Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: Daily treatment with IL1β decreases SHBG production over 5 d via MEK-1/2 and JNK MAPK by c-Jun activation in HepG2 cells. A, HepG2 cells were treated daily with vehicle or IL1β (50 ng/ml) for 5 d. SHBG accumulation in the medium was measured using an ELISA. B, Analysis of SHBG mRNA levels in HepG2 cells treated as in A. Human 18S (h18S) mRNA was amplified as a control. Data points are shown as mean ± sd of triplicates. **, P < 0.01 compared with the control. C, HepG2 cells were treated daily with vehicle, IL1β (50 ng/ml) alone or with U0126 (500 nm), and SP600125 (500 nm) or SB203580 (500 nm). SHBG accumulation in the medium was measured using an ELISA. D, Analysis of SHBG mRNA levels in HepG2 cells treated as in C. Human 18S mRNA was amplified as a control. Data points are shown as mean ± sd of triplicates. **, P < 0.01 compared with the control. E, Western blotting of phospho-c-Jun and PPIA in total cell protein extracts of HepG2 cells treated as in A. **, P < 0.01 compared with the control.

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Amplification, Control, Western Blot

Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: The SHBG promoter contains two AP1 binding sites, and it responds to IL1β in luciferase reporter gene assays. A, SHBG promoter scheme of the previously described FP regions and the TF that binds them. The putative AP1 binding sites are located within the FP 3 and FP 4 regions. B, The SHBG promoter activity was analyzed in HepG2 cells treated with vehicle or IL1β (50, 100, or 200 ng/ml) in luciferase reporter gene assays. Data points are mean ± sd of triplicate measurements. **, P < 0.01 compared with the control. C, The SHBG promoter activity of the wild type, FP 3 mutated, or FP 4 deleted was analyzed in HepG2 cells using treated with vehicle or IL1β (100 ng/ml) in luciferase reporter gene assays. Data points are mean ± sd of triplicate measurements. **, P < 0.01 compared with the control. USF, Upstream stimulatory factor; COUP-TF, chicken ovalbumin upstream promoter-TF.

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Binding Assay, Luciferase, Activity Assay, Control

Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: IL1β treatment decreases HNF-4α mRNA and protein levels via MEK-1/2 and JNK MAPK in HepG2 cells. A, Analysis of HNF-4α mRNA levels in HepG2 cells treated with vehicle or IL1β (50 ng/ml) over the course of 5 d. Human 18S (h18S) mRNA was amplified as a control. Data points are shown as mean ± sd of triplicates. **, P < 0.01 compared with the control. B, Western blotting of HNF-4α and PPIA in total cell protein extracts of HepG2 cells treated as in A. **, P < 0.01 compared with the control. C, Western blotting of HNF-4α and PPIA in total cell protein extracts of HepG2 cells treated with vehicle, IL1β (50 ng/ml) alone, or IL1β (50 ng/ml) in the presence of U0126 (500 nm), SP600125 (500 nm), or SB203580 (500 nm). **, P < 0.01 compared with the control. D, The SHBG promoter activity was analyzed in HepG2 cells cotransfected with an empty vector or a HNF-4α expression vector in the absence or presence of IL1β (200 ng/ml) in luciferase reporter gene assays. Data points are mean ± sd of triplicate measurements. **, P < 0.01 compared with the control.

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Amplification, Control, Western Blot, Activity Assay, Plasmid Preparation, Expressing, Luciferase

Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: IL1β treatment reduces HNF-4α mRNA and protein levels after 2–4 h via c-Jun activation. A, Analysis of SHBG and HNF-4α mRNA levels in HepG2 cells treated with vehicle or IL1β (100 ng/ml) at 0, 2, and 4 h. Human 18S (h18S) mRNA was amplified as a control. Data points are shown as mean ± sd of triplicates. **, P < 0.01 compared with the control. B, Western blotting of HNF-4α, phospho-c-Jun, and PPIA in total cell protein extracts of HepG2 cells treated as in A. C, Western blotting of HNF-4α and PPIA in total cell protein extracts of HepG2 cells treated with vehicle, IL1β (100 ng/ml) alone, or IL1β (50 ng/ml) with MG132 (10 μm).

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Activation Assay, Amplification, Control, Western Blot

Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: IL1β reduces HNF-4α mRNA and protein levels up to 12 h after treatment. A, Analysis of SHBG and HNF-4α mRNA levels in HepG2 cells treated with vehicle or IL1β (50, 100, and 200 ng/ml) at 6 and 12 h. Human 18S (h18S) mRNA was amplified as a control. Data points are shown as mean ± sd of triplicates. **, P < 0.01 compared with the control. B, Western blotting of HNF-4α and PPIA in total cell protein extracts of HepG2 cells treated as in A. C, The HNF4A promoter activity was analyzed in HepG2 cells treated with vehicle, IL1β (50 ng/ml) alone, or IL1β (50 ng/ml) with SP600125 (500 nm) or U0126 (500 nm) in luciferase reporter gene assays. Data points are mean ± sd of triplicate measurements. **, P < 0.01 compared with the control. DMSO, Dimethylsulfoxide.

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Amplification, Control, Western Blot, Activity Assay, Luciferase

Journal: Molecular Endocrinology

Article Title: IL1β Down-regulation of Sex Hormone-Binding Globulin Production by Decreasing HNF-4α Via MEK-1/2 and JNK MAPK Pathways

doi: 10.1210/me.2012-1152

Figure Lengend Snippet: Daily treatment with IL1β reduces plasma SHBG levels in human SHBG transgenic mice. A, Human SHBG transgenic mice (n = 3) were treated over 3 d with ip injections of PBS or IL1β (1.5 μg), and blood SHBG levels were measured by ELISA. The SHBG levels are expressed as mean ± sd relative to pretreatment levels to compensate for between-animal variability. B, SHBG and HNF-4α mRNA abundance was determined in relation to 18S RNA (mean ± sd) in liver of vehicle-treated (n = 3) and IL1β-treated (n = 3) mice. C, Liver HNF-4α and phospho-c-Jun protein levels were measured by Western blotting using PPIA as a housekeeping reference protein in liver of vehicle-treated (n = 3) and IL1β-treated (n = 3) mice. Data points are mean ± sd of triplicate measurements. **, P < 0.01 compared with the control.

Article Snippet: Eight-week male mice (n = 3) were treated with daily ip injections of PBS or 1.5 μg of human IL1β (Miltenyi Biotech S.L, Madrid, Spain).

Techniques: Clinical Proteomics, Transgenic Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control