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Crucial role of HSP90 in the Akt-dependent promotion of angiogenic-like effect of glucose-regulated protein94 (Grp94)-IgG complexes.

Tramentozzi E, Tibaldi E, Brunati AM, Pagetta A, Finotti P - J. Cell. Mol. Med. (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Largo E. Meneghetti, Padova, Italy.

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PU-H71 inhibitor inhibits the HSP90 expression and secretion stimulated by Grp94-IgG complexes. Cells (25 × 104) were seeded in duplicate wells (2 ml) in absence (control) and presence of Grp94-IgG complexes (10 ng/ml) and PU-H71 at the indicated concentrations as specified in ‘Materials and methods’. After 18 hrs incubation, supernatant was collected and cells lysed, as indicated in the legend to Figure 4, for obtaining whole lysates and measuring the expression of HSP90 in Western blotting (A). The same quantity of proteins was loaded in each lane without the addition of β-mercaptoethanol. (B) The media collected from cultured cells that were analysed for the expression of HSP90 (A), were also submitted to Western blotting for HSP90. Fifteen microlitres of media were loaded in each lane (without the addition of β-mercaptoethanol). In both (A) and (B), a representative blotting of three other independent analyses, is presented. Below each blotting is the graph with histograms representing the mean (±S.D.) of densitometric analysis of bands measured in all experiments (n = 3).
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fig06: PU-H71 inhibitor inhibits the HSP90 expression and secretion stimulated by Grp94-IgG complexes. Cells (25 × 104) were seeded in duplicate wells (2 ml) in absence (control) and presence of Grp94-IgG complexes (10 ng/ml) and PU-H71 at the indicated concentrations as specified in ‘Materials and methods’. After 18 hrs incubation, supernatant was collected and cells lysed, as indicated in the legend to Figure 4, for obtaining whole lysates and measuring the expression of HSP90 in Western blotting (A). The same quantity of proteins was loaded in each lane without the addition of β-mercaptoethanol. (B) The media collected from cultured cells that were analysed for the expression of HSP90 (A), were also submitted to Western blotting for HSP90. Fifteen microlitres of media were loaded in each lane (without the addition of β-mercaptoethanol). In both (A) and (B), a representative blotting of three other independent analyses, is presented. Below each blotting is the graph with histograms representing the mean (±S.D.) of densitometric analysis of bands measured in all experiments (n = 3).

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.


Crucial role of HSP90 in the Akt-dependent promotion of angiogenic-like effect of glucose-regulated protein94 (Grp94)-IgG complexes.

Tramentozzi E, Tibaldi E, Brunati AM, Pagetta A, Finotti P - J. Cell. Mol. Med. (2011)

PU-H71 inhibitor inhibits the HSP90 expression and secretion stimulated by Grp94-IgG complexes. Cells (25 × 104) were seeded in duplicate wells (2 ml) in absence (control) and presence of Grp94-IgG complexes (10 ng/ml) and PU-H71 at the indicated concentrations as specified in ‘Materials and methods’. After 18 hrs incubation, supernatant was collected and cells lysed, as indicated in the legend to Figure 4, for obtaining whole lysates and measuring the expression of HSP90 in Western blotting (A). The same quantity of proteins was loaded in each lane without the addition of β-mercaptoethanol. (B) The media collected from cultured cells that were analysed for the expression of HSP90 (A), were also submitted to Western blotting for HSP90. Fifteen microlitres of media were loaded in each lane (without the addition of β-mercaptoethanol). In both (A) and (B), a representative blotting of three other independent analyses, is presented. Below each blotting is the graph with histograms representing the mean (±S.D.) of densitometric analysis of bands measured in all experiments (n = 3).
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Related In: Results  -  Collection

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fig06: PU-H71 inhibitor inhibits the HSP90 expression and secretion stimulated by Grp94-IgG complexes. Cells (25 × 104) were seeded in duplicate wells (2 ml) in absence (control) and presence of Grp94-IgG complexes (10 ng/ml) and PU-H71 at the indicated concentrations as specified in ‘Materials and methods’. After 18 hrs incubation, supernatant was collected and cells lysed, as indicated in the legend to Figure 4, for obtaining whole lysates and measuring the expression of HSP90 in Western blotting (A). The same quantity of proteins was loaded in each lane without the addition of β-mercaptoethanol. (B) The media collected from cultured cells that were analysed for the expression of HSP90 (A), were also submitted to Western blotting for HSP90. Fifteen microlitres of media were loaded in each lane (without the addition of β-mercaptoethanol). In both (A) and (B), a representative blotting of three other independent analyses, is presented. Below each blotting is the graph with histograms representing the mean (±S.D.) of densitometric analysis of bands measured in all experiments (n = 3).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Largo E. Meneghetti, Padova, Italy.

Show MeSH
Related in: MedlinePlus