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Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

Thuringer D, Berthenet K, Cronier L, Jego G, Solary E, Garrido C - Oncotarget (2015)

Bottom Line: In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique.Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues.Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U866, Faculty of Medecine, Dijon, France.

ABSTRACT
High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

No MeSH data available.


Related in: MedlinePlus

The HSP70 release by monocytes alters their coupling with HMECA. M-CSF (100 nM) increases HSP70 expression in monocytes (representative of 5 experiments; Hsc70 as loading control). B. Cx43 expression in monocytes is not affected by 12h-treatment with M-CSF or rhHSP70 (representative of 3 experiments). C. M-CSF induced HSP70 release. Amounts of HSP70 measured by ELISA in supernatant of monocytes untreated (control) or treated with 100 nM M-CSF for 12 h (mean ± SD; n=4; **P-values <0.01). D. siRNA HSP70 knockdown. Cultured monocytes were transfected with HSP70 or control siRNA 48h prior to various analysis. Left, western blot analysis of protein extracts from cells treated with M-CSF for 12 h. Right, histogram shows HSP70 release by transfected monocytes in response to 100 nM M-CSF for 12 h (mean ± S.D., n=4; representative of 4 experiments). E. The ATP release by HMEC/monocyte cocultures is mainly due to HMEC (bioluminescence assay; means ± S.D. n=3; **P-values<0.01, *P-values<0.05 vs control). F. Functional GJIC between monocytes and HMEC. Monocytes (donors) were preloaded with calcein/AM and DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. Labelled monocytes are then plated with unlabeled HMEC monolayer (receivers). HMEC establishing GJIC with monocytes become fluorescent by calcein diffusion. Only siRNA HSP70-transfected monocytes establish GJIC with HMEC and exogenously added rhHSP70 (5μg/ml) improved it. Phase-contrast microphotographs after 3 h of culture (representative of 6 experiments; Bar 100 μm).
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Figure 6: The HSP70 release by monocytes alters their coupling with HMECA. M-CSF (100 nM) increases HSP70 expression in monocytes (representative of 5 experiments; Hsc70 as loading control). B. Cx43 expression in monocytes is not affected by 12h-treatment with M-CSF or rhHSP70 (representative of 3 experiments). C. M-CSF induced HSP70 release. Amounts of HSP70 measured by ELISA in supernatant of monocytes untreated (control) or treated with 100 nM M-CSF for 12 h (mean ± SD; n=4; **P-values <0.01). D. siRNA HSP70 knockdown. Cultured monocytes were transfected with HSP70 or control siRNA 48h prior to various analysis. Left, western blot analysis of protein extracts from cells treated with M-CSF for 12 h. Right, histogram shows HSP70 release by transfected monocytes in response to 100 nM M-CSF for 12 h (mean ± S.D., n=4; representative of 4 experiments). E. The ATP release by HMEC/monocyte cocultures is mainly due to HMEC (bioluminescence assay; means ± S.D. n=3; **P-values<0.01, *P-values<0.05 vs control). F. Functional GJIC between monocytes and HMEC. Monocytes (donors) were preloaded with calcein/AM and DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. Labelled monocytes are then plated with unlabeled HMEC monolayer (receivers). HMEC establishing GJIC with monocytes become fluorescent by calcein diffusion. Only siRNA HSP70-transfected monocytes establish GJIC with HMEC and exogenously added rhHSP70 (5μg/ml) improved it. Phase-contrast microphotographs after 3 h of culture (representative of 6 experiments; Bar 100 μm).

Mentions: The release of ATP and subsequent activation of endothelial intracellular Ca2+ signalling are reported to modulate monocyte adhesion to endothelial cells and their transendothelium migration [23, 51]. Exposure of human, peripheral blood monocytes to M-CSF for 12 hours increased the expression and release of HSP70, without affecting Cx43 expression and phosphorylation (Fig. 6A, B, C). The amount of HSP70 secreted by monocytes seems very low compared with the exogenously added in HMEC cultures. However this was a dosage for the whole fluid bathing the cells whereas the secretion by monocytes must be considered in their closed vicinity near the endothelial cell. So the real quantity of HSP70 secreted by the monocyte and collected by the endothelial cell is certainly much higher that the dose measured (diluted) in the whole bath. Knocking down HSP70 with a specific siRNA reduced by about 80% the amount of HSP70 found after 12 h into the bath of M-CSF-treated monocytes (Fig. 6D). M-CSF-stimulated monocytes increased ATP release by HMEC, which could be mediated by HSP70 released from these monocytes (Fig. 6E).


Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

Thuringer D, Berthenet K, Cronier L, Jego G, Solary E, Garrido C - Oncotarget (2015)

The HSP70 release by monocytes alters their coupling with HMECA. M-CSF (100 nM) increases HSP70 expression in monocytes (representative of 5 experiments; Hsc70 as loading control). B. Cx43 expression in monocytes is not affected by 12h-treatment with M-CSF or rhHSP70 (representative of 3 experiments). C. M-CSF induced HSP70 release. Amounts of HSP70 measured by ELISA in supernatant of monocytes untreated (control) or treated with 100 nM M-CSF for 12 h (mean ± SD; n=4; **P-values <0.01). D. siRNA HSP70 knockdown. Cultured monocytes were transfected with HSP70 or control siRNA 48h prior to various analysis. Left, western blot analysis of protein extracts from cells treated with M-CSF for 12 h. Right, histogram shows HSP70 release by transfected monocytes in response to 100 nM M-CSF for 12 h (mean ± S.D., n=4; representative of 4 experiments). E. The ATP release by HMEC/monocyte cocultures is mainly due to HMEC (bioluminescence assay; means ± S.D. n=3; **P-values<0.01, *P-values<0.05 vs control). F. Functional GJIC between monocytes and HMEC. Monocytes (donors) were preloaded with calcein/AM and DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. Labelled monocytes are then plated with unlabeled HMEC monolayer (receivers). HMEC establishing GJIC with monocytes become fluorescent by calcein diffusion. Only siRNA HSP70-transfected monocytes establish GJIC with HMEC and exogenously added rhHSP70 (5μg/ml) improved it. Phase-contrast microphotographs after 3 h of culture (representative of 6 experiments; Bar 100 μm).
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Related In: Results  -  Collection

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Figure 6: The HSP70 release by monocytes alters their coupling with HMECA. M-CSF (100 nM) increases HSP70 expression in monocytes (representative of 5 experiments; Hsc70 as loading control). B. Cx43 expression in monocytes is not affected by 12h-treatment with M-CSF or rhHSP70 (representative of 3 experiments). C. M-CSF induced HSP70 release. Amounts of HSP70 measured by ELISA in supernatant of monocytes untreated (control) or treated with 100 nM M-CSF for 12 h (mean ± SD; n=4; **P-values <0.01). D. siRNA HSP70 knockdown. Cultured monocytes were transfected with HSP70 or control siRNA 48h prior to various analysis. Left, western blot analysis of protein extracts from cells treated with M-CSF for 12 h. Right, histogram shows HSP70 release by transfected monocytes in response to 100 nM M-CSF for 12 h (mean ± S.D., n=4; representative of 4 experiments). E. The ATP release by HMEC/monocyte cocultures is mainly due to HMEC (bioluminescence assay; means ± S.D. n=3; **P-values<0.01, *P-values<0.05 vs control). F. Functional GJIC between monocytes and HMEC. Monocytes (donors) were preloaded with calcein/AM and DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. Labelled monocytes are then plated with unlabeled HMEC monolayer (receivers). HMEC establishing GJIC with monocytes become fluorescent by calcein diffusion. Only siRNA HSP70-transfected monocytes establish GJIC with HMEC and exogenously added rhHSP70 (5μg/ml) improved it. Phase-contrast microphotographs after 3 h of culture (representative of 6 experiments; Bar 100 μm).
Mentions: The release of ATP and subsequent activation of endothelial intracellular Ca2+ signalling are reported to modulate monocyte adhesion to endothelial cells and their transendothelium migration [23, 51]. Exposure of human, peripheral blood monocytes to M-CSF for 12 hours increased the expression and release of HSP70, without affecting Cx43 expression and phosphorylation (Fig. 6A, B, C). The amount of HSP70 secreted by monocytes seems very low compared with the exogenously added in HMEC cultures. However this was a dosage for the whole fluid bathing the cells whereas the secretion by monocytes must be considered in their closed vicinity near the endothelial cell. So the real quantity of HSP70 secreted by the monocyte and collected by the endothelial cell is certainly much higher that the dose measured (diluted) in the whole bath. Knocking down HSP70 with a specific siRNA reduced by about 80% the amount of HSP70 found after 12 h into the bath of M-CSF-treated monocytes (Fig. 6D). M-CSF-stimulated monocytes increased ATP release by HMEC, which could be mediated by HSP70 released from these monocytes (Fig. 6E).

Bottom Line: In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique.Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues.Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U866, Faculty of Medecine, Dijon, France.

ABSTRACT
High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

No MeSH data available.


Related in: MedlinePlus