Abstract
Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5'-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.
Highlights
Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA
Our results support the hypothesis that regulation of the TH gene by Ca2؉ is mediated by mechanisms involving both the TH cAMPresponsive element (CRE) and TH activating protein-1 (AP1) sites and that transcription factors other than or in addition to cAMP-responsive element binding protein (CREB) participate in this response
Our results indicate that CREB is not required for Ca2ϩ-dependent regulation of the TH gene, supporting the hypothesis that multiple promoter elements are involved in regulation of the TH gene promoter in response to Ca2ϩ influx
Summary
Materials—RPMI 1640 medium and penicillin/streptomycin were purchased from Life Technologies, Inc. Separate dishes of cells were transfected with CAT-Basic (a plasmid containing the CAT gene without a promoter to drive its expression). Approximately 0.2 pmol (50,000 cpm) of 32P-radiolabeled TH AP1 oligonucleotide probe was incubated with 10 g of nuclear protein extract on ice for 20 min in a reaction buffer containing 25 mM HEPES (pH 7.5), 6% glycerol, 0.1 M NaCl, 0.1% Triton X-100, 2 mM EDTA, 0.6 mM spermidine, 0.2 mM spermine, 0.1 mM phenylmethylsulfonyl fluoride, 0.25 g/ml leupeptin, 0.25 g/ml pepstatin, 10 g/ml poly(dI-dC), and 0.4 mM dithiothreitol. Control cell lines were constructed by stably transfecting PC12 cells with the null vector, pBK-RSV These clonal cell lines were transiently transfected with TH(Ϫ272/ϩ27)-CAT and treated with appropriate drugs as described above. A p value less than 0.05 was considered statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
