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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.


The effect of TSH on HNF-4α phosphorylation is dependent on TSHR.(A) Total protein (500 μg) from Tshr(+/+) and Tshr(−/−) mouse liver extracts were purified by IP with the HNF-4α antibody and subjected to WB with the p-Ser antibody. Total lysates were analyzed by WB with antibody against HNF-4α as indicated. Normal mouse IgG was used as a negative control. (B) Protein levels of cytoplasmic (-P) and nuclear (-N) HNF-4α were detected in Tshr(+/+) and Tshr(−/−) 8-week old littermate mice. 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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f3: The effect of TSH on HNF-4α phosphorylation is dependent on TSHR.(A) Total protein (500 μg) from Tshr(+/+) and Tshr(−/−) mouse liver extracts were purified by IP with the HNF-4α antibody and subjected to WB with the p-Ser antibody. Total lysates were analyzed by WB with antibody against HNF-4α as indicated. Normal mouse IgG was used as a negative control. (B) Protein levels of cytoplasmic (-P) and nuclear (-N) HNF-4α were detected in Tshr(+/+) and Tshr(−/−) 8-week old littermate mice. 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: The characteristics of Tshr(−/−) mice are described in Table 1 and the experimental procedures section. The serum total T4 (TT4), free T4 (FT4) and TSH levels showed no differences between the wild-type and the Tshr-KO littermate mice (all p > 0.05).Compared with the littermate wild-type mice, the phosphorylation of HNF-4α decreased in the Tshr-KO mice (Fig. 3A). The cytoplasmic HNF-4α decreased, while the nuclear HNF-4α increased (Fig. 3B).


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 effect of TSH on HNF-4α phosphorylation is dependent on TSHR.(A) Total protein (500 μg) from Tshr(+/+) and Tshr(−/−) mouse liver extracts were purified by IP with the HNF-4α antibody and subjected to WB with the p-Ser antibody. Total lysates were analyzed by WB with antibody against HNF-4α as indicated. Normal mouse IgG was used as a negative control. (B) Protein levels of cytoplasmic (-P) and nuclear (-N) HNF-4α were detected in Tshr(+/+) and Tshr(−/−) 8-week old littermate mice. 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

f3: The effect of TSH on HNF-4α phosphorylation is dependent on TSHR.(A) Total protein (500 μg) from Tshr(+/+) and Tshr(−/−) mouse liver extracts were purified by IP with the HNF-4α antibody and subjected to WB with the p-Ser antibody. Total lysates were analyzed by WB with antibody against HNF-4α as indicated. Normal mouse IgG was used as a negative control. (B) Protein levels of cytoplasmic (-P) and nuclear (-N) HNF-4α were detected in Tshr(+/+) and Tshr(−/−) 8-week old littermate mice. 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: The characteristics of Tshr(−/−) mice are described in Table 1 and the experimental procedures section. The serum total T4 (TT4), free T4 (FT4) and TSH levels showed no differences between the wild-type and the Tshr-KO littermate mice (all p > 0.05).Compared with the littermate wild-type mice, the phosphorylation of HNF-4α decreased in the Tshr-KO mice (Fig. 3A). The cytoplasmic HNF-4α decreased, while the nuclear HNF-4α increased (Fig. 3B).

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.