The Transcription Factor C/EBP-β Mediates Constitutive and LPS-Inducible Transcription of Murine SerpinB2

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.

丝氨酸蛋白酶抑制剂B2（SerpinB2，亦称纤溶酶原激活物抑制剂2型，PAI-2）可在炎症刺激下于巨噬细胞中被高度诱导，并与固有免疫调控、巨噬细胞存活及纤溶酶原激活剂抑制活性密切相关。脂多糖（LPS）作为强效细菌内毒素，可通过Toll样受体4（TLR4）诱导小鼠巨噬细胞中SerpinB2的表达，在24小时内其表达水平可上调约1000倍。为定位受LPS调控的SerpinB2启动子区域，我们将小鼠SerpinB2基因约5kb的5'侧翼序列及其多个缺失突变体驱动的报告基因构建体转染至小鼠巨噬细胞中。此外，我们将小鼠5'侧翼序列的DNA序列与人类同源基因序列进行比对以鉴定同源功能性调控元件，并发现人类SERPINB2启动子内的多个调控顺式作用元件在小鼠中具有保守性。突变分析结果显示，小鼠SerpinB2近端启动子中的CCAAT增强子结合蛋白（C/EBP）结合元件、环腺苷酸应答元件（CRE）以及两个激活蛋白1（AP-1）应答元件，是实现最佳LPS诱导表达活性所必需的。电泳迁移率变动分析（EMSA）与染色质免疫沉淀（ChIP）实验证实，LPS可诱导形成包含CCAAT增强子结合蛋白β（C/EBP-β）的复合物并结合至SerpinB2启动子区域。尤为重要的是，在C/EBP-β基因敲除的小鼠胚胎成纤维细胞（MEFs）以及原代C/EBP-β缺陷型腹腔巨噬细胞中，组成型表达与LPS诱导的SerpinB2表达均被显著抑制。综上，本研究数据为阐释炎症相关SerpinB2表达的C/EBP-β依赖性调控机制提供了新的见解。