Fig. 5

eHSP90α induces a feedforward loop of macrophage HSP90α secretion. a, b Abundant HSP90α secretion from the THP-1-derived macrophages after EndoMT CM and rHSP90α stimulation. THP-1-derived macrophages were pre-incubated with 1% FBS-containing medium for 16 h. The medium was then added with PBS or 15 μg/ml of rHSP90α, or replaced with Ctrl, Endo CM, or EndoMT CM for another 24 h. The treated macrophages were further incubated with 5 ml of fresh 1% FBS-containing medium for 24 h. The media were finally collected and subjected to immunoblot analysis (a) and quantitative measurement (b) of HSP90α. ^αP < 0.05 when compared with “Ctrl-1” group. ^βP < 0.05 when compared with “Mø(Ctrl-1) CM” group. ^γP < 0.05 when compared with “Mø(PBS) CM” group. c eHSP90α binds macrophage TLR4 and CD91 receptors. PLAs showed red fluorescent dots in rHSP90α-treated macrophages by using the antibody combinations detecting the physical interactions of TLR4–HSP90α, CD91–HSP90α, TLR4–MyD88, and CD91–MyD88, suggesting that HSP90α binds to TLR4 and CD91 which could further recruit MyD88. d mRNA levels of HSP90α, TNF-α, IL-1β, CD163, CD204, IL-10, and TGF-β in the macrophages treated with PBS or rHSP90α in the absence or presence of control IgG or the antibody against CD91 or TLR4. Phenomena induced by rHSP90α such as downregulation of TNF-α and IL-1β mRNA levels and upregulation of HSP90α, IL-10, and TGF-β mRNA level were drastically abolished by CD91 and TLR4-antagonizing antibodies, whereas rHSP90α-induced CD163 and CD204 mRNA levels were repressed by the antibody antagonizing TLR4 but not CD91. e eHSP90α induces associations of MyD88 with JAK2 and TYK2. PLAs showed red fluorescent dots in rHSP90α-treated macrophages when using the antibody combinations detecting the interactions of MyD88–JAK2 and MyD88–TYK2 but not the antibody combinations detecting CD91–JAK2 and CD91–TYK2 interactions, suggesting that eHSP90α induced physical associations of JAK2 and TYK2 with MyD88 but not CD91. f Levels of phosphorylated/activated JAK2, TYK2, and STAT-3 in the macrophages treated with PBS or rHSP90α in the absence or presence of control IgG or anti-CD91 or -TLR4 antibody. Phosphorylation/activation of JAK2, TYK2, and STAT-3 was detected in rHSP90α-treated macrophages. Both TLR4 and CD91-antagonizing antibodies could inhibit the phosphorylation of JAK2 and STAT-3. However, anti-TLR4 antibody but not anti-CD91 antibody inhibited rHSP90α-induced TKY2 phosphorylation. g Levels of phosphorylated/activated STAT-3 in the macrophages treated with PBS or rHSP90α in the absence or presence of 10 μM JAK2/TYK2 inhibitor (JSI-124) or 10 nM JAK2 inhibitor (JAKi). h ChIP assay showed that rHSP90α induced binding of STAT-3 to HSP90α gene promoter in macrophages. i mRNA level of HSP90α in the macrophages treated with PBS or rHSP90α in the absence or presence of JSI-124 or JAKi. rHSP90α-induced macrophage HSP90α expression was effectively prevented by JSI-124 and JAKi