C with 3 μg antibody and ∼8 μg chromatin, and rabbit pre-immune immunoglobulin served as a negative control. Bound chromatin was washed and the recovered DNA was assayed for enrichment of the human ABCA1 promoter by PCR using the following primers: forward 5′-CCC AAC TCC CTA GAT GTG TC-3′; reverse 5′-CCA CTC ACT CTC GCT CGC A-3′. The PCR contained 5 μl eluted chromatin, 2.5 μl PCR buffer, 10 pmol/μl primers (forward and reverse), 1 U/reaction platinum Taq polymerase (Invitrogen) and H2O to 25 μl. The PCR program consisted of 34 cycles of 94 °C for 20 s, 58 °C for 30 s, and 72 °C for 30 s. Reactions were separated on a 1.5% agarose gel and stained with SYBR-green. Gels were imaged on a Typhoon 9200 Variable Mode Imager (Molecular Dynamics, Amersham Biosciences, Baie D'urfée, QC, Canada).

In vitro kinase assay

In vitro kinase assays were performed by mixing 0. 4 μg purified LXRα (ProteinOne, Bethesda, MD, USA) in 20 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 0.2 mM CaCl2, 5 μg/ml phosphatidylserine, 0.5 μg/ml diolein, 5 μM ATP, and 5 μCi/μl [32P]ATP along with 50 ng purified PKCα (Calbiochem) in a final reaction volume of 20 μl. The reaction was incubated at 30 °C for 30 min and products were analyzed by sodium dodecyl sulfate PAGE and detected by autoradiography.

Western blot analysis

Western blot analysis was carried out with commercially available kits (Amersham) according to manufacturer's instructions. Briefly, 25 μg protein isolated from the Cos-1 cells were subjected to PAGE and transferred to a nitrocellulose membrane. Blots were incubated with mouse anti-LXRα (1:1000) or rabbit anti-RXRα (1:200) antibodies for 1 h, followed by 2° goat HRP-conjugated antibody (1:5000) for an additional hour and visualized by enhanced chemiluminescence. Anti-β-actin was used as a loading control.

Real-time PCR

Total RNA was isolated using RNeasy mini kits (Qiagen), and cDNA was prepared from 1 μg RNA using the quantitect reverse transcription kit (Qiagen) according to the manufacturer's instructions. The real-time PCR was performed using platinum SYBR green supermix-UDG with ROX PCR mix (Invitrogen) according to manufacturer's instructions and as described previously (Bookout & Mangelsdorf 2003). Briefly, 5 μl SYBR-green Supermix, 2.5 μl H2O, 1.25 μl primer sets (1.25 μM each forward and reverse primer; specific for human ABCA1, ABCG1, and SREBP1c respectively), and 1.25 μl cDNA was mixed with a final reaction volume of 10 μl. The PCR amplification was carried out in 384-well plates in an Applied Biosystems 7900HT real-time PCR machine (Applied Biosystems, Foster City, CA, USA). Values were normalized to β-actin levels and relative abundance of transcripts was calculated using the ΔΔCt method, as described previously (Bookout & Mangelsdorf 2003).

Statistical analysis

Unpaired t-tests were used for comparison of groups. P values <0.05 were considered significant. Samples were compared with the corresponding samples treated without PMA.

Results

PMA treatment downregulates LXRα transactivation

To determine whether PKC signaling alters LXRα transactivation, we carried out transient transfection assays with LXR-responsive luciferase reporter genes. As expected, cells co-transfected with expression plasmids for human LXRα and RXRα showed a 12- and 20-fold increase in luciferase activity over basal activity of the reporter gene alone when treated in the absence or presence of the LXR agonist GW3965 respectively (Fig. 1A). Addition of PMA, a PKC activator that mimics DAG in the cellular membrane, inhibited the ligand-independent effect by ∼30–50% and completely eliminated the ligand-dependent effect (Fig. 1A). The PMA-mediated decrease in transactivation was not due to increased degradation or decreased expression of LXRα or RXRα as determined by western blot analysis (Fig. 1A). The repressive effect of PMA was blocked by co-treatment with bisindolylmaleimide, a PKC inhibitor (Fig. 1B). Similar effects on LXR transactivation were observed, when 22R-HC was used in place of GW3965 (Fig. 1C). Furthermore, the inactive enantiomer of PMA, 4α-PMA, did not decrease LXR-mediated transactivation (Fig. 1C).

Figure 1

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Figure 1

PMA modulates LXR transactivation. Cos-1 cells were transfected with luciferase reporter genes containing a consensus LXRE (panels A–C) or (D) an ABCA1 promoter, in the presence or absence of expression vectors for human LXRα and RXRα and treated with GW3965 (1 μM), 22R-hydroxycholesterol (22R-HC; 10 μM), 9-cis-retinoic acid (9-cisRA; 10 μM), Bisindolylmaleimide (Bis; 50 nM), PMA (1 nM) or 4α-PMA (PMA4α; 1 nM) for 48 h as indicated, and luciferase activity was measured. In panel A, total protein extracts (25 μg) from the corresponding transfections were analyzed by western blot with antibodies specific for human LXRα and RXRα. β-actin levels served as a loading control. (E) Cos-1 cells were transfected with a PPRE-luciferase construct in the presence or absence of expression vectors for human PPARα and RXRα in the presence of WY14,643 (WY; 5 μM) and PMA (100 nM) as indicated, and luciferase activity was measured. (F) Cos-1 cells were transfected with an ABCA1 promoter reporter vector in the presence or absence of expression vectors for human LXRβ and RXRα and treated with GW3965 (1 μM) and/or PMA (1 nM) for 48 h as indicated, and luciferase activity was measured. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PMA indicated by †; **P<0.02 versus corresponding sample minus PMA indicated by #; In panel B, not significant (N.S.) difference versus sample treated with GW3965 alone indicated by #. In panel C, not significant (N.S.) difference versus corresponding samples minus 4α-PMA indicated by † and #.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

The PMA-mediated repressive effects on the LXR activity were also observed on a natural LXR target promoter, the human ABCA1 promoter. As shown in Fig. 1D, the PMA treatment inhibited 22R-HC and 9-cisRA mediated transactivation of an ABCA1-linked reporter gene by 50–70%.

We next tested the effects of PMA on PPARα transactivation to determine whether PKC activation resulted in a general decrease in reporter activity in Cos-1 cells. In contrast to LXRα, transactivation by the related nuclear receptor PPARα is increased under similar conditions. As shown in Fig. 1E, PPARα-mediated induction of a PPRE-reporter gene in the presence of the PPAR ligand WY14-643 was increased by co-treatment with PMA in concordance with previous findings (Gray et al. 2005).

Finally, we tested the effects of PKC signaling on LXRβ-mediated transactivation of the ABCA1 luciferase reporter construct. LXRβ activity was also decreased by PMA treatment similar to LXRα (Fig. 1F). Our results indicate that under our experimental conditions, PKC activation represses LXR activity in contrast to the related nuclear hormone receptor PPARα.

To determine whether the repressive effects of PMA on LXR activity was a general phenomenon of LXRα or specific to Cos-1 cells, we performed similar transfection experiments in HEK293T and HepG2 cells. PMA treatment decreased LXRα transactivation in HEK293T (Fig. 2A) as well as HepG2 cell lines (Fig. 2B).

Figure 2

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Figure 2

PMA decreases LXRα transactivation in HEK293T cells and HepG2 cell lines. (A) HEK293T cells or (B) HepG2 cells were transfected with ABCA1-luciferase plasmid in the presence or absence of expression vectors for human LXRα and RXRα and treated as indicated. Following transfection, cells were treated with 22R-hydroxycholesterol (22R-HC; 10 μM), 9-cis-retinoic acid (9-cisRA; 10 μM) or GW3965 (2 μM) and PMA (1 nM for HEK293 cells; 80 nM for HepG2 cells) for 48 h as indicated and luciferase activity was measured. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized to β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PMA indicated by †; **P<0.02 versus corresponding sample minus PMA indicated by #.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

The repressive effects of PMA on LXR activity was dose-dependent and saturated at 1–2 nM, a concentration that is within the physiological range that activates PKC (Fig. 3A and B). This PMA dose–response profile was similar when LXR agonist 22R-HC was used in place of GW3965 (not shown). Extended treatment of PMA (>24 h) can cause depletion of PKC and, therefore, the response to PMA may not involve signaling via PKC but rather a lack of PKC kinase activity (Ohno et al. 1990). To explore this, we examined the time course of PMA-mediated inhibition. As shown in Fig. 3B, the inhibitory effects on LXR activity were observed between 3 and 4 h following PMA addition, implying that PKC depletion is likely not the cause of the decrease in LXR function (Fig. 3B). No decrease in protein expression of LXR or RXR was observed throughout the time trial (Fig. 3C), as determined by western blot analysis.

Figure 3

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Figure 3

PMA exhibits a dose- and time-dependent effect on LXR transactivation. Cos-1 cells were transfected with (A) an LXRE-luciferase reporter gene or (B) an ABCA1-luciferase reporter gene and human LXRα and RXRα expression plasmids in the presence of GW3965 (1 μM) and increasing levels of PMA (0.25–2 nM) as indicated. Cells were lysed 24 h post-transfection and luciferase activity was measured. (C) Cos-1 cells were transfected with an LXRE-luciferase reporter gene along with expression plasmids for human LXRα and RXRα. GW3965 (1 μM) or both GW3965 and PMA (1 nM) were added to the cells 24 h post-transfection and luciferase activity was measured at the indicated times. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. (D) Extracts (25 μg) from samples in (C) were analyzed by western blot with antibodies specific for LXRα and RXRα. Antibodies to β-actin served as a loading control. *P<0.05; **P<0.05 versus corresponding sample minus PMA indicated by #.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

Constitutively active PKCα mimics the effects of PMA in Cos-1 cells

To further confirm the role of PKC signaling in LXR function, we carried out transfection experiments with a constitutively active PKCα expression vector. Constitutively active PKCα decreased ligand-dependent and ligand-independent LXR transactivation with the LXRE-reporter plasmid (Fig. 4A) and with the ABCA1-luciferase reporter construct (not shown). This finding further confirms that PMA does not act by depleting PKCα. In contrast to the effects observed with LXR, the expression of constitutively active PKCα increased PPARα ligand-independent transactivation but had minimal effect on the ligand-dependent activation (Fig. 4B).

Figure 4

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Figure 4

Constitutively active PKCα mimics the effects of PMA. (A) Cos-1 cells were transfected with an LXRE-luciferase reporter plasmid and expression vectors for human LXRα and RXRα and constitutively active PKCα as indicated, in the presence of GW3965 (1 μM). Cells were lysed 24 h post-transfection and luciferase activity was measured. (B) Cos-1 cells were transfected with a PPRE-luciferase plasmid and expression vectors for PPARα, RXRα and constitutively active PKCα in the presence of Wy14,643 (WY; 100 μM) as indicated, and luciferase activity was measured as above. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PKCα indicated by †; **P<0.02 versus corresponding sample minus PKCα indicated by #.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

PMA treatment does not decrease LXRα/RXRα DNA binding

PKC-mediated phosphorylation of the VDR causes decreased DNA binding (Hsieh et al. 1993) to target sites. To determine whether this is also the case for LXR, Cos-1 cells were transfected with LXRα and RXRα expression plasmids and treated with PMA. Nuclear extracts were prepared and used in gel retardation assays with a radiolabeled LXRE probe. As shown in Fig. 5A, nuclear extracts prepared from cells transfected with LXRα and RXRα treated in the presence or absence of PMA formed a protein/DNA complex of similar intensity, and with migration similar to that formed with in vitro-synthesized LXR/RXR translated proteins. We also performed ChIP analysis of endogenous LXR protein bound to the ABCA1 promoter in HEK293 cells. As shown in Fig. 5B, HEK293 cells treated with PMA+GW3965 show similar enrichment of the ABCA1 promoter following LXR immunoprecipitation when compared with GW3965 alone. These findings indicate that PMA-treated cells do not decrease the binding of LXR/RXR heterodimers to DNA.

Figure 5

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Figure 5

PMA does not block LXR/RXR heterodimer DNA binding. (A) Gel retardation analysis. Cos-1 cells were transfected with expression plasmids for human LXRα and RXRα and treated with PMA (1 nM) for an additional 24 h. Nuclear extracts (NE) were prepared and 10 μg were incubated with a P32 labeled LXRE DNA probe. Complexes were resolved by native PAGE and detected by autoradiography. Arrow indicates the LXR/RXR protein DNA complex. In vitro translated (ivt) LXRα and RXRα served as a positive control. (B) Chromatin immunoprecipitation (ChIP) analysis of LXRα/β binding to the ABCA1 promoter following PMA treatment. HEK293 cells were treated with GW3965 (2 μM) and/or PMA (80 nM) for 2 h prior to ChIP analysis, as described in Materials and Methods. Rabbit pre-immune immunoglobulin G (IgG) or antibodies specific for LXRα/β were used to immunoprecipitate the ABCA1 promoter region. Precipitated DNA was used as template in PCR with primers specific for the human ABCA1 promoter region containing the putative LXRE. SYBR-green-stained bands representing ABCA1 are shown after 36 cycles. Lanes marked ‘input’ represent DNA isolated prior to immunoprecipitation.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

PKCα phosphorylates LXRα in vitro

To determine whether PKC could indeed phosphorylate LXR, purified LXRα was added to an in vitro kinase assay with PKCα. As shown in Fig. 6A, PKCα was able to phosphorylate LXRα in vitro indicating a possible direct modulation of LXR activity in vivo.

Figure 6

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Figure 6

PKCα phosphorylates LXRα in vitro. Recombinant PKCα and purified LXRα were incubated alone or in combination in PKC buffer for 30 min as described in experimental procedures in the presence of P32-labeled ATP. Proteins were separated by SDS-PAGE and visualized by autoradiography.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

LXR transactivation of endogenous target genes is repressed by PKC signaling

The above experiments were carried out using transient transfection assays. To determine whether PKC activation downregulates endogenous LXR target genes, we treated HEK293 cells with LXR agonist±PMA for 24 h and analyzed expression of bona fide LXR target genes by real-time PCR. As shown in Fig. 7A and B, PMA treatment repressed ligand-induced expression of endogenous LXR target genes ABCA1 and SREBP1c. The repressive effects of PMA were blocked by the PKC inhibitor bisindolylmaleimide (Fig. 7A and B). The results indicate that PKC signaling can downregulate LXR activation of endogenous target genes.

Figure 7

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Figure 7

PMA downregulates LXR target genes in HEK293 cells. HEK293 cells were treated with GW3965 (2 μM), PMA (80 nM), and/or bisindolylmaleimide (Bis; 50 nM) as indicated for 24 h and RNA was isolated. One microgram RNA was reverse transcribed and levels of (A) ABCA1 and (B) SREBP1c were determined by real-time PCR, as described in the material and methods. Relative expression was normalized to β-actin levels. Values represent the average of triplicates from two independent experiments ±s.d. *P<0.01 versus corresponding sample minus PMA indicated by †; **P<0.01 versus corresponding sample minus PMA indicated by #.

Citation: Journal of Endocrinology 197, 1; 10.1677/JOE-07-0525

Discussion

Post-translational modification of nuclear hormone receptors provides the cell with important modes of regulation and allows for rapid regulation of receptor function in response to intra- and extracellular stimuli. In particular, phosphorylation of nuclear receptors has been shown to affect their sub-cellular localization, stability, ability to bind DNA, affinity for co-activator or co-repressor proteins, and/or alter their affinity for ligands (Tahayato et al. 1993, Diradourian et al. 2005, Xu & Koenig 2005, Khan et al. 2006).

The findings reported here show that PKC activation, as shown through activation of endogenous PKC isoforms by PMA as well as by over-expression of constitutively active PKCα and the use of specific PKC inhibitors, downregulates the activity of human LXRα in transient transfection assays. The effect was observed on a consensus LXRE-luciferase construct as well as a luciferase construct harboring the human ABCA1 promoter region that contains the reported LXRE in multiple cell types (Costet et al. 2000). Moreover, in contrast to LXR, experiments done under similar conditions with PPARα and PPRE-reporter plasmids indicate that PKC signaling does not decrease PPARα activity, a result reported by others as well (Gray et al. 2005).

The mechanisms by which the PKC signaling pathway alters LXR function in vivo remains to be determined. We demonstrate here that PKCα can phosphorylate LXRα in vitro; however, it is not yet clear whether LXR is directly phosphorylated by PKC in vivo or whether this in fact correlates with the observed attenuation of receptor function.

Chen et al. (2006), using over-expression studies with FLAG-tagged LXR, have recently shown that LXR is constitutively phosphorylated in HEK293 cells and further demonstrated that S198, which is part of a consensus mitogen-activated protein kinase phosphorylation sequence, is the major phosphorylated residue. However, the physiological consequence of this modification is not apparent. Mutation of S198 did not affect the ability of LXR to bind to DNA, its ability to activate target genes, its sub-cellular distribution, nor its response to ligand (Chen et al. 2006).

Similarly, while PKA signaling in liver cells has been reported to decrease LXR/RXR heterodimer binding to DNA (Yamamoto et al. 2007), we did not observe any changes in LXR/RXR DNA complex formation using nuclear extracts or ChIP analysis from cells stimulated with the PKC activator PMA. Therefore, a potential conformational modification of LXR/RXR heterodimers induced by PKC signaling may alter LXR function. LXR is known to undergo ‘heterodimerization-induced activation’ when heterodimerized to RXR, a mechanism not reported for other nuclear receptors (Wiebel & Gustafsson 1997, Wiebel et al. 1999, Son et al. 2007). This response is dependent on the activation domain-2 (AF-2) of LXR and is the result of a conformational change in LXR induced allosterically by RXR (Wiebel & Gustafsson 1997). Indeed, we observe high basal activity when LXRα and RXRα are co-expressed in Cos-1 cells in the absence of ligand (see Fig. 1A). Our findings show that PKC signaling reduces both basal and ligand-induced activation on both a consensus LXRE-reporter plasmid as well as the natural ABCA1-reporter construct, while maintaining the ability of LXR/RXR heterodimers to bind DNA (see Fig. 5). Therefore, PKC activation could potentially prevent the allosteric modulation of LXR induced by RXR or alternatively, mask the transcriptionally active structure via post-translational modification. At present, it cannot be ruled out that RXR itself is post-translationally modified thereby blocking its ability to allosterically modulate LXR. Ongoing studies to determine the phosphorylation status of LXR and RXR in vivo, specific residues phosphorylated by PKC and domains responsible for activation/repression in response to PKC may potentially uncover a direct link between PKC, LXR and RXR phosphorylation and function.

Alternatively, it is also possible that the PKC pathway modulates LXR activity through indirect mechanisms in response to extracellular cues. For instance, it is possible that PKC signaling modulates the recruitment, function, and/or affinity of co-repressor/co-activator complexes to LXR. Altered co-factor recruitment has been reported for the related nuclear receptor PXR in response to PKC activation (Ding & Staudinger 2005), and with LXR in liver cells through PKA activation (Yamamoto et al. 2007). At present, it cannot be ruled out that co-activator or co-repressor molecules are themselves post-translationally modified by PKC, thereby blocking LXR-mediated transactivation of reporter genes (Rochette-Egly 2003). Indeed, co-activators specifically modulating the function of LXR have been identified such as activating signal cointegrator-2 (Lee et al. 2001). More recent findings have demonstrated that glucose signaling alters the sub-cellular distribution of LXR, although there is no evidence that this is phosphorylation dependent (Helleboid-Chapman et al. 2006).

Tranheim Kase et al. (2006) have also demonstrated that the synthetic LXR agonist T0901317 can induce DAG formation in cultured myoblasts. However, it has yet to be tested whether GW3965 induces similar changes in DAG levels and, furthermore, physiological LXR ligands such as 22R-HC did not alter DAG levels. In the studies presented here, we show that PKC activation has similar effects on LXRα transactivation in the presence of GW3965 or 22R-HC, and thus the physiological relevance of DAG formation by T0901317 remains to be determined.

Our findings also show that attenuation of LXR activity by PKC occurs in multiple cells types. In addition to HepG2 cells, PMA decreased LXRα transactivation in Cos-1 cells and HEK293T cells, two kidney-derived cell lines. This points to a possible role for PKC modulation of LXR activity in the kidney. Indeed, LXR plays an important role in regulating cholesterol efflux in the kidney as well as controlling the expression of renin in juxtaglomerular cells (Wu et al. 2004, Morello et al. 2005, Wang et al. 2005, Zhang et al. 2005). Interestingly, renin promoter activity is increased by cAMP/PKA signaling, a response dependent on LXRα expression. Consistent with this, we also observed an increase in LXRα activity when Cos-1 cells are treated with 8Br-cAMP, which activates the PKA pathway, on an LXRE-luciferase construct (not shown). Other reports indicate that angiotensin II, a product of renin-induced cleavage in the blood plasma, decreases renin expression via a PKC-dependent pathway (Muller et al. 2002). A possible negative feedback loop may therefore exist to regulate blood pressure by modulating LXRα activity. Indeed, LXR has recently been reported to play a role in regulating blood pressure in male Sprague–Dawley rats by modulating the angiotensin-II receptor gene in vasculature (Leik et al. 2007).

In summary, we demonstrate that PKC signaling can attenuate LXRα transactivation in Cos-1, HEK293T, and HepG2 cells. The findings reveal a potentially important mechanism of regulation of LXR that warrants further study as abnormal PKC signaling has been observed in diabetes, atherosclerosis, renin expression and glucose metabolism in the liver, all conditions in which LXR is known to play an important role (Collins 2004, Grefhorst et al. 2005, Aiello et al. 2006, Dey et al. 2006).

Acknowledgements

This work was supported by grants from the Heart and Stroke Foundation of Ontario (J P C). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

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  • BookoutALMangelsdorfDJ2003Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nuclear Receptor Signaling1e012.

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  • CostetPLuoYWangNTallAR2000Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. Journal of Biological Chemistry2752824028245.

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  • DelmotteMHTahayatoAFormstecherPLefebvreP1999Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity. Journal of Biological Chemistry2743822538231.

  • DempseyECNewtonACMochly-RosenDFieldsAPReylandMEInselPAMessingRO2000Protein kinase C isozymes and the regulation of diverse cell responses. American Journal of Physiology. Lung Cellular and Molecular Physiology279L429L438.

  • DeyDBasuDRoySSBandyopadhyayABhattacharyaS2006Involvement of novel PKC isoforms in FFA induced defects in insulin signaling. Molecular and Cellular Endocrinology2466064.

  • DingXStaudingerJL2005Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochemical Pharmacology69867873.

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  • Helleboid-ChapmanAHelleboidSJakelHTimmermanCSergheraertCPattouFFruchart-NajibJFruchartJC2006Glucose regulates LXRalpha subcellular localization and function in rat pancreatic beta-cells. Cell Research16661670.

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  • HsiehJCJurutkaPWNakajimaSGalliganMAHausslerCAShimizuYShimizuNWhitfieldGKHausslerMR1993Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. Journal of Biological Chemistry2681511815126.

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  • TahayatoALefebvrePFormstecherPDautrevauxM1993A protein kinase C-dependent activity modulates retinoic acid-induced transcription. Molecular Endocrinology716421653.

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    PMA modulates LXR transactivation. Cos-1 cells were transfected with luciferase reporter genes containing a consensus LXRE (panels A–C) or (D) an ABCA1 promoter, in the presence or absence of expression vectors for human LXRα and RXRα and treated with GW3965 (1 μM), 22R-hydroxycholesterol (22R-HC; 10 μM), 9-cis-retinoic acid (9-cisRA; 10 μM), Bisindolylmaleimide (Bis; 50 nM), PMA (1 nM) or 4α-PMA (PMA4α; 1 nM) for 48 h as indicated, and luciferase activity was measured. In panel A, total protein extracts (25 μg) from the corresponding transfections were analyzed by western blot with antibodies specific for human LXRα and RXRα. β-actin levels served as a loading control. (E) Cos-1 cells were transfected with a PPRE-luciferase construct in the presence or absence of expression vectors for human PPARα and RXRα in the presence of WY14,643 (WY; 5 μM) and PMA (100 nM) as indicated, and luciferase activity was measured. (F) Cos-1 cells were transfected with an ABCA1 promoter reporter vector in the presence or absence of expression vectors for human LXRβ and RXRα and treated with GW3965 (1 μM) and/or PMA (1 nM) for 48 h as indicated, and luciferase activity was measured. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PMA indicated by †; **P<0.02 versus corresponding sample minus PMA indicated by #; In panel B, not significant (N.S.) difference versus sample treated with GW3965 alone indicated by #. In panel C, not significant (N.S.) difference versus corresponding samples minus 4α-PMA indicated by † and #.

  • View in gallery

    PMA decreases LXRα transactivation in HEK293T cells and HepG2 cell lines. (A) HEK293T cells or (B) HepG2 cells were transfected with ABCA1-luciferase plasmid in the presence or absence of expression vectors for human LXRα and RXRα and treated as indicated. Following transfection, cells were treated with 22R-hydroxycholesterol (22R-HC; 10 μM), 9-cis-retinoic acid (9-cisRA; 10 μM) or GW3965 (2 μM) and PMA (1 nM for HEK293 cells; 80 nM for HepG2 cells) for 48 h as indicated and luciferase activity was measured. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized to β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PMA indicated by †; **P<0.02 versus corresponding sample minus PMA indicated by #.

  • View in gallery

    PMA exhibits a dose- and time-dependent effect on LXR transactivation. Cos-1 cells were transfected with (A) an LXRE-luciferase reporter gene or (B) an ABCA1-luciferase reporter gene and human LXRα and RXRα expression plasmids in the presence of GW3965 (1 μM) and increasing levels of PMA (0.25–2 nM) as indicated. Cells were lysed 24 h post-transfection and luciferase activity was measured. (C) Cos-1 cells were transfected with an LXRE-luciferase reporter gene along with expression plasmids for human LXRα and RXRα. GW3965 (1 μM) or both GW3965 and PMA (1 nM) were added to the cells 24 h post-transfection and luciferase activity was measured at the indicated times. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. (D) Extracts (25 μg) from samples in (C) were analyzed by western blot with antibodies specific for LXRα and RXRα. Antibodies to β-actin served as a loading control. *P<0.05; **P<0.05 versus corresponding sample minus PMA indicated by #.

  • View in gallery

    Constitutively active PKCα mimics the effects of PMA. (A) Cos-1 cells were transfected with an LXRE-luciferase reporter plasmid and expression vectors for human LXRα and RXRα and constitutively active PKCα as indicated, in the presence of GW3965 (1 μM). Cells were lysed 24 h post-transfection and luciferase activity was measured. (B) Cos-1 cells were transfected with a PPRE-luciferase plasmid and expression vectors for PPARα, RXRα and constitutively active PKCα in the presence of Wy14,643 (WY; 100 μM) as indicated, and luciferase activity was measured as above. The values presented represent the average (±s.d.) relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for β-galactosidase and protein levels. *P<0.02 versus corresponding sample minus PKCα indicated by †; **P<0.02 versus corresponding sample minus PKCα indicated by #.

  • View in gallery

    PMA does not block LXR/RXR heterodimer DNA binding. (A) Gel retardation analysis. Cos-1 cells were transfected with expression plasmids for human LXRα and RXRα and treated with PMA (1 nM) for an additional 24 h. Nuclear extracts (NE) were prepared and 10 μg were incubated with a P32 labeled LXRE DNA probe. Complexes were resolved by native PAGE and detected by autoradiography. Arrow indicates the LXR/RXR protein DNA complex. In vitro translated (ivt) LXRα and RXRα served as a positive control. (B) Chromatin immunoprecipitation (ChIP) analysis of LXRα/β binding to the ABCA1 promoter following PMA treatment. HEK293 cells were treated with GW3965 (2 μM) and/or PMA (80 nM) for 2 h prior to ChIP analysis, as described in Materials and Methods. Rabbit pre-immune immunoglobulin G (IgG) or antibodies specific for LXRα/β were used to immunoprecipitate the ABCA1 promoter region. Precipitated DNA was used as template in PCR with primers specific for the human ABCA1 promoter region containing the putative LXRE. SYBR-green-stained bands representing ABCA1 are shown after 36 cycles. Lanes marked ‘input’ represent DNA isolated prior to immunoprecipitation.

  • View in gallery

    PKCα phosphorylates LXRα in vitro. Recombinant PKCα and purified LXRα were incubated alone or in combination in PKC buffer for 30 min as described in experimental procedures in the presence of P32-labeled ATP. Proteins were separated by SDS-PAGE and visualized by autoradiography.

  • View in gallery

    PMA downregulates LXR target genes in HEK293 cells. HEK293 cells were treated with GW3965 (2 μM), PMA (80 nM), and/or bisindolylmaleimide (Bis; 50 nM) as indicated for 24 h and RNA was isolated. One microgram RNA was reverse transcribed and levels of (A) ABCA1 and (B) SREBP1c were determined by real-time PCR, as described in the material and methods. Relative expression was normalized to β-actin levels. Values represent the average of triplicates from two independent experiments ±s.d. *P<0.01 versus corresponding sample minus PMA indicated by †; **P<0.01 versus corresponding sample minus PMA indicated by #.

References

AielloLPClermontAAroraVDavisMDSheetzMJBursellSE2006Inhibition of PKC beta by oral administration of ruboxistaurin is well tolerated and ameliorates diabetes-induced retinal hemodynamic abnormalities in patients. Investigative Ophthalmology and Visual Science478692.

AndrewsNCFallerDV1991A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Research192499.

BookoutALMangelsdorfDJ2003Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nuclear Receptor Signaling1e012.

ChenMBradleyMNBeavenSWTontonozP2006Phosphorylation of the liver X receptors. FEBS Letters58048354841.

CollinsJL2004Therapeutic opportunities for liver X receptor modulators. Current Opinion in Drug Discovery and Development7692702.

CostetPLuoYWangNTallAR2000Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. Journal of Biological Chemistry2752824028245.

DarneCVeyssiereGJeanC1998Phorbol ester causes ligand-independent activation of the androgen receptor. European Journal of Biochemistry256541549.

DelmotteMHTahayatoAFormstecherPLefebvreP1999Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity. Journal of Biological Chemistry2743822538231.

DempseyECNewtonACMochly-RosenDFieldsAPReylandMEInselPAMessingRO2000Protein kinase C isozymes and the regulation of diverse cell responses. American Journal of Physiology. Lung Cellular and Molecular Physiology279L429L438.

DeyDBasuDRoySSBandyopadhyayABhattacharyaS2006Involvement of novel PKC isoforms in FFA induced defects in insulin signaling. Molecular and Cellular Endocrinology2466064.

DingXStaudingerJL2005Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochemical Pharmacology69867873.

DiradourianCGirardJPegorierJP2005Phosphorylation of PPARs: from molecular characterization to physiological relevance. Biochimie873338.

GrayJPBurnsKALeasTLPerdewGHVanden HeuvelJP2005Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C. Biochemistry441031310321.

GrefhorstAvan DijkTHHammerAvan der SluijsFHHavingaRHavekesLMRomijnJAGrootPHReijngoudDJKuipersF2005Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice. American Journal of Physiology. Endocrinology and Metabolism289E829E838.

Helleboid-ChapmanAHelleboidSJakelHTimmermanCSergheraertCPattouFFruchart-NajibJFruchartJC2006Glucose regulates LXRalpha subcellular localization and function in rat pancreatic beta-cells. Cell Research16661670.

HsiehJCJurutkaPWGalliganMATerpeningCMHausslerCASamuelsDSShimizuYShimizuNHausslerMR1991Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. PNAS8893159319.

HsiehJCJurutkaPWNakajimaSGalliganMAHausslerCAShimizuYShimizuNWhitfieldGKHausslerMR1993Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. Journal of Biological Chemistry2681511815126.

HuEKimJBSarrafPSpiegelmanBM1996Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARgamma. Science27421002103.

JanowskiBAWillyPJDeviTRFalckJRMangelsdorfDJ1996An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature383728731.

JosephSBCastrilloALaffitteBAMangelsdorfDJTontonozP2003Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nature Medicine9213219.

JosephSBBradleyMNCastrilloABruhnKWMakPAPeiLHogeneschJO'ConnellRMChengGSaezE2004LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell119299309.

Juge-AubryCEHammarESiegrist-KaiserCPerninATakeshitaAChinWWBurgerAGMeierCA1999Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor alpha by phosphorylation of a ligand-independent trans-activating domain. Journal of Biological Chemistry2741050510510.

KhanSAParkSWHuqMDWeiLN2006Ligand-independent orphan receptor TR2 activation by phosphorylation at the DNA-binding domain. Proteomics6123130.

LandisMSPatelHVCaponeJP2002Oxysterol activators of liver X receptor and 9-cis-retinoic acid promote sequential steps in the synthesis and secretion of tumor necrosis factor-alpha from human monocytes. Journal of Biological Chemistry27747134721.

LeeSKJungSYKimYSNaSYLeeYCLeeJW2001Two distinct nuclear receptor-interaction domains and CREB-binding protein-dependent transactivation function of activating signal cointegrator-2. Molecular Endocrinology15241254.

LeikCECarsonNLHennanJKBassoMDLiuQYCrandallDLNambiP2007GW3965, a synthetic liver X receptor (LXR) agonist, reduces angiotensin II-mediated pressor responses in Sprague–Dawley rats. British Journal of Pharmacology151450456.

MakPAKast-WoelbernHRAnisfeldAMEdwardsPA2002aIdentification of PLTP as an LXR target gene and apoE as an FXR target gene reveals overlapping targets for the two nuclear receptors. Journal of Lipid Research4320372041.

MakPALaffitteBADesrumauxCJosephSBCurtissLKMangelsdorfDJTontonozPEdwardsPA2002bRegulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta. Journal of Biological Chemistry2773190031908.

MeertensLMMiyataKSCechettoJDRachubinskiRACaponeJP1998A mitochondrial ketogenic enzyme regulates its gene expression by association with the nuclear hormone receptor PPARalpha. EMBO Journal1769726978.

MitroNMakPAVargasLGodioCHamptonEMolteniVKreuschASaezE2007The nuclear receptor LXR is a glucose sensor. Nature445219223.

MiyataKSMcCawSEMeertensLMPatelHVRachubinskiRACaponeJP1998Receptor-interacting protein 140 interacts with and inhibits transactivation by, peroxisome proliferator-activated receptor alpha and liver-X-receptor alpha. Molecular and Cellular Endocrinology1466976.

MorelloFde BoerRASteffensenKRGnecchiMChisholmJWBoomsmaFAndersonLMLawnRMGustafssonJKLopez-IlasacaM2005Liver X receptors alpha and beta regulate renin expression in vivo. Journal of Clinical Investigation11519131922.

MullerMWTodorovVKramerBKKurtzA2002Angiotensin II inhibits renin gene transcription via the protein kinase C pathway. Pflugers Archiv444499505.

NewtonAC2003Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochemical Journal370361371.

OhnoSKonnoYAkitaYYanoASuzukiK1990A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. Journal of Biological Chemistry26562966300.

Rask-MadsenCKingGL2005Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. 25487496.

RigasACOzanneDMNealDERobsonCN2003The scaffolding protein RACK1 interacts with androgen receptor and promotes cross-talk through a protein kinase C signaling pathway. Journal of Biological Chemistry2784608746093.

Rochette-EglyC2003Nuclear receptors: integration of multiple signalling pathways through phosphorylation. Cellular Signalling15355366.

SonYLParkOGKimGSLeeJWLeeYC2007RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal cointegrator-2. Biochemical Journal(In Press).

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