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Thyroid-Stimulating Hormone Increases HNF-4α Phosphorylation via cAMP/PKA Pathway in the Liver.

Song Y, Zheng D, Zhao M, Qin Y, Wang T, Xing W, Gao L, Zhao J - Sci Rep (2015)

Bottom Line: Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression.Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased.In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong, 250021, China.

ABSTRACT
Hepatocyte nuclear factor-4 alpha (HNF-4α) is an orphan nuclear receptor with important roles in hepatic metabolism. Protein phosphorylation plays a functional role in its nuclear localization, DNA binding, and transactivation. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland, whose direct effect on the metabolic pathway has been observed. Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression. However, whether TSH can influence HNF-4α phosphorylation is unclear. Here, we discovered that TSH can increase HNF-4α phosphorylation and modulate its subcellularlocalization. When HepG2 cells were treated with TSH, the phosphorylation of HNF-4α increased and its nuclear localization was interrupted. Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased. When the cAMP/PKA pathway was inhibited by the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536, the TSH-mediated phosphorylation of HNF-4α was disrupted. When Tshr was silenced in mice, the phosphorylation of HNF-4α decreased, and cytoplasmic HNF-4α decreased while nuclear HNF-4α increased. In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes.

No MeSH data available.


Related in: MedlinePlus

The TSH-mediated phosphorylation of HNF-4α via the cAMP/PKA pathway is responsible for its reduced nuclear translocation and transcriptional activity.(A) cAMP content and PKA activity in HepG2 cells were assayed. (B) HepG2 cells treated with bTSH for 48 hours in the absence or presence of PKA inhibitor (H89) and AC inhibitor (SQ22536). Cell lysates were purified by immunoprecipitation (IP) with an anti-HNF-4α antibody and were subjected to WB with antibody against phosphorylated-Serine (p-Ser) which represents the phosphorylated levels of HNF-4α. Total lysates were analyzed by WB with anti- HNF-4α antibody as indicated. Normal mouse IgG was used as a negative control. (C) The cytoplasmic and nuclear HNF-4α protein levels in HepG2 cells treated with bTSH for 48 hours in the absence or presence of H89 and SQ22536. Representative images from 3 ~ 5 independent experiments are shown. All data are expressed as the mean±standard deviations. **P < 0.01 versus the control (con). The error bars represent the standard deviations. All panels above are representative of 3 independent experiments. All the gels were run under the same experimental conditions, and key data cropped blots are used here. The full-length gel images are available in the Supplementary Figures.
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f2: The TSH-mediated phosphorylation of HNF-4α via the cAMP/PKA pathway is responsible for its reduced nuclear translocation and transcriptional activity.(A) cAMP content and PKA activity in HepG2 cells were assayed. (B) HepG2 cells treated with bTSH for 48 hours in the absence or presence of PKA inhibitor (H89) and AC inhibitor (SQ22536). Cell lysates were purified by immunoprecipitation (IP) with an anti-HNF-4α antibody and were subjected to WB with antibody against phosphorylated-Serine (p-Ser) which represents the phosphorylated levels of HNF-4α. Total lysates were analyzed by WB with anti- HNF-4α antibody as indicated. Normal mouse IgG was used as a negative control. (C) The cytoplasmic and nuclear HNF-4α protein levels in HepG2 cells treated with bTSH for 48 hours in the absence or presence of H89 and SQ22536. Representative images from 3 ~ 5 independent experiments are shown. All data are expressed as the mean±standard deviations. **P < 0.01 versus the control (con). The error bars represent the standard deviations. All panels above are representative of 3 independent experiments. All the gels were run under the same experimental conditions, and key data cropped blots are used here. The full-length gel images are available in the Supplementary Figures.

Mentions: We also demonstrated the possible mechanism that TSH modulates the subcellular localization of HNF-4α. A recent report suggested that phosphorylation of a highly conserved serine (Ser78) in HNF-4α resulted in its impaired nuclear localization15. Furthermore, our previous study showed that TSH increases cAMP production via TSHR21, and cAMP represses CYP7A1 via the PKA-mediated phosphorylation of HNF-4α22. The system used in this study showed that TSH caused an evident increase in the phosphorylation of HNF-4α; indeed, this phenomenon was disrupted using the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536 in HepG2 cells (Fig. 2A–C). To confirm that the TSH-mediated effect on HNF-4α nuclear localization was due to its phosphorylation, we also observed the subcellular localization of HNF-4α by treating HepG2 cells with H89; the levels of nuclear HNF-4α were partially but not completely reversed by H89 and SQ22536 (Fig. 2D), which suggests that pathways other than the cAMP/PKA pathway may also be involved in the TSH-mediated repression of nuclear HNF-4α, for example, the PI3K/Akt pathway20. Together, these results confirm that TSH increases HNF4 phosphorylation through the activation of cAMP/PKA pathways. This is responsible for the reduced nuclear localization and transcription factor activity of HNF-4α.


Thyroid-Stimulating Hormone Increases HNF-4α Phosphorylation via cAMP/PKA Pathway in the Liver.

Song Y, Zheng D, Zhao M, Qin Y, Wang T, Xing W, Gao L, Zhao J - Sci Rep (2015)

The TSH-mediated phosphorylation of HNF-4α via the cAMP/PKA pathway is responsible for its reduced nuclear translocation and transcriptional activity.(A) cAMP content and PKA activity in HepG2 cells were assayed. (B) HepG2 cells treated with bTSH for 48 hours in the absence or presence of PKA inhibitor (H89) and AC inhibitor (SQ22536). Cell lysates were purified by immunoprecipitation (IP) with an anti-HNF-4α antibody and were subjected to WB with antibody against phosphorylated-Serine (p-Ser) which represents the phosphorylated levels of HNF-4α. Total lysates were analyzed by WB with anti- HNF-4α antibody as indicated. Normal mouse IgG was used as a negative control. (C) The cytoplasmic and nuclear HNF-4α protein levels in HepG2 cells treated with bTSH for 48 hours in the absence or presence of H89 and SQ22536. Representative images from 3 ~ 5 independent experiments are shown. All data are expressed as the mean±standard deviations. **P < 0.01 versus the control (con). The error bars represent the standard deviations. All panels above are representative of 3 independent experiments. All the gels were run under the same experimental conditions, and key data cropped blots are used here. The full-length gel images are available in the Supplementary Figures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548215&req=5

f2: The TSH-mediated phosphorylation of HNF-4α via the cAMP/PKA pathway is responsible for its reduced nuclear translocation and transcriptional activity.(A) cAMP content and PKA activity in HepG2 cells were assayed. (B) HepG2 cells treated with bTSH for 48 hours in the absence or presence of PKA inhibitor (H89) and AC inhibitor (SQ22536). Cell lysates were purified by immunoprecipitation (IP) with an anti-HNF-4α antibody and were subjected to WB with antibody against phosphorylated-Serine (p-Ser) which represents the phosphorylated levels of HNF-4α. Total lysates were analyzed by WB with anti- HNF-4α antibody as indicated. Normal mouse IgG was used as a negative control. (C) The cytoplasmic and nuclear HNF-4α protein levels in HepG2 cells treated with bTSH for 48 hours in the absence or presence of H89 and SQ22536. Representative images from 3 ~ 5 independent experiments are shown. All data are expressed as the mean±standard deviations. **P < 0.01 versus the control (con). The error bars represent the standard deviations. All panels above are representative of 3 independent experiments. All the gels were run under the same experimental conditions, and key data cropped blots are used here. The full-length gel images are available in the Supplementary Figures.
Mentions: We also demonstrated the possible mechanism that TSH modulates the subcellular localization of HNF-4α. A recent report suggested that phosphorylation of a highly conserved serine (Ser78) in HNF-4α resulted in its impaired nuclear localization15. Furthermore, our previous study showed that TSH increases cAMP production via TSHR21, and cAMP represses CYP7A1 via the PKA-mediated phosphorylation of HNF-4α22. The system used in this study showed that TSH caused an evident increase in the phosphorylation of HNF-4α; indeed, this phenomenon was disrupted using the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536 in HepG2 cells (Fig. 2A–C). To confirm that the TSH-mediated effect on HNF-4α nuclear localization was due to its phosphorylation, we also observed the subcellular localization of HNF-4α by treating HepG2 cells with H89; the levels of nuclear HNF-4α were partially but not completely reversed by H89 and SQ22536 (Fig. 2D), which suggests that pathways other than the cAMP/PKA pathway may also be involved in the TSH-mediated repression of nuclear HNF-4α, for example, the PI3K/Akt pathway20. Together, these results confirm that TSH increases HNF4 phosphorylation through the activation of cAMP/PKA pathways. This is responsible for the reduced nuclear localization and transcription factor activity of HNF-4α.

Bottom Line: Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression.Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased.In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong, 250021, China.

ABSTRACT
Hepatocyte nuclear factor-4 alpha (HNF-4α) is an orphan nuclear receptor with important roles in hepatic metabolism. Protein phosphorylation plays a functional role in its nuclear localization, DNA binding, and transactivation. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland, whose direct effect on the metabolic pathway has been observed. Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression. However, whether TSH can influence HNF-4α phosphorylation is unclear. Here, we discovered that TSH can increase HNF-4α phosphorylation and modulate its subcellularlocalization. When HepG2 cells were treated with TSH, the phosphorylation of HNF-4α increased and its nuclear localization was interrupted. Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased. When the cAMP/PKA pathway was inhibited by the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536, the TSH-mediated phosphorylation of HNF-4α was disrupted. When Tshr was silenced in mice, the phosphorylation of HNF-4α decreased, and cytoplasmic HNF-4α decreased while nuclear HNF-4α increased. In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes.

No MeSH data available.


Related in: MedlinePlus