title: Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease.
abstract: We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5'-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the -250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Over-expression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3, and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells. Previously, we demonstrated that protein kinase D1 (PKD1) plays a survival role during the early stage of oxidative stress in dopaminergic neuronal cells.
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Muhammet
authors.initials: M
authors.lastname: Ay
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Huajun
authors.initials: H
authors.lastname: Jin
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Dilshan S
authors.initials: DS
authors.lastname: Harischandra
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Arunkumar
authors.initials: A
authors.lastname: Asaithambi
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Arthi
authors.initials: A
authors.lastname: Kanthasamy
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Vellareddy
authors.initials: V
authors.lastname: Anantharam
authors.affiliation: Parkinson's Disorder Research Laboratory, Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, Iowa, USA.
authors.forename: Anumantha G
authors.initials: AG
authors.lastname: Kanthasamy
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D000077209:Decitabine; D059290:Dopaminergic Neurons / Q000473:pathology*; D044127:Epigenesis, Genetic / Q000235:genetics*; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010300:Parkinson Disease / Q000235:genetics* / Q000473:pathology*; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D000077209:Decitabine; D014446:Tyrosine 3-Monooxygenase; C089212:protein kinase D; D011493:Protein Kinase C; D001374:Azacitidine
keywords: PKD1; Parkinson's disease; epigenetic regulation; neurodegeneration; promoter function; redox signaling