Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter

Abstract

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction.

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

“…The direct activation of PKC with PMA increased PAI-2 expression 33.3±+16.0-and 81.8±16.4-fold in RME cells and RASMC, respectively. This is consistent with previous reports that have demonstrated that PKC increases the transcription of PAI-2 via two AP1 binding sites located in the 5 ' flanking promoter region of the gene (35). The .…”

“…The effects of PMA on PAI-I and PAI-2 mRNA expression were also compared in these two cell types. Previous reports have demonstrated that phorbol ester stimulates the expression of PAI-2 in a number of other cell types (34)(35)(36). This analysis revealed that the PMA-stimulated expression of PAI-2 was 33.3+16.0 (SEM, n = 5) in RME cells and 81.8-+16.4 (SEM, n = 4) in RASMC.…”

“…Tyrosine kinase inhibitors have been shown to block the endothelin-l-stimulated induction of cfos and activation of AP-1 cis-element activity in glomerular mesangial cells (51). Since Cousins et al (35) have shown that two AP-1 binding sites in the 5' flanking region of the PAI-2 promoter are required for the PKC-stimulated transcription of PAI-2, this effect of tyrosine kinase inhibition by genistein may also apply to the PKC-dependent mechanism of All-stimulated PAI-2 expression. Second, in addition to the PKC-dependent mechanism of All-stimulated PAI-2 expression that is observed in both RME cells and RASMC, All also stimulates a PKCindependent pathway that increases PAI-2 mRNA in RME cells.…”

Transcriptional and posttranscriptional regulation of the plasminogen activator system

“…Va riousagents controlling PA I-2 geneexpression have beenreported,including growth factors(TGFβ ,EGF,M-CSF and GM-CSF), hormones (retinoic acid,dexamethasoneand vitamin D3), cytokines (TNF α ,IL-1 and IL-2),vasoactivepeptides(angiotensin II), toxins (dioxin and endotoxin) and tumor promoters (phorbol esters and okadaic acid) (172).Ofthese regulators, the most investigated are tumor promoters and TNFα .I nt he promoter proximal region therea re twoA P1-likee lements, AP1a (TGAATCA: -103 to -97)a nd AP1b (TGAGTAA: -114t o -108), and one CRE-likeelement (TGACCTCA:-187 to -182) (173).Itw as shown in humanHT1080 fibrosarcoma cells that AP1a and CRE-likesitesare required forboth basaland PMAinducedP AI-2 gene activation (173). It seemst hat c-Jun and JunD aret he major components binding to the AP1a element under both basala nd PMA-treatedc onditions (174).I nterestingly, basaland PMA-inducedtranscription of the PA I-2 genein HT1080 and U937 cells wassignificantly greater with a-219-bp than with a-1100-bp promoter construct, suggesting the presenceofarepressor site between -219 and -1100 (174).…”

Urokinase‐mediated transactivation of the plasminogen activator inhibitor type 2 (PAI‐2) gene promoter in HT‐1080 cells utilises AP‐1 binding sites and potentiates phorbol ester‐mediated induction of endogenous PAI‐2 mRNA

“…Analysis of the proximal PAI-2 gene promoter has identified at least three functionally relevant regulatory sites which are responsible for constitutive and inducible expression. Two of these sites are related to the AP-1 binding site consensus sequence, while the other site is related to the cyclic AMP response element [11].…”