The role of c -Fos in regulation of tyrosine hydroxylase gene expression

Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of catecholamines. Gene expression of TH is regulated by many pharmacological and physiological processes. The AP1 site at position -205 to -199 in the TH gene proximal promoter is necessary for optimal stimulation of TH gene promoter activity in response to calcium influx or PKC activation in PC12 cells. Moreover, these stimuli dramatically induce c-Fos and increase formation of the TH AP1 complex. Results of supershift assays using specific c-Fos antibodies demonstrate that c-Fos is present in the induced TH AP1 complex. These observations support the hypothesis that c-Fos plays an important role in regulating TH gene expression in response to calcium influx or PKC activation. In this thesis, I directly test this hypothesis.;PC18 cells are a subclone of PC12 cells. These cells do not normally express c-Fos under either basal or stimulated conditions. By using the tetracycline-regulated inducible system, I have established clonal PC18 cell lines in which c-Fos expression is under the control of the tet operon. Results of nuclear run-on assays demonstrate that after c-Fos induction, TH gene transcription rate is dramatically increased, and this increase is dependent on the extent of c-Fos expression. Promoter analysis studies show that the transactivational activity of the AP1 site in the TH gene proximal promoter is also increased after c-Fos induction, but to a much lesser degree than the c-Fos-mediated stimulation of TH gene transcription rate. These results suggest that an increase in c-Fos protein is sufficient to stimulate TH gene transcription rate, but that this response is only partially dependent on the AP1 site in the TH gene proximal promoter.;By utilizing an antisense RNA strategy, c-Fos down-regulated clonal PC12 cell lines have been generated. In these cell lines, c-Fos protein induction elicited by either calcium influx or TPA treatment is dramatically inhibited. Nuclear run-on assays and promoter analyses demonstrate that stimulation of TH gene transcription rate and TH gene promoter activity in response to PKC activation or calcium influx are almost totally blocked in these c-Fos down-regulated cells. In contrast, the absence of c-Fos induction does not block the effect of PKA activation on either TH gene transcription rate or TH gene promoter activity. These results support the hypothesis that c-Fos expression is essential for maximal stimulation of the TH gene in response to either calcium influx or PKC activation, but not to PKA activation.