NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen.

Sang Jin Lee, KangRyul Lee, Xiumei Yang, Chaeyong Jung, Thomas Gardner, Hong Sup Kim, Meei Huey Jeng, Chinghai Kao

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40% higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell.

Original languageEnglish
Pages (from-to)749-760
Number of pages12
JournalJournal of Molecular Biology
Volume330
Issue number4
StatePublished - Jul 18 2003

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Binding Sites
Gene Expression
Transcription Factor AP-1
Prostate
Protein Isoforms
Transcription Factors
Ionomycin
Calcium Ionophores
Site-Directed Mutagenesis
Sepharose
Introns
Transfection
human glutamate carboxypeptidase II
Prostatic Neoplasms
Epithelium
Calcium
Antibodies
DNA
Genes
Proteins

ASJC Scopus subject areas

  • Virology

Cite this

NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen. / Lee, Sang Jin; Lee, KangRyul; Yang, Xiumei; Jung, Chaeyong; Gardner, Thomas; Kim, Hong Sup; Jeng, Meei Huey; Kao, Chinghai.

In: Journal of Molecular Biology, Vol. 330, No. 4, 18.07.2003, p. 749-760.

Research output: Contribution to journalArticle

Lee, Sang Jin ; Lee, KangRyul ; Yang, Xiumei ; Jung, Chaeyong ; Gardner, Thomas ; Kim, Hong Sup ; Jeng, Meei Huey ; Kao, Chinghai. / NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen. In: Journal of Molecular Biology. 2003 ; Vol. 330, No. 4. pp. 749-760.
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abstract = "Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40{\%} higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell.",
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