Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, Georgia, USA.
PLoS One. 2013 Aug 13;8(8):e71366. doi: 10.1371/journal.pone.0071366. eCollection 2013.
We have previously reported that free Heme generated during experimental cerebral malaria (ECM) in mice, is central to the pathogenesis of fatal ECM. Heme-induced up-regulation of STAT3 and CXCL10 promotes whereas up-regulation of HO-1 prevents brain tissue damage in ECM. We have previously demonstrated that Heme is involved in the induction of apoptosis in vascular endothelial cells. In the present study, we further tested the hypothesis that Heme reduces blood-brain barrier integrity during ECM by induction of apoptosis of brain vascular endothelial cells through STAT3 and its target gene matrix metalloproteinase three (MMP3) signaling.
Genes associated with the JAK/STAT3 signaling pathway induced upon stimulation by Heme treatment, were assessed using real time RT(2) Profile PCR arrays. A human MMP3 promoter was cloned into a luciferase reporter plasmid, pMMP3, and its activity was examined following exposure to Heme treatment by a luciferase reporter gene assay. In order to determine whether activated nuclear protein STAT3 binds to the MMP3 promoter and regulates MMP3 gene, we conducted a ChIP analysis using Heme-treated and untreated human brain microvascular endothelial cells (HBVEC), and determined mRNA and protein expression levels of MMP3 using qRT-PCR and Western blot. Apoptosis in HBVEC treated with Heme was evaluated by MTT and TUNEL assay.
The results show that (1) Heme activates a variety of JAK/STAT3 downstream pathways in HBVEC. STAT3 targeted genes such as MMP3 and C/EBPb (Apoptosis-related genes), are up regulated in HBVEC treated with Heme. (2) Heme-induced HBVEC apoptosis via activation of STAT3 as well as its downstream signaling molecule MMP3 and upregulation of CXCL10 and HO-1 expressions. (3) Phosphorylated STAT3 binds to the MMP3 promoter in HBVEC cells, STAT3 transcribed MMP3 and induced MMP3 protein expression in HBVEC cells.
Activated STAT3 binds to the MMP3 promoter region and regulates MMP3 in Heme-induced endothelial cell apoptosis.
我们之前报道过，在实验性脑疟疾（ECM）中产生的游离血红素是导致致命性 ECM 发病机制的关键。血红素诱导的 STAT3 和 CXCL10 的上调促进了脑组织损伤，而 HO-1 的上调则阻止了 ECM 中的脑组织损伤。我们之前已经证明血红素参与了血管内皮细胞凋亡的诱导。在本研究中，我们通过 STAT3 及其靶基因基质金属蛋白酶 3（MMP3）信号通路进一步验证了血红素通过诱导脑血管内皮细胞凋亡导致 ECM 期间血脑屏障完整性降低的假设。
使用实时 RT(2) Profile PCR 阵列评估血红素处理后诱导的与 JAK/STAT3 信号通路相关的基因。将人 MMP3 启动子克隆到荧光素酶报告质粒 pMMP3 中，并通过荧光素酶报告基因测定法检测血红素处理后其活性。为了确定激活的核蛋白 STAT3 是否与 MMP3 启动子结合并调节 MMP3 基因，我们使用血红素处理和未处理的人脑血管内皮细胞（HBVEC）进行了 ChIP 分析，并使用 qRT-PCR 和 Western blot 测定 MMP3 的 mRNA 和蛋白表达水平。通过 MTT 和 TUNEL 测定评估血红素处理的 HBVEC 中的细胞凋亡。
结果表明：（1）血红素在 HBVEC 中激活多种 JAK/STAT3 下游途径。血红素处理的 HBVEC 中，STAT3 靶向基因如 MMP3 和 C/EBPb（凋亡相关基因）上调。（2）血红素通过激活 STAT3 及其下游信号分子 MMP3 以及上调 CXCL10 和 HO-1 的表达，诱导 HBVEC 细胞凋亡。（3）磷酸化 STAT3 与 HBVEC 细胞中的 MMP3 启动子结合，STAT3 转录 MMP3 并诱导 HBVEC 细胞中 MMP3 蛋白表达。
激活的 STAT3 与血红素诱导的内皮细胞凋亡中的 MMP3 启动子区域结合并调节 MMP3。
