Abstract
Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.
Highlights
Glioma ranges from slowly growing low grade tumors to rapidly growing high grade tumors, including anaplastic astrocytoma and glioblastoma [1, 2]
An additive effect of the activation of cell invasion was observed when both agents were added together into the cell bath medium. These results suggested that extracellular heat shock protein 90 (HSP90)␣ exerted an additive effect on the ability of EGF to promote U87 cell migration
The transcription factors, NFB p65 and IRF-3, related to TLR4 activation, were phosphorylated within the first 10 min of HSP90␣ application and maintained for at least 6 h. (Intracellular calcium is required for NFB p65 phosphorylation; see supplemental Fig. S8.) Because clathrin-mediated endocytosis is the major pathway of EGFR internalization, we examined the co-localization of internalized receptor with clathrin-HC
Summary
Glioma ranges from slowly growing low grade tumors to rapidly growing high grade tumors, including anaplastic astrocytoma and glioblastoma [1, 2]. EGFR (ErbB1, or HER1) is overexpressed in 60% of multiform glioblastomas [13, 14] and promotes invasion, proliferation, and metastasis [15,16,17] in response to several ligands known as EGF-related peptide growth factors [18]. Binding of these ligands to the extracellular domain of EGFR leads to the formation of homo- and heterodimers, which triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR, inducing signaling cascades. We show that exogenously applied HSP90␣ mediates a cross communication between TLR4 and EGFR and accelerates cell migration
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