Interaction between signaling molecules and HSP90 on apoptosis pathway in HSG cells

近年来，有报道称HSP家族蛋白作为分子伴侣蛋白调控了许多信号分子。本研究主要研究了信号分子与HSP90在人唾液腺腺癌细胞（HSG）凋亡通路中的相互作用。在HSG细胞中，TNF-α单独不诱导细胞凋亡。另一方面，单独使用激动性Fas抗体诱导30%的细胞凋亡，TNF-α治疗后再使用激动性Fas抗体诱导60%的细胞凋亡。TNF-α处理后凋亡信号分子的RT-PCR分析显示Fas高表达。因此，TNF-α增加Fas可增强Fas抗体介导的细胞凋亡。从凋亡通路中起重要作用的caspase抑制剂的实验结果来看，caspase-8和caspase-3都参与了激动性Fas抗体诱导的细胞凋亡。用热休克蛋白90特异性抑制剂格尔达霉素处理HSG细胞。Fas抗体阳性的细胞凋亡率为30%，而格尔达霉素联合Fas抗体阳性的细胞凋亡率为90%。提示HSP90参与了凋亡信号分子的调控。激动性Fas抗体刺激后的Western blotting分析显示，HSP90β亚型的蛋白水平升高，而HSP90α亚型的蛋白水平没有变化。此外，对HSG细胞进行蛋白质组分析。在双向凝胶电泳上观察到的HSP90蛋白点在激动性Fas抗体刺激后呈偏酸性，提示HSP90被磷酸化。

Recently, it has been reported that a number of signaling molecules are regulated by the HSP family proteins which function as molecular chaperones. The present study focused on the interaction between signaling molecules and HSP90 on apoptosis pathway in the human salivary gland adenocarcinoma cell line (HSG).In HSG cells, TNF-α alone did not induce apoptosis. On the other hand, agonistic Fas antibody alone induced apoptosis in 30 % of the cells and TNF-α treatment followed by agonistic Fas antibody induced apoptosis in 60 % of the cells. RT-PCR analysis of apoptotic signaling molecules after TNF-α treatment revealed the highly expression of Fas. Thus, it was suggested that Fas increased by TNF-α enhanced the agonistic Fas antibody-mediated apoptosis. From the results of the experiment with inhibitors for caspases which play important roles for apoptotic pathway, both caspase-8 and caspase-3 are involved in the agonistic Fas antibody-induced apoptosis.HSG cells were treated with geldanamycin which is a specific inhibitor for HSP90. While agonistic Fas antibody showed apoptosis in 30 % of the cells, the combination of geldanamycin and agonistic Fas antibody increased to 90 % of the cells. This result suggested that HSP90 participates in the regulation of apoptotic signaling molecules. Western blotting analysis after the agonistic Fas antibody stimulation showed that the protein level of HSP90β isoform increased while that of HSP90α did not changed. Furthermore, proteome analysis of HSG cells was performed. Protein spots of HSP90 observed on the two-dimensional gel electrophoresis were shifted to the more acidic after the agonistic Fas antibody stimulation, suggesting the phosphorylation of HSP90.