Neural and hormonal regulation of the tyrosine hydroxylase gene in adrenal medullary cells: Participation of c-fos and AP1 factors.
Previous studies identified nicotine and angiotensin receptors as major trans-synaptic and hormonal inputs controlling activities and mRNA levels of the catecholamine biosynthetic enzymes in adrenal medullary (AM) cells. The purpose of this study was to further explore the molecular mechanisms underlying these modes of regulations. Incubation of cultured bovine AM cells with nicotine or a stable analog of angiotensin II, sar1-angiotensin II, increased synthesis of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNAs (2- to 3-fold), suggesting transcriptional activation of these genes. No effects of nicotine or sar1-angiotensin II on TH and PNMT mRNA levels could be demonstrated in cells treated with cycloheximide or puromycin, suggesting regulation by inducible proteins. However, translational inhibitors produced small increases in basal TH mRNA levels (1.5- to 1.9-fold), indicating that repressor-like mechanisms participate in the regulation of the TH gene. Sequence analysis of the bovine TH promoter (1.6 kb) revealed the presence of several putative regulatory elements including four nonidentical AP1-like sites, potential targets for c-Fos/c-Jun/AP1 regulation. Formation of sequence-specific complexes between nuclear proteins and five fragments of the TH promoter (200-400 nt) was demonstrated by gel mobility shift assay (one to three complexes per fragment). Binding of proteins to three promoter fragments containing single AP1-like elements was antagonized by an oligodeoxynucleotide-containing AP1 consensus sequence (oligo-AP1), but not by other unrelated DNA sequences. Oligo-AP1 inhibited the formation of one or two complexes with individual DNA fragments. These observations suggest formation of different AP1-like complexes on individual promoter fragments. c-Fos antibody inhibited the formation of three oligo-AP1-displaceable complexes. The formation of the majority of retarded bands, including all AP1-like complexes, was increased by incubation of cells with sar1-angiotensin II or nicotine. Western analysis demonstrated the presence of c-Fos (p54), c-Jun (p36-38), and several c-Fos- and c-Jun-related antigens in control and stimulated AM cells. Sar1-angiotensin II and nicotine increased steady-state levels of c-Fos (1.5- to 2-fold) and c-fos mRNA (10- to 14-fold) and the levels of several c-Fos-related antigens (FRA) (2- to 7-fold). Some of FRA showed differential regulation by angiotensin II and nicotinic receptors. The levels of p36-38 c-Jun and c-jun mRNA were increased by sar1-angiotensin II (2- to 5-fold) and were less affected by nicotine. In conclusion, our results suggest transcriptional regulation of the TH gene by angiotensin II and nicotinic receptors, through partially different nuclear pathways. The regulation may involve multiple target sites in the TH promoter, inducible proteins, AP1-like factors, c-Fos, and c-Fos-related antigens.
Stachowiak
M K
MK
Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, USA.
Goc
A
A
Hong
J S
JS
Kaplan
B B
BB
Stachowiak
E K
EK