Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells

in Journal of Molecular Endocrinology
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  • 1 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Florida Jacksonville College of Medicine, Jacksonville, Florida, USA
  • 2 Department of Medicine, Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1–3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1β, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

Abstract

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1–3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1β, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

Introduction

The vitamin D metabolite 24,25-[OH]2D3 is generated from 25-hydroxyvitamin D3 (25-OHD3) and 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3) by the activity of the cytochrome P450 24-hydroxylase (24A2) that is expressed in many tissues, including the liver (Chen et al. 1993). Once produced in peripheral tissues, 24,25-[OH]2D3 circulates in plasma and is taken up by the liver where it accumulates until it subjected to further hydroxylation and excretion in the bile and the feces. Though it was once thought to be inactive, studies during the past 20 years have shown that 24,25-[OH]2D3 has biological activity, stimulating the resting zone chondrocytes and promoting the maturation of cartilage to bone (Dickson & Maher 1985, Wientroub et al. 1987, Miyahara et al. 1994). Recently, our laboratory demonstrated that 24,25-[OH]2D3 is a potent inhibitor of apo A-I synthesis and HDL secretion by hepatocytes (Wehmeier et al. 2011). This suggests that this compound may have other unanticipated biologic effects that remain to be elucidated.

Many of the cellular effects of vitamin D are mediated by the vitamin D receptor (VDR), a member of the steroid hormone superfamily of nuclear receptors. After ligand binding, VDR and its partner retinoid x receptor (RXR) mediate the transcriptional effects of the hormone on target genes (Kimmel-Jehan et al. 1997). However, recent studies have shown that vitamin D exhibits nongenomic signaling activity including activation of ion channels and activation of various mitogen-activated protein (MAP) kinases and pp60c-src (Chappel et al. 1997, Gniadecki 1998, Zhang et al. 2007, Buitrago et al. 2013). Unlike 1,25-[OH]2D3, which has high affinity for the VDR (Makin et al. 1989), 24,25-[OH]2D3 binds the VDR poorly if at all (Uchida et al. 1997) but in resting zone chondrocytes and other cells, stimulates various intracellular protein kinases including pp60c-src, PKCα, and MAP kinase, and phospholipase A2 activity (van Leeuwen et al. 2001). Since several of these proteins are involved in signaling pathways shown to mediate TNF α-related suppression of apo A-I expression (Haas et al. 2003, Beers et al. 2006, ­Parseghian et al. 2014) and since 24,25-[OH]2D3 is a known and potent inhibitor of apo A-I synthesis (Wehmeier et al. 2011), we investigated whether some of these pathways are modulated by 24,25-[OH]2D3 in hepatocytes. The amount of 24,25-[OH]2D3 required for repressing apo A-I gene expression (Wehmeier et al. 2011) was significantly lower than media concentrations required for the activation of various PPARs or the bile acid receptor farnesoid-X receptor (FXR) (Vu-Dac et al. 1998, Sakamoto et al. 2000, Claudel et al. 2002). However, it is possible that 24,25-[OH]2D3 down-regulates the expression of various nuclear receptors that regulate lipid and cholesterol metabolism. Therefore, we investigated the effect of 24,25-[OH]2D3 on kinase signaling, nuclear receptor expression, and proinflammatory cytokine expression in HepG2 cells.

Materials and methods

Materials

Phorbol-12-myristate-13-acetate (PMA), gemfibrozil, and the vitamin D metabolite 24,25-[OH]2D3 was purchased from Sigma Chemical Company. Immobilon-P for Western blotting was purchased from Millipore and enhanced chemiluminescence materials were purchased from Pierce Biotechnology. Thin layer chromatography plates and antibodies to PKCα and phosphorylated PKCα were obtained from Millipore. Antibodies to phosphorylated and unphosphorylated forms of P38 MAP kinase, JNK, ERK1/2, and c-jun were purchased from Cell Signaling, Inc., as was an antibody to β-actin. Antibodies specific for human apo A-I and albumin were purchased from Thermo Fisher Scientific. Control and c-jun-specific siRNA and antibodies to PPARα and RXRα were purchased from Santa Cruz Biotechnology. Secondary antibodies were purchased from Southern Biotech (Birmingham, AL, USA). 14C-chloramphenicol was purchased from Perkin-Elmer and reagents for luciferase assays were purchased from Promega. Enzyme-linked immunosorbent assay (ELISA) kits (DuoSets) for measuring interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factorα(TNF α) were purchased from R&D Systems. Lipopolysaccharide (E. coli O127:B8) (LPS) was purchased from Thermo Fisher Scientific. All other reagents were from Sigma Chemical Company or Thermo Fischer Scientific.

Western blotting

HepG2 cells were treated as described in each figure and protein samples were prepared by lysing the cells in buffer containing 50mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 7.4), 1% sodium dodecylsulfate (SDS), and 1mM ethylenediaminetetraacetic acid (EDTA). For measurement of apo A-I and albumin levels, conditioned medium was obtained from the cells after 24-h of treatment. Protein content was measured using the bicinchoninic acid assay with a kit from Pierce Biotechnology. Fifty microgram of protein was fractionated by electrophoresis on a 10% SDS polyacrylamide gel and transferred to nitrocellulose as described (Towbin et al. 1979). The nitrocellulose was blocked by shaking in 5% nonfat dry milk in phosphate-buffered saline (PBS) (50mM sodium phosphate, 150mM NaCl, pH 7.4) containing 0.05% Tween-20 for 2h at room temperature, before addition of the primary antibody (diluted in 1% nonfat dry milk in PBS) diluted according to the manufacturer’s recommendations, and incubated overnight at 4°C. After 4–5min washes in PBS-T, the membranes were incubated with either a goat-anti-mouse or a goat-­anti-rabbit secondary antibody (diluted 1:5000 in PBS). After 4–5min washes, the binding was visualized by enhanced ­chemiluminescence and autoradiography. The signal for each band was quantified using NIH Image J software.

Transient transfection

Apo A-I promoter activity was measured in HepG2 cells transfected with pAI.474.CAT, which contains the apo A-I gene promoter fused to the chloramphenicol acetyltransferase (CAT) gene (Gorman et al. 1982). Collagenase promoter activity was measured in HepG2 cells transfected with phu.collase-73/+63-CAT, which contains the AP-1-responsive collagenase promoter fused to CAT (Mitchell & Cheung 1991), while the plasmid pJ6 TK pG13, containing six copies of a PPARα response element driving luciferase expression, was obtained from Dr. Bart Staels (Institut Pasteur de Lille, Lille, France). The plasmid pCMV.SPORT.β-gal, which contains the β-galactosidase gene fused to the cytomegalovirus promoter, was used to normalize transfection efficiency (Herbomel et al. 1984). Cells were transfected with 1μg of each plasmid using Lipofectamine and 24h later treated as indicated in each figure for 24h.

Inhibition of c-jun expression via siRNA

HepG2 cells were transfected with either control or c-jun-specific inhibitory RNA (siRNA) using lipofectamine in the presence of various plasmids as described below. Previous studies in our laboratory have shown that this methodology knocks down c-jun expression over 80% (Parseghian et al. 2014).

Proinflammatory cytokine measurement by ELISA

IL-1β, IL-2, IL-6, IL-8, and TNFαlevels were measured in 100 μL of conditioned medium from cells treated with solvent, 50nM 24,25-[OH]2D3, 50nM PMA, or 10ng/mL LPS for 24-h. The immunoassays were performed essentially as described by the manufacturer’s general ELISA protocol though two changes were incorporated. First, the samples and standards were allowed to bind the capture antibody by incubating the plates at 4°C overnight. Second, detection antibody binding was also at 4°C overnight. Optical densities at 450nm (signal) and 570nm (background) were measured with an ELx800 microplate spectrophotometer (BioTek Instruments) and exported to Microsoft Excel. Cytokine levels were calculated from the linear region of the standard curves using Microsoft Excel.

Statistics

Measurements are reported as the mean ± s.d. Analysis of variance (ANOVA) and the Students t-test for independent variables were performed with Statistica for Windows (Statsoft Inc, Tulsa, OK, USA). Statistical significance was defined as a two-tailed P<0.05.

Results

The effect of 24,25-[OH]2D3 on PPARα and RXRα expression

Previous studies suggested that 10–100 nM 24,25-[OH]2D3 were sufficient to maximally suppress hepatic apo A-I gene expression (Wehmeier et al. 2011). Therefore, in the following experiments, we chose to use 50 nM 24,25-[OH]2D3. To assess whether or not 24,25-[OH]2D3 affects expression of PPARα and RXRα, HepG2 cells were treated with 50 nM 24,25-[OH]2D3 for 0, 1, 2, 6, and 24 h. PPARα and RXRα were measured by Western blot (Fig. 1A) and normalized to β-actin levels (Fig. 1B and C). Treatment with 24,25-[OH]2D3 suppressed both PPARα and RXRα expression in a time-dependent fashion, though PPARα levels were much lower than RXRα levels by 24h. In contrast, β-actin levels did not change in 24,25-[OH]2D3-treated cells. Since PPARα and RXRα regulate apo A-I gene expression (Zhang et al. 1992, Vu-Dac et al. 1998, Sakamoto et al. 2000), these results suggest that 24,25-[OH]2D3 may suppress apo A-I gene expression by inhibiting expression of these nuclear receptors.

Figure 1
Figure 1

The effect of 24,25-[OH]2D3 on PPARα and RXRα expression. HepG2 cells were treated with 24,25-[OH]2D3 for 0, 1, 3, 6, and 24 h, and PPARα, RXRα, and β-actin expression were measured by Western blot (A) and normalized to β-actin levels (B, C). Treatment with 24,25-[OH]2D3 suppressed both PPARα and RXRα expression. (B) N=6; *P<0.008, P<0.0003, and P<0.0001, respectively, relative to time-zero. (C) N=6; *P<0.03 and P<0.009, respectively, relative to time-zero.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

The effect of 24,25-[OH]2D3 on PPARα activity

HepG2 cells were transfected with the plasmids pJ6 TK pG13, containing six consensus PPARα binding sites, and pCMV.SPORT.β-gal and treated with the PPARα ligand gemfibrozil (50μM) or 50nM 24,25-[OH]2D3, or both gemfibrozil and 24,25-[OH]2D3 for 24h and luciferase activity was measured and normalized to β-galactosidase activity (Fig. 2). Treatment with gemfibrozil increased PPARα-dependent luciferase activity 2.6-fold (P<0.0008), while treatment with 24,25-[OH]2D3 suppressed PPARα-dependent luciferase activity 79.0% (P<0.0007). Furthermore, treatment with gemfibrozil was unable to reverse the effects of 24,25-[OH]2D3 on luciferase activity; luciferase activity decreased 76.9% relative to control cells (P<0.0001) in cells treated with gemfibrozil and 24,25-[OH]2D3. These results suggest that treatment with 24,25-[OH]2D3 may suppress PPARα-dependent gene expression in hepatocytes.

Figure 2
Figure 2

The effect of 24,25-[OH]2D3 on PPARα activity. HepG2 cells were transfected with the plasmids J6 TK pG13 and pCMV.SPORT.β-gal and treated with solvent, 50μM gemfibrozil, 50nM 24,25-[OH]2D3, or both gemfibrozil and 24,25-[OH]2D3 for 24h and luciferase activity was measured and normalized to β-galactosidase activity. Treatment with gemfibrozil induced PPARα-driven luciferase activity while treatment with 24,25-[OH]2D3 suppressed activity. Luciferase activity in cells treated with both gemfibrozil and 24,25-[OH]2D3 was similar to luciferase activity in cells treated with 24,25-[OH]2D3 alone. N=6; *P<0.0008, P<0.0007, and P<0.0001, respectively, relative to control cells.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

The effect of 24,25-[OH]2D3 on PKCα, JNK1, ERK1/2, and c-jun expression and phosphorylation

HepG2 cells were treated with 50nM 24,25-[OH]2D3 for 0, 1, 3, 6, and 24h and protein extracts were prepared as described above. PKCα activity, as assessed by phosphorylation on serine 3 (Fig. 3A and B), increased by 3.5-fold (P<0.0001) at 1h and this was essentially maintained throughout the 24h. These results suggest that 24,25-[OH]2D3 induces PKCα-mediated signaling in hepatocytes. JNK1 phosphorylation (C and D) increased 2.1-fold at 3h (P<0.0004 relative to phospho-JNK1 levels at time-zero) but decreased to control levels by 24h. ERK1/2 phosphorylation (E and F) increased 1.8-fold at 3h (P<0.0006 relative to phospho-ERK1/2 levels at time-zero) and decreased slightly from peak levels at 24h. Phosphorylation of c-jun (G and H) reached a maximum at 1h (2.9-fold) (P<0.0005 relative to phospho-c-jun levels at time-zero) and was sustained at 24h. Total PKCα, JNK1, ERK1/2, and c-jun levels did not change with 24,25-[OH]2D3 treatment. These results suggest that 24,25-[OH]2D3 increases c-jun phosphorylation, correlating with changes in PKCα, JNK1, and ERK1/2 phosphorylation.

Figure 3
Figure 3

The effect of 24,25-[OH]2D3 on PKCα, JNK1, ERK1/2, and c-jun expression and phosphorylation. HepG2 cells were treated with 24,25-[OH]2D3 for 0, 1, 3, 6, and 24h, and PKCα (A and B), JNK1 (C and D), ERK1/2 (E and F), and c-jun (G and H) phosphorylation and expression were measured by Western blot. Treatment with 24, 25-[OH]2D3 resulted in rapid PKCα phosphorylation (1h) which correlated with changes in c-jun phosphorylation (also 1 h). Phosphorylation of both JNK1 and ERK1/2 was elevated only at 3h post-treatment. (B) N=6; *P<0.0001, P<0.0001, P<0.0001, and P<0.0001 respectively, relative to time-zero. (D) N=6; *P<0.0004 and P<0.001, respectively, relative to time-zero. (F) N=6; *P<0.0006, P<0.0004, and P<0.0002, respectively, relative to time-zero. (H) N=6; *P<0.0005, P<0.0003, P<0.0003, and P<0.0001, respectively, relative to time-zero.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

The effect of 24,25-[OH]2D3 on AP1 activity

Since the c-jun component of the transcription factor AP1 is a substrate for PKCα and the MAP kinases ERK1/2 and JNK1, and 24,25-[OH]2D3 induced PKCα, JNK1, and ERK1/2 activity, we investigated whether or not 24,25-[OH]2D3 induces AP1-dependent transcriptional activity. HepG2 cells were transfected with a plasmid containing the AP1-dependent collagenase promoter and treated with either 100nM PMA or 50nM 24,25-[OH]2D3 for 24-h (Fig. 4A). Treatment with PMA induced collagenase promoter activity 2.7-fold (P<0.001). Likewise, treatment with 24,25-[OH]2D3 induced collagenase promoter activity 2.2-fold (P<0.008). These results suggest that 24,25-[OH]2D3, like PMA, induces AP1 activity in HepG2 cells.

Figure 4
Figure 4

The effect of 24,25-[OH]2D3 on AP1 activity. HepG2 cells were transfected with the plasmids hu.collase −73/+63.CAT and pCMV.SPORT.β-gal with either the control siRNA or a siRNA-targeting c-jun. Forty-eight hours later, the cells were treated with solvent, 100nM PMA, or 50nM 24,25-[OH]2D3, and CAT and β-galactosidase activity were measured 24h later. In the presence of the control siRNA, treatment with PMA and 24,25-[OH]2D3 induced reporter gene expression. In contrast, in cells transfected with the c-jun-specific siRNA, treatment with PMA and 24,25-[OH]2D3 had no effect on reporter gene expression. N=6; *P<0.0001 and P<0.0005, respectively, relative to control cells transfected with the control siRNA. P<0.0001 and P<0.0008, respectively, relative to cells transfected with the control siRNA and treated with PMA and 24,25-[OH]2D3, respectively.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

We next determined if the c-jun component of AP1 was required for PMA and 24,25-[OH]2D3 to induce collagenase promoter activity. HepG2 cells were transfected with the collagenase reporter plasmid in the presence of a control siRNA or a c-jun-specific siRNA. After 48-h, the cells were treated with 100 nM PMA or 50 nM 24,25-[OH]2D3 for 24 h and CAT activity was measured (Fig. 4B). In the presence of the control siRNA, PMA induced collagenase reporter gene expression 2.4-fold (P<0.0001) and 24,25-[OH]2D3 increased collagenase reporter gene expression 2.1-fold (P<0.0005). In contrast, in cells transfected with the c-jun-specific siRNA, both PMA and 24,25-[OH]2D3 induced collagenase reporter gene expression by only 1.2-fold (both NS relative to control cells). Furthermore, collagenase promoter activity was significantly lower in cells transfected with the c-jun siRNA and treated with PMA relative to cells transfected with the control siRNA and treated with PMA (P<0.0001). Likewise, collagenase promoter activity was significantly lower in cells transfected with the c-jun-specific siRNA and treated with 24,25-[OH]2D3 relative to cells transfected with the ­control siRNA and treated with 24,25-[OH]2D3 (P<0.0008). These results suggest that 24,25-[OH]2D3 induces AP1 activity by activating endogenous c-jun.

The effect of 24,25-[OH]2D3 on apo A-I promoter activity

HepG2 cells were transfected with the plasmid pAI.474.CAT and either a c-jun-specific siRNA or a control siRNA and treated with either 100nM PMA or 50nM 24,25-[OH]2D3 for 24h (Fig. 5). In the presence of the control siRNA, treatment of HepG2 cells with PMA decreased apo A-I promoter activity by 41.2% (P<0.002), while treatment with 24,25-[OH]2D3 decreased apo A-I promoter activity by 38.7% (P<0.002). However, in cells depleted of c-jun via siRNA-mediated knockdown, neither PMA nor 24,25-[OH]2D3 suppressed apo A-I promoter activity significantly (only 5.9 and 7.7%, respectively, relative to control; both changes were not statistically significant). Furthermore, apo A-I promoter activity was significantly higher in cells transfected with the c-jun siRNA and treated with PMA relative to cells transfected with the control siRNA and treated with PMA (P<0.001). Likewise, apo A-I promoter activity was significantly higher in cells transfected with the c-jun-specific siRNA and treated with 24,25-[OH]2D3 relative to cells transfected with the control siRNA and treated with 24,25-[OH]2D3 (P<0.002). These results suggest that 24,25-[OH]2D3 suppresses apo A-I gene expression by inducing AP1 activity.

Figure 5
Figure 5

The effect of 24,25-[OH]2D3 on apo A-I promoter activity. HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.β-gal with either the control siRNA or a siRNA-targeting c-jun. Forty-eight hours later, the cells were treated with solvent, 100 nM PMA, or 50nM 24,25-[OH]2D3, and CAT and β-galactosidase activity were measured 24h later. In the presence of the control siRNA, treatment with PMA and 24,25-[OH]2D3 suppressed apo A-I promoter activity. In contrast, in the presence of the c-jun-specific siRNA, treatment with PMA and 24,25-[OH]2D3 had no effect on apo A-I promoter activity. N=6; *P<0.002 and P<0.002, respectively, relative to control cells transfected with the control siRNA. P<0.001 and P<0.002, respectively, relative to cells transfected with the control siRNA and treated with PMA and 24,25-[OH]2D3, respectively.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

The effect of 24,25-[OH]2D3 on proinflammatory cytokine expression in HepG2 cells

HepG2 cells were treated as described in Materials and methods, and IL-1β (A), IL-2 (B), IL-6 (C), IL-8 (D), and TNFαlevels were measured by ELISA and apo A-I and albumin levels were measured by Western blot of the conditioned medium (Fig. 6). TNFαlevels were below the level of detection in all treatment groups. IL-1β, IL-6, and IL-8 levels were all increased in cells treated with 24,25-[OH]2D3, but not IL-2. Both PMA and LPS treatment induced IL-1β, Il-2, IL-6, and IL-8 secretion from HepG2 cells. Apo A-I levels, but not albumin levels, were significantly lower in cells treated with 24,25-[OH]2D3, PMA, and LPS as reported previously (Morishima et al. 2003, Wehmeier et al. 2011). These results suggest that 24,25-[OH]2D3 has pro­inflammatory effects in HepG2 cells.

Figure 6
Figure 6

The effect of 24,25-[OH]2D3 on proinflammatory cytokine expression in HepG2 cells. HepG2 cells were treated with solvent (control cells, C), 50nM 24,25-[OH]2D3, 50nM PMA, or 10ng/mL LPS for 24h, and IL-1β (A), IL-2 (B), IL-6 (C), IL-8 (D), TNF α, apo A-I, and albumin levels were measured either by ELISA (IL-1β, IL-2, IL-6, IL-8, and TNF α) or Western blot (apo A-I and albumin) (E, F). TNFαlevels were below the level of detection in all treatment groups. While PMA and LPS treatment induced expression of all the other proinflammatory cytokine levels, 24,25-[OH]2D3 increased only IL-1β, IL-6, and IL-8. (A) N=3; *P<0.0007, P<0.0002, and P<0.0002, relative to control cells. (B) N=3; *P<0.03 and P<0.01, relative to control cells. (C) N=3; *P<0.0003, P<0.02, and P<0.001, relative to control cells. (D) N=3; *P<0.0001, P<0.0008, and P<0.0001, relative to control cells. (E). Apo A-I and albumin expression in control cells and cells treated with 24,25-[OH]2D3, PMA, and LPS. (F) Apo A-I levels were quantified and normalized to albumin expression. N=3; *P<0.02, P<0.02, and P<0.01, relative to control cells.

Citation: Journal of Molecular Endocrinology 57, 2; 10.1530/JME-16-0009

Discussion

Previous studies in our laboratory have shown that 1,25-[OH]2D3 inhibits apo A-I gene expression via a VDR-mediated mechanism (Wehmeier et al. 2005). In contrast, 24,25-[OH]2D3-mediated suppression of apo A-I did not require VDR expression since knockdown of VDR expression with siRNA had no effect on the efficacy of 24,25-[OH]2D3 to inhibit apo A-I gene expression (Wehmeier et al. 2011). Therefore, we examined the potential of 24,25-[OH]2D3 to induce various signaling pathways that are independent of VDR and account for its apo A-I suppressive effect.

Since nuclear receptors PPARα and RXRα have been shown to be important in regulating apo A-I gene expression (Beigneux et al. 2000, Wang & Wan 2008), we examined the effects of 24,25-[OH]2D3 on their expression and activity. Treatment with 24,25-[OH]2D3 suppressed both PPARα and RXRα expression in HepG2 cells, though PPARα levels were affected more so than RXRα. These changes in PPARα and RXRα expression affected the capacity of PPARα to stimulate a PPARα-responsive reporter gene, even in the presence of a potent PPARα ligand gemfibrozil. It is not clear how 24,25-[OH]2D3 reduces PPARα and RXRα levels, though the rapid kinetics suggests that it occur, protein degradation may be involved (Genini & Catapano 2006, Lefebvre et al. 2010). PPARα is phosphorylated by PCKα in PMA-treated cells (Gray et al. 2005), though this post-translational modification resulted in increased transcriptional activity. Likewise, ERK1/2 has been shown to phosphorylate PPARα in insulin-treated cells, also enhancing its transcriptional activity (Shalev et al. 1996). In contrast, JNK1 has been shown to suppress PPARα activity in hepatocytes, though this effect was mediated by the corepressors nuclear corepressor 1 (NCoR1) and nuclear receptor interacting protein 1 (NRIP1), without changes in PPARα expression (Vernia et al. 2014). Additional studies will be required to determine the mechanism. However, these results suggest that 24,25-[OH]2D3 may suppress apo A-I gene expression in part by reducing the expression of these two key transcriptional regulators.

It is noteworthy that proinflammatory cytokines including tumor necrosis factorαinhibit apo A-I gene expression by down-regulating the expression of various steroid hormone nuclear receptors including PPARα and RXRα (Beigneux et al. 2000, Wang & Wan 2008). This effect is in part due to induction of ERK1/2 and JNK1 branches of the MAP kinase pathway, leading to the activation of c-jun and squelching of apo A-I promoter activity (Haas et al. 2003, Beers et al. 2006, Parseghian et al. 2014). Since 24,25-[OH]2D3 has been shown to stimulate MAP kinase activity in other tissues (van Leeuwen et al. 2001), we determined the response and kinetics of PKCα, JNK1, and ERK1/2 activation in HepG2 cells. Treatment with 24,25-[OH]2D3 rapidly stimulated (within 1h) PKCα phosphorylation as well as c-jun phosphorylation, whereas JNK1 and ERK1/2 required longer (3h) leading to the rapid activation of AP1 activity by stimulating serine 63 phosphorylation on c-jun (Davis 2000). Furthermore, treatment with 24,25-[OH]2D3 induced the AP1-responsive collagenase reporter gene expression to a similar extent as the phorbol ester PMA. However, this response was inhibited by the addition of a c-jun siRNA but not a control siRNA. Likewise, treatment with 24,25-[OH]2D3 inhibited apo A-I promoter activity as did the phorbol ester PMA, to a similar extent, which was inhibited by the c-jun-specific siRNA but not the control siRNA. This data suggests that of the two potential pathways involved in suppressing apo A-I gene expression (nuclear receptor expression or AP1-dependent inhibition), AP1-dependent inhibition is the most likely pathway involved. If both pathways were involved, 24,25-[OH]2D3 would inhibit apo A-I gene expression to a greater extent than observed with PMA treatment.

Finally, treatment with 24,25-[OH]2D3 increased IL-1β, IL-6, and IL-8 levels in the conditioned medium, similar to cells treated with PMA and LPS. While TNFαlevels were undetectable under all treatment conditions, both PMA and LPS treatment, but not 24,25-[OH]2D3, increased IL-2 levels. Of the cytokines that were induced by 24,25-[OH]2D3, the most is known about IL-1β inflammasome activity in the liver. Though liver immune cells such as Kupffer cells express most of the IL-1β released by inflammasome activation (Petrasek et al. 2012), hepatocytes, including HepG2 cells, also express the cytokine (­Palabiyik et al. 2016, Zhang et al. 2016). Once released, IL-1β has been shown to act in both autocrine and paracrine fashions to increase its own expression (Szabo & Petrasek 2015) as well as the expression of other proinflammatory cytokines, including TNFα(Mandrekar et al. 2011), though we did not observe the latter in our studies. IL-1β activates hepatic stellate cells leading to fibrosis (Miura et al. 2010) and increases triglyceride accumulation in hepatocytes (Miura et al. 2010, Petrasek et al. 2012). IL-6 is also expressed in HepG2 cells (Deng et al. 2014) and is important in promoting the acute phase response as well as liver regeneration and obesity and insulin resistance. IL-6 induces the expression of c-reactive protein, serum amyloid A, fibrinogen, and haptoglobin (Bode et al. 2012), while IL-6 knockout mice have impaired liver regeneration (Cressman et al. 1996). The effect of IL-6 on obesity and glucose metabolism is more complex. While IL-6 administration decreased insulin action in skeletal muscle and liver in hyperinsulinemic-euglycemic clamp studies (Kim et al. 2004), IL-6-deficient mice displayed hyperleptinemia and hyperinsulinemia, liver inflammation, and steatohepatitis (Wallenius et al. 2002, Matthews et al. 2010). Similar to IL-1β and IL-6, IL-8 is also expressed in HepG2 cells (Wang et al. 2016). IL-8 is an angiogenic factor for human microvascular cells (Heidemann et al. 2003) and has chemoattractant and neutrophil-activating activity (Stillie et al. 2009). These results suggest that 24,25-[OH]2D3 may enhance hepatic inflammation in part by stimulating IL-1β, IL-6, and IL-8 expression.

In conclusion, the results of our studies strongly suggest that the principle vitamin D metabolite 24,25-[OH]2D3 has proinflammatory effects in HepG2 cells. Serum 24,25-[OH]2D3 levels in healthy individuals has been reported to be 2.3ng/mL (6.2nM) (Tartarotti et al. 1984), and though it can rapidly concentrate in vitamin D-responsive tissues as well as the liver, to our knowledge, there have been no studies examining tissue levels in humans; nor have there been studies examining how changes in physiological state may lead to changes in tissue levels. These aspects, as well as additional studies examining the molecular ­mechanism by which 24,25-[OH]2D3 enhances inflammation remain for investigation both in vitro and in vivo.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

Funding was provided by a Dean’s Grant from the University of Florida Jacksonville to K Wehmeier.

Author contributions

All of the authors contributed significantly to the experimental design, carrying out the studies, and writing and editing the finished manuscript.

Acknowledgments

The authors wish to thank Jonathan Schneider for his assistance in the laboratory.

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    The effect of 24,25-[OH]2D3 on PPARα and RXRα expression. HepG2 cells were treated with 24,25-[OH]2D3 for 0, 1, 3, 6, and 24 h, and PPARα, RXRα, and β-actin expression were measured by Western blot (A) and normalized to β-actin levels (B, C). Treatment with 24,25-[OH]2D3 suppressed both PPARα and RXRα expression. (B) N=6; *P<0.008, P<0.0003, and P<0.0001, respectively, relative to time-zero. (C) N=6; *P<0.03 and P<0.009, respectively, relative to time-zero.

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    The effect of 24,25-[OH]2D3 on PPARα activity. HepG2 cells were transfected with the plasmids J6 TK pG13 and pCMV.SPORT.β-gal and treated with solvent, 50μM gemfibrozil, 50nM 24,25-[OH]2D3, or both gemfibrozil and 24,25-[OH]2D3 for 24h and luciferase activity was measured and normalized to β-galactosidase activity. Treatment with gemfibrozil induced PPARα-driven luciferase activity while treatment with 24,25-[OH]2D3 suppressed activity. Luciferase activity in cells treated with both gemfibrozil and 24,25-[OH]2D3 was similar to luciferase activity in cells treated with 24,25-[OH]2D3 alone. N=6; *P<0.0008, P<0.0007, and P<0.0001, respectively, relative to control cells.

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    The effect of 24,25-[OH]2D3 on PKCα, JNK1, ERK1/2, and c-jun expression and phosphorylation. HepG2 cells were treated with 24,25-[OH]2D3 for 0, 1, 3, 6, and 24h, and PKCα (A and B), JNK1 (C and D), ERK1/2 (E and F), and c-jun (G and H) phosphorylation and expression were measured by Western blot. Treatment with 24, 25-[OH]2D3 resulted in rapid PKCα phosphorylation (1h) which correlated with changes in c-jun phosphorylation (also 1 h). Phosphorylation of both JNK1 and ERK1/2 was elevated only at 3h post-treatment. (B) N=6; *P<0.0001, P<0.0001, P<0.0001, and P<0.0001 respectively, relative to time-zero. (D) N=6; *P<0.0004 and P<0.001, respectively, relative to time-zero. (F) N=6; *P<0.0006, P<0.0004, and P<0.0002, respectively, relative to time-zero. (H) N=6; *P<0.0005, P<0.0003, P<0.0003, and P<0.0001, respectively, relative to time-zero.

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    The effect of 24,25-[OH]2D3 on AP1 activity. HepG2 cells were transfected with the plasmids hu.collase −73/+63.CAT and pCMV.SPORT.β-gal with either the control siRNA or a siRNA-targeting c-jun. Forty-eight hours later, the cells were treated with solvent, 100nM PMA, or 50nM 24,25-[OH]2D3, and CAT and β-galactosidase activity were measured 24h later. In the presence of the control siRNA, treatment with PMA and 24,25-[OH]2D3 induced reporter gene expression. In contrast, in cells transfected with the c-jun-specific siRNA, treatment with PMA and 24,25-[OH]2D3 had no effect on reporter gene expression. N=6; *P<0.0001 and P<0.0005, respectively, relative to control cells transfected with the control siRNA. P<0.0001 and P<0.0008, respectively, relative to cells transfected with the control siRNA and treated with PMA and 24,25-[OH]2D3, respectively.

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    The effect of 24,25-[OH]2D3 on apo A-I promoter activity. HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.β-gal with either the control siRNA or a siRNA-targeting c-jun. Forty-eight hours later, the cells were treated with solvent, 100 nM PMA, or 50nM 24,25-[OH]2D3, and CAT and β-galactosidase activity were measured 24h later. In the presence of the control siRNA, treatment with PMA and 24,25-[OH]2D3 suppressed apo A-I promoter activity. In contrast, in the presence of the c-jun-specific siRNA, treatment with PMA and 24,25-[OH]2D3 had no effect on apo A-I promoter activity. N=6; *P<0.002 and P<0.002, respectively, relative to control cells transfected with the control siRNA. P<0.001 and P<0.002, respectively, relative to cells transfected with the control siRNA and treated with PMA and 24,25-[OH]2D3, respectively.

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    The effect of 24,25-[OH]2D3 on proinflammatory cytokine expression in HepG2 cells. HepG2 cells were treated with solvent (control cells, C), 50nM 24,25-[OH]2D3, 50nM PMA, or 10ng/mL LPS for 24h, and IL-1β (A), IL-2 (B), IL-6 (C), IL-8 (D), TNF α, apo A-I, and albumin levels were measured either by ELISA (IL-1β, IL-2, IL-6, IL-8, and TNF α) or Western blot (apo A-I and albumin) (E, F). TNFαlevels were below the level of detection in all treatment groups. While PMA and LPS treatment induced expression of all the other proinflammatory cytokine levels, 24,25-[OH]2D3 increased only IL-1β, IL-6, and IL-8. (A) N=3; *P<0.0007, P<0.0002, and P<0.0002, relative to control cells. (B) N=3; *P<0.03 and P<0.01, relative to control cells. (C) N=3; *P<0.0003, P<0.02, and P<0.001, relative to control cells. (D) N=3; *P<0.0001, P<0.0008, and P<0.0001, relative to control cells. (E). Apo A-I and albumin expression in control cells and cells treated with 24,25-[OH]2D3, PMA, and LPS. (F) Apo A-I levels were quantified and normalized to albumin expression. N=3; *P<0.02, P<0.02, and P<0.01, relative to control cells.

  • Beers A, Haas MJ, Wong NCW & Mooradian AD 2006 Inhibition of apolipoprotein AI gene expression by tumor necrosis factor α: roles for MEK/ERK and JNK signaling. Biochemistry 45 24082413. (doi:10.1021/bbib518040)

    • Search Google Scholar
    • Export Citation
  • Beigneux AP, Moser AH, Shigenega JK, Grunfeld C & Feingold KR 2000 The acute phase response is associated with retinoid X receptor repression in rodent liver. Journal of Biological Chemistry 275 1639016399. (doi:10.1074/jbc.M000953200)

    • Search Google Scholar
    • Export Citation
  • Bode JG, Albrecht U, Haussinger D, Heinrich PC & Schaper F 2012 Hepatic acute phase proteins-regulation by IL-6 and IL-1-type cytokines involving STAT3 and its crosstalk with NF-κB-dependent signaling. European Journal of Cell Biology 91 496505. (doi:10.1016/j.ejcb.2011.09.008)

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