Wang Zhenhui, Li Chenghua, Xing Ronglian, Shao Yina, Zhao Xuelin, Zhang Weiwei, Guo Ming
School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province, 315211, PR China.
School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province, 315211, PR China; College of Life Sciences, Yantai University, Yantai, 264005, PR China.
Dev Comp Immunol. 2019 Feb;91:26-36. doi: 10.1016/j.dci.2018.10.004. Epub 2018 Oct 16.
Lipopolysaccharides (LPS) can induce the apoptosis of coelomocytes in Apostichopus japonicus (A. japonicus), and β-integrin serves as an apoptotic inhibitor during this process. However, the underlying mechanism in invertebrates is largely unknown. Integrin/focal adhesion kinase (FAK) signaling pathway modulates the apoptosis in vertebrates. In this study, a novel FAK was identified from A. japonicus (designated as AjFAK) by β-integrin (designated as AjITGB) -mediated GST-pull down assay. This interaction was further validated in the LPS-exposed coelomocytes through co-immunoprecipitation and immunofluorescence analyses. To investigate the functional role of AjFAK in AjITGB-mediated coelomocyte apoptosis, we cloned the full-length cDNA of AjFAK and characterized its relationship with AjITGB through real-time PCR. The mRNA expression levels of AjFAK exhibited consistent expression patterns with those of AjITGB in our previous work with 0.48- and 0.22-fold decreases at 12 and 96 h in LPS-exposed coelomocytes and in Vibrio splendidus challenged sea cucumber, respectively. Moreover, the expression level of AjFAK decreased to 0.35-fold in AjITGB knockdown treatment by specific small interference RNA (siRNA). We further performed an assay for the apoptotic rate of coelomocytes in AjITGB, AjFAK, and AjITGB/AjFAK silencing conditions and found that their apoptotic percentages increased to 26%, 25%, and 30%, respectively, compared with those of the control. Finally, the expression levels of four caspases from A. japonicus were also investigated to determine the apoptotic effector. After AjITGB or AjFAK was silenced, the mRNA levels of caspase-3 were 6.6-fold and 2.5-fold higher than those of the control, respectively. In addition, the enzymatic activity of caspase-3 was enhanced to 1.82- and 1.79-fold that of the control in the two groups. However, no significant changes were detected in caspase-2/6/8. All our results supported that β-integrin mediated the LPS-induced coelomocyte apoptosis in sea cucumber via the integrin/FAK/caspase-3 signaling pathway.
脂多糖（LPS）可诱导刺参体腔细胞凋亡，而β-整合素在此过程中作为凋亡抑制剂。然而，无脊椎动物中的潜在机制在很大程度上尚不清楚。整合素/粘着斑激酶（FAK）信号通路调节脊椎动物的细胞凋亡。在本研究中，通过β-整合素（命名为AjITGB）介导的GST下拉实验，从刺参中鉴定出一种新型FAK（命名为AjFAK）。通过免疫共沉淀和免疫荧光分析，在暴露于LPS的体腔细胞中进一步验证了这种相互作用。为了研究AjFAK在AjITGB介导的体腔细胞凋亡中的功能作用，我们克隆了AjFAK的全长cDNA，并通过实时PCR表征了其与AjITGB的关系。在我们之前的研究中，AjFAK的mRNA表达水平与AjITGB呈现一致的表达模式，在暴露于LPS的体腔细胞中，12小时和96小时时分别下降0.48倍和0.22倍，在灿烂弧菌攻击的海参中也有类似情况。此外，通过特异性小干扰RNA（siRNA）敲低AjITGB后，AjFAK的表达水平降至0.35倍。我们进一步检测了在AjITGB、AjFAK和AjITGB/AjFAK沉默条件下体腔细胞的凋亡率，发现与对照组相比，它们的凋亡百分比分别增加到26%、25%和30%。最后，还研究了刺参中四种半胱天冬酶的表达水平以确定凋亡效应器。在AjITGB或AjFAK沉默后，caspase-3的mRNA水平分别比对照组高6.6倍和2.5倍。此外，两组中caspase-3的酶活性分别增强到对照组的1.82倍和1.79倍。然而，caspase-2/6/8未检测到显著变化。我们所有的结果都支持β-整合素通过整合素/FAK/caspase-3信号通路介导海参中LPS诱导的体腔细胞凋亡。
