Crucial role of HSP90 in the Akt-dependent promotion of angiogenic-like effect of glucose-regulated protein94 (Grp94)-IgG complexes.
Bottom Line: CCT failed to inhibit any morphological alteration induced by Grp94-IgG on HUVECs, on its own displaying a paradoxical angiogenic-like activity.CTT appeared to enhance the angiogenic-like effect of Grp94-IgG by increasing the rate of secretion of both HSP90 and MMP-9.Results reveal a fundamental role of HSP90 in the PI3K/Akt pathway-mediated angiogenic-like effect of Grp94-IgG, also questioning the capacity of CTT to serve as an effective inhibitor of the angiogenic effect.
Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Largo E. Meneghetti, Padova, Italy.Show MeSH
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Mentions: Because CTT was devoid of any inhibitory effect on angiogenic-like activity of Grp94-IgG, being in addition on its own a promoter of angiogenesis, and because this effect appeared to be mediated by an HSP90-dependent activation of the Akt pathway, we searched whether an inhibitor of this pathway could effectively antagonize the major Grp94-IgG effect on HUVECs. To this aim, we chosen an inhibitor of the HSP90 function that causes down-regulation of the Akt kinase-dependent pathway instead of the classic inhibitor LY294002 that in our cell cultures reduced cell viability by 40% in control HUVECs even at concentrations as low as 5 μM (data not shown). We thus employed the novel pan-HSP90 inhibitor, the non-quinone PU-H71, already successfully used in cancer cell cultures and in vivo to reduce up-regulation of HSP90 and to inhibit the expression of Akt in a dose-dependent manner [26, 27]. PU-H71 was added to cell cultures 15 min. before the addition of Grp94-IgG at the final concentrations of both 50 nM (IC50) and 150 nM, to measure to what extent effects of Grp94-IgG were dependent on the PI3K/Akt pathway activation. Stimulation of HSP90 expression by Grp94-IgG was inhibited by PU-H71 in a dose-dependent manner, as demonstrated by immunoblotting on whole cell lysates (Fig. 6A). A much higher, dose-dependent inhibitory effect was observed with PU-H71 on the HSP90 secretion in culture media where HSP90 was barely detectable already at 50 nM PU-H71 (Fig. 6B). It is worth noting that the same effect was also present with PU-H71 alone, as if the inhibition of HSP90 as such could lead to the block of HSP90 secretion.
Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Largo E. Meneghetti, Padova, Italy.