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. 2012 Mar;26(3):438-46.
doi: 10.1210/me.2011-1321. Epub 2012 Feb 2.

Molecular Mechanism of TNFα-Induced Down-Regulation of SHBG Expression

Rafael Simó  1 , Anna Barbosa-Desongles, Cristina Sáez-Lopez, Albert Lecube, Cristina Hernandez, David M Selva
Affiliations

Molecular Mechanism of TNFα-Induced Down-Regulation of SHBG Expression

Rafael Simó et al. Mol Endocrinol. 2012 Mar.

Abstract

The reason why obesity (a chronic low-grade inflammatory disease) is associated with low levels of sex hormone-binding globulin (SHBG) remains to be elucidated. The present study provides evidence that TNFα (a proinflammatory cytokine increased in obesity) reduces SHBG production by human HepG2 hepatoblastoma cells. Although the human SHBG promoter contains one nuclear factor-κB (NF-κB) binding site, the human SHBG promoter activity did not change after TNFα treatment or transfection with either small interfering RNA against p65 or a p65 expression vector in luciferase reporter gene assays. The effect of TNFα on human SHBG expression was indirect, and it was mediated by NF-κB through the down-regulation of hepatocyte nuclear factor (HNF)-4A: a key SHBG transcriptional regulator. Furthermore, the HNF-4A proximal promoter contains three putative NF-κB binding sites. The HNF-4A promoter activity was decreased by the treatment with TNFα or the transfection of a p65 expression vector, and it was increased by the treatment with small interfering RNA against NF-κB in luciferase reporter gene assays. Finally, the TNFα treatment promotes the NF-κB binding to the HNF-4A promoter in chromatin immunoprecipitation assays. We conclude that sustained exposition to elevated levels of TNFα decreases SHBG production by reducing hepatic HNF-4α levels via NF-κB activation in HepG2 cells.

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Figures

Fig. 1.
Fig. 1.
Daily treatment with TNFα decreases SHBG production in a 5 d time course study in HepG2 cells. A, HepG2 cells were treated daily with vehicle or TNFα (50 ng/ml) for 5 d. Daily SHBG accumulation in the medium was measured using an ELISA. B, Analysis of SHBG mRNA levels in HepG2 cells treated as in panel A. Human 18S (h18S) mRNA was amplified as a control. Data are shown as the mean ± sd (n = 3). **, P < 0.01 compared with the control.
Fig. 2.
Fig. 2.
The SHBG promoter contains a NF-κB binding site but does not respond to TNFα in luciferase resporter gene assays. A, Scheme of the SHBG promoter previously described FP and the new putative NF-κB binding site. B, SHBG promoter activity was analyzed in HepG2 cells treated with vehicle or TNFα (50, 100, or 200 ng/ml) in luciferase reporter gene assays. C, Using a NF-κB siRNA there were no differences in the luciferase reporter gene assay when compared with the siRNA control (left panel). Western blot of HepG2 cells treated with the siRNA control and NF-κB (right panel). D, SHBG proximal promoter did not respond when cotransfected with a p65 expression vector (right panel) whereas the NF-κB-responsive promoter showed a 10 times increase activity (left panel). Data are expressed as the mean ± sd (n = 3). **, P < 0.01 compared with the control.
Fig. 3.
Fig. 3.
TNFα reduces HNF-4α mRNA and protein levels in a 5-d time course study in HepG2 cells. A, Analysis of HNF-4α mRNA levels in HepG2 cells treated daily with vehicle or TNFα (50 ng/ml). Human 18S mRNA was amplified as a control. Data are expressed as the mean ± sd (n = 3). *, P < 0.05 and **, P < 0.01 compared with the control. B, Western blot of HNF-4α and PPIA in total cell protein extracts of HepG2 cells treated as in panel A.
Fig. 4.
Fig. 4.
TNFα-induced reduction of SHBG expression is mediated by the reduction in HNF4α levels via NF-κB. A, Analysis of HNF-4α mRNA levels in HepG2 cells transfected with a GFP expression vector or a p65 (NF-κB subunit)-GFP (GFP-p65) expression vector and treated for 48 h with vehicle or TNFα (50 ng/ml). Human 18S mRNA was amplified as an internal control. Data are expressed as the mean ± sd (n = 3); **, P < 0.01. B, Western blot of HNF-4α, p65, and PPIA in total cell protein extracts of HepG2 cells treated as in panel A. Data are expressed as the mean ± sd (n = 3); **, P < 0.01. C, SHBG promoter activity cotransfected with an empty vector or an HNF4α expression vector was analyzed in HepG2 cells treated with vehicle or TNFα (50 ng/ml) for 4 d in luciferase reporter gene assays. Data are expressed as the mean ± sd (n = 3); *, P < 0.05 and **, P < 0.01 compared with the control.
Fig. 5.
Fig. 5.
The HNF-4A P1 promoter respond to TNFα or NF-κB treatment in luciferase reporter gene assays in HepG2 cells. A, HNF-4A P1 promoter contains three putative NF-κB binding sites. B, TNFα (100 ng/ml) reduces the HNF-4A P1 promoter in luciferase reporter gene assays when compared with vehicle-treated HepG2 cells. C, Using NF-κB siRNA there was an increase in HNF-4A P1 promoter activity in luciferase reporter gene assays when compared with the siRNA control. D, The HNF-4A P1 promoter activity decreased when cotransfected with a p65 expression vector (right panel) whereas the NF-κB responsive promoter showed a 10 times increased activity (left panel). E, Single mutants of the putative NF-κB binding sites of the HNF-4A P1 promoter were analyzed in luciferase reporter gene assays in HepG2 cells treated with vehicle or TNFα (100 ng/ml). F, Single mutants of the putative NF-κB binding sites of the HNF-4A P1 promoter were analyzed in luciferase reporter gene assays in HepG2 cells cotransfected with an empty vector or p65 expression vector. G, ChIP assays of NF-κB binding to the human HNF-4A P1 promoter in HepG2 cells treated with vehicle or TNFα for 5 d. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and E-selectin promoters were used as a negative and positive control, respectively. A nonspecific mouse IgG was used in ChIP reactions to control for nonspecific immunoprecipitation. Positive PCR controls of sheared genomic DNA templates indicated the integrity of the input DNA used in the ChIP reactions. Data are expressed as the mean ± sd (n = 3); **, P < 0.01 compared with the control.
Fig. 6.
Fig. 6.
Mechanism by which TNFα leads to a low serum levels of SHBG. TNFα reduces the expression of HNF-4α via NF-κB activation which in turn reduces SHBG expression in the liver.
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