COLD-INDUCED ALTERATIONS IN THE BINDING OF ADRENOMEDULLARY NUCLEAR PROTEINS TO THE PROMOTER REGION OF THE TYROSINE-HYDROXYLASE GENE

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland. Formation of the cold-induced DNA/nucleoprotein complex was inhibited by competition with a synthetic oligode-oxyribonucleotide containing a consensus sequence for the AP1 binding site. Pretreatment of adrenomedullary nuclear extracts with antibodies to either c-Fos or c-Jun also inhibited the cold-induced alteration in DNA/nucleoprotein binding. Heat denaturation of the anti-Fos or anti-Jun antibodies, or preadsorption of the antibodies with their cognate antigenic peptides eliminated the inhibitory effect. Taken together, these findings suggest that the cold-induced increases in adrenomedullary TH gene expression are mediated, at least in part, through the interaction of a phorbol ester-responsive element and proto-oncogene transcription factors.