H2S硫巯基化修饰GRP78调控ERS-IRE1途径介导TAM-M1/M2表型转化作用机制

The phenotypic switch in tumor-associated macrophage (TAM) mediates immunity escape of carcinoma. Hydrogen sulfide (H2S) , which are known as the third gasotransmitters after the nitric oxide (NO) and carbon monoxide (CO) , involved in regulation of macrophage polarization and its mediated inflammatory response. And yet, the function of H2S and the underlying mechanisms in the phenotype switch of TAMs are still unclear. Applicants have found that H2S can induce the reduction of M2-type markers and the increase of M1-type markers of TAMs. And we detected synchronous GRP78 S-sulfhydration and the increase of IRE1 phosphorylation which is the marker of ERS-IRE1 pathway. Furthermore, the result of theoretical prediction shows the active site of IRE1 inhibitor-GRP78 contain one free cysteine residues. These above-mentioned findings contribute an understanding that H2S may play a role in the activation of ERS-IRE1 pathway and in the following TAM-M1/M2 phenotype switch through GRP78 S-sulfhydration. Through studying the correlation between TAM-M1/M2 phenotype switch mediated by H2S and GRP78 S-sulfhydration, the project intends to clarify the the mechanism of activation of ERS-IRE1 pathway by H2S mediated GRP78 S-sulfhydration and illustrate the key roles of ERS-IRE1 pathway in the regulation of TAM-M1/M2 phenotype switch by H2S. The study will establish the experimental basis for further research which explore the processes and mechanisms of tumor immune escape mediated by TAM-M1/M2 phenotype switch regulating inflammatory tumor microenvironment.

肿瘤相关巨噬细胞（TAM）表型转化介导肿瘤免疫逃逸。H2S是继NO、CO后发现的第三种气体信号分子，参与调控巨噬细胞极化及其介导的炎症反应，但在TAM表型转化中作用与机制不清。申请者发现H2S可诱导TAM的M2型标志物减少，M1型标志物增加；同步检测到GRP78硫巯基化及ERS-IRE1途径标志分子IRE1磷酸化增加；理论预测发现IRE1抑制因子GRP78活性部位包含1个游离半胱氨酸残基，提示H2S可能通过GRP78硫巯基化修饰活化ERS-IRE1途径介导TAM-M1/M2表型转化。本项目拟通过检测H2S介导的TAM-M1/M2表型转化与GRP78硫巯基化的相关性，明确H2S硫巯基化修饰GRP78关键位点活化ERS-IRE1途径分子机制，阐明ERS-IRE1途径是H2S调控TAM-M1/M2表型转化的关键通路，为深入研究TAM-M1/M2表型转化调控肿瘤炎性微环境介导免疫逃逸奠定实验基础。

本项目拟研究H2S调控肿瘤相关巨噬细胞（TAM-M1/M2）表型转化的作用机制。通过TCGA数据库发现，乳腺癌中CSE表达与CD163表达呈负相关；H2S处理TAM细胞后，M1表型相关标志物表达升高，M2表型相关标志物表达降低；H2S处理后的TAM上清液诱导乳腺癌细胞侵袭迁移能力减弱；PPG处理TAM细胞后，M1表型相关标志物表达降低，M2表型相关标志物表达升高；PPG处理后的TAM上清液诱导乳腺癌细胞侵袭迁移能力增强。为考察H2S调控TAM表型转化与ERS-IRE1 途径相关性，以H2S处理TAM，发现p-IRE-1α、p-P65和p-JNK蛋白表达水平增加，IRE-1α和其他内质网受体的表达无显著变化；抑制IRE-1α活化降低p-IRE-1α、p-P65和p-JNK表达； CoIP实验证实H2S抑制IRE-1α与GRP78结合；硫巯基化水平检测发现H2S诱导GRP78硫巯基化修饰，将GRP78-cys41位点突变为丙氨酸后，H2S诱导GRP78硫巯基化修饰水平降低，H2S介导的GRP78与IRE-1解离作用被抑制，p-IRE-1α、p-P65、p-JNK和M1标志物表达水平降低。构建GRP78野生型和GRP78-cys41位点突变的过表达载体及TAM稳转细胞株，利用H2S处理后，将TAM与MCF-7混合并尾静脉注射入裸鼠体内，发现H2S可抑制TAM-GRP78野生型组小鼠MCF-7肺转移发生, 而对GRP78-cys41突变型组小鼠MCF-7肺转移无显著影响。上述研究结果提示，H2S通过硫巯基化修饰GRP78-cys41位点促进ERS-IRE1 途径活化介导TAM细胞M1表型，可能是H2S调控TAM表型转化的新机制。