Abstract
SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by ∼1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the ∼5 kb 5'-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5′ flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-β containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-β-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-β-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-β-dependent regulation of inflammation-associated SerpinB2 expression.
Highlights
The inflammatory response is a double-edged sword
PMAinduced SerpinB2 gene regulation has been extensively studied in human macrophage cell lines [45,46,47], and has been shown to occur through several proximal and distal activator protein 1 (AP-1) responsive elements [30;37;47,48,49,50,51]
The results showed that C/EBP-b is constitutively present at the SerpinB2 promoter as demonstrated by its association with the promoter in unstimulated macrophages and increased temporally in response to LPS reaching as much as 10-fold over unstimulated cells after 8 hrs (Fig. 7)
Summary
The inflammatory response is a double-edged sword. Properly orchestrated, it results in the clearing of foreign molecules and invading pathogens from the body. Many of the pathological manifestations of the inflammatory response are mediated by cytokines and other inducible gene products expressed by macrophages upon exposure to the gram-negative bacterial cell wall component LPS. The SerpinB2 gene is highly regulated in a cell type specific manner analogous to that of cytokines and oncogenes [29;30] It is one of the most responsive genes known [31], and can be induced over 1000-fold by LPS [31,32], and is up-regulated by a range of inflammatory mediators [9]. A C/EBP-b phosphoacceptor site was found to negatively regulate LPS-induced SerpinB2 promoter activity Together, these findings provide new insight into the transcriptional regulation of the SerpinB2 gene
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