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. No rthern blot analysis of hepatic MT-I mRNA in interleukin (IL)-6 or tumour n ecrosis 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 exp ression in vivo after LPS injection. In vivo genomic footprinting of the MT -I promoter demonstrated a rapid increase, after LPS injection, in the prot ection of several guanine residues in the -250 to -300 bp region of the MT- I promoter. The protected bases were within sequences which resemble bindin g sites for the signal transducers and activators of transcription (STAT) t ranscription factor family. Electrophoretic mobility-shift assays using oli gonucleotides from foot-printed MT-I promoter regions showed that injection of LPS resulted in a rapid increase in the specific, high-affinity, in vit ro binding of STAT1 and STAT3 to a binding site at -297 bp (TTCTCGTAA), Wes tern blotting of hepatic nuclear proteins showed that the time-course for c hanges of total nuclear STAT1 and STAT3 after LPS injection paralleled the increased complex formation in vitro using this oligonucleotide, and bindin g was specifically competed for by a functional STAT-binding site from the rat a,macroglobulin promoter. Furthermore, the MT-I promoter -297 bp STAT-b inding site conferred IL-6 responsiveness in the context of a minimal promo ter ill transient transfection assays using HepG2 cells. This study suggest s that the effects of LPS on hepatic MT-I gene expression are mediated by I L-6 and involve the activation of STAT-binding to the proximal promoter.