Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif

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-α (TNFα). Here we have examined whether PAI-2 gene transcription in response to TNFα may be mediated through a regulatory pathway involving the transcription factor, NF-κB. We have tested the function of two potential NF-κB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFα using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFα regulation of the PAI-2 gene through either of these two sites, one of the NF-κB-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-κB 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

Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells