title: Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif.
abstract: Induction of human plasminogen activator inhibitor type-2 (PAI-2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor-alpha (TNFalpha). Here we have examined whether PAI-2 gene transcription in response to TNFalpha may be mediated through a regulatory pathway involving the transcription factor, NF-kappaB. We have tested the function of two potential NF-kappaB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFalpha using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFalpha regulation of the PAI-2 gene through either of these two sites, one of the NF-kappaB-like motifs, transcriptional regulatory motif (TRM), present at position -400 was found to be essential for constitutive PAI-2 transcription, as mutation of this motif abolished basal PAI-2 promoter activity in both monocyte-like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM-binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF-kappaB regulatory complex. Expression screening of a HeLa cell cDNA library using the -400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto-oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI-2 transcription, and identify potential DNA-binding proteins associated with this element that may play a role in PAI-2 gene regulation.
authors.affiliation: Cellular Oncology Laboratory, Queensland Institute of Medical Research,University of Queensland, Brisbane, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Mahony
authors.forename: B
authors.initials: B
authors.lastname: Kalionis
authors.forename: T M
authors.initials: TM
authors.lastname: Antalis
mesh_terms: D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005354:Fibrosarcoma; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016328:NF-kappa B / Q000378:metabolism*; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
chemical_list: C000715470:MAS1 protein, human; D016328:NF-kappa B; D017396:Plasminogen Activator Inhibitor 2; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase