title: Stat3 and CCAAT/enhancer binding protein beta (C/EBP-beta) regulate Jab1/CSN5 expression in mammary carcinoma cells.
abstract: BACKGROUND
  The c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells.
  
  
  METHODS
  To identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling.
  
  
  RESULTS
  We identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3.
  
  
  CONCLUSIONS
  These findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.
authors.affiliation: Department of Systems Biology, University of Texas - MD Anderson Cancer Center, Houston, 77030, USA.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Shackleford
authors.forename: Qingxiu
authors.initials: Q
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Tian
authors.forename: Thuy T
authors.initials: TT
authors.lastname: Vu
authors.forename: Anita L
authors.initials: AL
authors.lastname: Korapati
authors.forename: Angela M
authors.initials: AM
authors.lastname: Baumgartner
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Le
authors.forename: Warren S
authors.initials: WS
authors.lastname: Liao
authors.forename: Francois X
authors.initials: FX
authors.lastname: Claret
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D000075686:COP9 Signalosome Complex; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D050982:GATA1 Transcription Factor / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering; D012045:Regulatory Sequences, Nucleic Acid; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C545012:CEBPB protein, human; D004268:DNA-Binding Proteins; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D019061:src-Family Kinases; D010447:Peptide Hydrolases; C501488:COPS5 protein, human; D000075686:COP9 Signalosome Complex