The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1

Deborah L. Galson, Terumasa Tsuchiya, Drory S. Tendler, L. Eric Huang, Yan Ren, Toshihiko Ogura, H. Franklin Bunn

研究成果: Article査読

174 被引用数 (Scopus)

抄録

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis- acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, RORα1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for RORα1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4ΔC, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4ΔC. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue- specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

本文言語English
ページ(範囲)2135-2144
ページ数10
ジャーナルMolecular and cellular biology
15
4
DOI
出版ステータスPublished - 1995 4月
外部発表はい

ASJC Scopus subject areas

  • 分子生物学
  • 細胞生物学

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