JFC1 is transcriptionally activated by nuclear factor-kappaB and up-regulated by tumour necrosis factor alpha in prostate carcinoma cells.
The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549 bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide -321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-kappaB (NF-kappaB). We analysed the three putative NF-kappaB binding sites by gel retardation and supershift assays. Each of the putative NF-kappaB sites interacted specifically with recombinant NF-kappaB p50, and the complexes co-migrated with those formed by the NF-kappaB consensus sequence and p50. An antibody to p50 generated a supershifted complex for these NF-kappaB sites. These sites formed specific complexes with nuclear proteins from tumour necrosis factor alpha (TNFalpha)-treated WEHI 231 cells, which were supershifted with antibodies against p50 and p65. The jfc1 promoter was transcriptionally active in various cell lines, as determined by luciferase reporter assays following transfection with a jfc1 promoter luciferase vector. Co-transfection with NF-kappaB expression vectors or stimulation with TNFalpha resulted in significant transactivation of the jfc1 promoter construct, although transactivation of a mutated jfc1 promoter was negligible. The expression of a dominant negative IkappaB (inhibitor kappaB) decreased basal jfc1 promoter activity. The cell lines PC-3, LNCaP and DU-145, but not Epstein-Barr virus-transformed lymphocytes, showed a dramatic increase in the expression of JFC1 after treatment with TNFalpha, suggesting that transcriptional activation of JFC1 by the TNFalpha/NF-kappaB pathway is significant in prostate carcinoma cell lines.
Biochemistry Division, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Sergio D
SD
Catz
Bernard M
BM
Babior
Jennifer L
JL
Johnson
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011994:Recombinant Proteins / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology*
D008565:Membrane Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; C431146:SYTL1 protein, human; D014409:Tumor Necrosis Factor-alpha; D004247:DNA