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In Vivo Identification of Multiple Response Elements Mediating Thyroid Hormone Stimulation of Hepatic HMG‐CoA Reductase Transcription
Author(s) -
Boone Lindsey R.,
Ness Gene C.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.689.10
Subject(s) - transcription factor , electroporation , luciferase , reductase , transcription (linguistics) , response element , medicine , promoter , in vivo , endocrinology , microbiology and biotechnology , biology , messenger rna , deiodinase , sterol regulatory element binding protein , chemistry , triiodothyronine , enzyme , transfection , hormone , biochemistry , gene expression , gene , genetics , linguistics , philosophy
Hepatic HMG‐CoA reductase (HMGR) is the enzyme that catalyzes the rate‐limiting step in cholesterol biosynthesis. Hypothyroidism is a known risk factor for cardiovascular disease. HMGR transcription is decreased in hypothyroid mammals. Treatment of hypothyroid (Hx) Sprague Dawley rats with triiodothyronine (T 3 ) increased HMGR mRNA by approximately 2.7‐fold. We have utilized in vivo electroporation to introduce HMGR promoter‐luciferase constructs directly into the livers of Hx Sprague Dawley rats treated ±T 3 in an effort to identify the regions of the HMGR promoter responsible for this T 3 ‐mediated regulation. Electroporation of an HMGR promoter construct inclusive of the ‐325/+70 promoter region resulted in a 2.6‐fold induction of promoter activity in the T 3 ‐treated animal. Mutations in the sterol response element (SRE), nuclear factor‐y site (NF‐Y), and the sequence AGGTCA at ‐316/‐321 identified the necessity of these elements for the T 3 response. EMSAs with liver nuclear extracts from +T 3 rats showed strong shifted bands with probes to each of these elements. Antibody supershift assays revealed that SREBP‐2 binds to the SRE, NF‐Y binds to the NF‐Y site, and USF‐2 binds to the AGGTCA element at ‐316/‐321. In vivo electroporation of siRNA to SREBP‐2 was shown to reduce HMGR mRNA in normal Sprague Dawley rats to an average of 60% of control. These data suggest that the SRE, NF‐Y site, ‐316/‐321 element and their corresponding binding proteins appear to mediate the T 3 response. (This investigation was supported by NIH grant DK075414).