Identification of a signal transducer and activator of transcription (STAT) binding site in the mouse metallothionein-I promoter involved in interleukin-6-induced gene expression.
Mechanisms of regulation of mouse metallothionein (MT)-I gene expression in response to bacterial endotoxin-lipopolysaccharide (LPS) were examined. Northern blot analysis of hepatic MT-I mRNA in interleukin (IL)-6 or tumour necrosis factor (TNF)-receptor type I knock-out mice demonstrated that IL-6, not TNF-alpha, is of central importance in mediating hepatic MT-I gene expression in vivo after LPS injection. In vivo genomic footprinting of the MT-I promoter demonstrated a rapid increase, after LPS injection, in the protection of several guanine residues in the -250 to -300 bp region of the MT-I promoter. The protected bases were within sequences which resemble binding sites for the signal transducers and activators of transcription (STAT) transcription factor family. Electrophoretic mobility-shift assays using oligonucleotides from footprinted MT-I promoter regions showed that injection of LPS resulted in a rapid increase in the specific, high-affinity, in vitro binding of STAT1 and STAT3 to a binding site at -297 bp (TTCTCGTAA). Western blotting of hepatic nuclear proteins showed that the time-course for changes of total nuclear STAT1 and STAT3 after LPS injection paralleled the increased complex formation in vitro using this oligonucleotide, and binding was specifically competed for by a functional STAT-binding site from the rat alpha2-macroglobulin promoter. Furthermore, the MT-I promoter -297 bp STAT-binding site conferred IL-6 responsiveness in the context of a minimal promoter in transient transfection assays using HepG2 cells. This study suggests that the effects of LPS on hepatic MT-I gene expression are mediated by IL-6 and involve the activation of STAT-binding to the proximal promoter.
Lee
D K
DK
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS 66160-7421, USA.
Carrasco
J
J
Hidalgo
J
J
Andrews
G K
GK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D016209:Interleukin-8 / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D018124:Receptors, Tumor Necrosis Factor; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D008668:Metallothionein