Cell Type-dependent Regulation of the Hypoxia-responsive Plasminogen Activator Inhibitor-1 Gene by Upstream Stimulatory Factor-2

Elitsa Y Dimova,Thomas Kietzmann

doi:10.1074/jbc.m512078200

Abstract

Transcriptional regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene is an important issue since PAI-1 plays a crucial role in various pathological conditions. The transcription factor USF-2 was shown to be a negative regulator for rat PAI-1 expression, and therefore it was the aim of this study to evaluate the role of USF-2 for human PAI-1 expression. We found in human hepatoma cells (HepG2) that USF-2 induced human PAI-1 expression via two classical E-boxes and the hypoxia-responsive element (HRE) within the promoter. Gel-shift analyses showed that E-box 4 and E-box 5 bound USFs, and although the HRE contributed to the USF-dependent effects, it did not bind them. By contrast, USF-2 inhibited PAI-1 promoter activity in primary rat hepatocytes suggesting that PAI-1 expression depends on either the promoter context or USF activity which might be cell type-specific. Cotransfection of human or rat PAI-1 promoter luciferase constructs with expression vectors for wild-type USF-2 or USF-2 mutants in human HepG2 and rat H4IIE cells as well as in primary rat hepatocytes revealed that the effects of USF on PAI-1 expression depend on the cell type rather than the promoter context and that the USF-specific region domain of USF accounts for the observed cell type-specific effects.

Highlights

USF-1 and USF-2 are members of the basic helix-loop-helix leucine zipper transcription factor family and were first identified by their abil
To investigate the involvement of the hypoxia-responsive element (HRE) as well as E4 and E5 in the USF-2-dependent induction of human plasminogen activator inhibitor type-1 (PAI-1) expression, the Ϫ806 bp wild-type human PAI-1 promoter Luc construct and its derivatives mutated in the HRE as well as in the classical E-boxes were cotransfected with the USF-2 expression vector or an empty vector into HepG2 cells
Regulation of PAI-1 Promoter Luciferase Gene Constructs by Wildtype and Mutant USF-2 in Different Cell Lines—Since our results from the transfection studies and the results of our previous study with the rat PAI-1 promoter [6] implicate that the regulation by USF depends on the promoter context, different domains of USF, or the cell type, we investigated the different regulation of human and rat PAI-1 promoter Luc constructs together with plasmids expressing various USF-2 mutants in human (HepG2), rat (H4IIE) hepatoma cells, and primary rat hepatocytes

Summary

Introduction

USF-1 and USF-2 are members of the basic helix-loop-helix leucine zipper transcription factor family and were first identified by their abil-. Induction of Human PAI-1 mRNA and Protein Expression by USF-2 under Normoxia and Hypoxia—In HepG2 cells transfected with the empty control vector hypoxia enhanced PAI-1 mRNA by about 3.5-fold (Fig. 1). To investigate the involvement of the HRE as well as E4 and E5 in the USF-2-dependent induction of human PAI-1 expression, the Ϫ806 bp wild-type human PAI-1 promoter Luc construct (pGl3hPAI-806) and its derivatives mutated in the HRE as well as in the classical E-boxes were cotransfected with the USF-2 expression vector or an empty vector into HepG2 cells.

Results

Conclusion