Insulin Represses Transcription of the Thyroid Stimulating Hormone β-Subunit Gene through Increased Recruitment of Nuclear Factor I

Kee Kwang Kim,Key Sun Park,Seok Bean Song,Kyoon Eon Kim

doi:10.1074/jbc.m110.107573

Kee Kwang Kim, Key Sun Park + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.107573

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Abstract

Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the -615/-516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the -588/-560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the -588/-560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the -588/-560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Highlights

National University in 2008. 1 Both authors contributed to this work. 2 Present address: Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, MD 20892. 3 Present address: Institute of Drug Research and Development, Chungnam
We focused on the nuclear factor I (NFI)-B for further study as it strongly binds to the TSH␤ promoter
Because NFI has been reported to be phosphorylated in adipocytes treated with insulin [15], we examined the effects of genistein, an inhibitor of tyrosine protein kinase, on TSH␤ promoter activity in the presence of NFI-B or insulin

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—T␣T1, L␤T2, and ␣T1-1 cells, which were derived from thyrotroph, gonadotroph, and progenitor of thyrotroph cells in mouse anterior pituitary, respectively, were generous gifts from Dr Pamela Mellon (University of California, San Diego) These cells were grown in DMEM supplemented with 10% fetal bovine serum. KpnI to EcoRI fragment spanning Ϫ795 to Ϫ580 region was PCR-amplified using forward primer (5Ј-CCAGGTACCTTAGATAAACAGTGATC-3Ј) and reverse primer (5Ј-CCCGAATTCAACTGACCTGAGATCAAAT-3Ј). EcoRI to NheI fragment spanning Ϫ568 to ϩ13 region was PCR-amplified using forward primer (5Ј-CCCGAATTCGATTTAGCCACGCTATCAG-3Ј) and reverse primer (5Ј-CATGCCTGCAGGTCGACTGGCTAGCCAGG-3Ј). These two PCR products were simultaneously ligated into the pLuc-link vector, which was cut with KpnI and NheI restriction enzymes.

RESULTS

DISCUSSION

According to our EMSA and