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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 endothelial Cx43 expression is required for the transendothelial migration of monocytesA. The adenosine-derived inhibitor of HSP70, VER155008 (10 μM) favors the establishment of GJIC between monocytes (M-CSF stimulated) and HMEC within 3 h. Phase-contrast microphotographs are representative of 4 experiments. Bar 100 μm. Right, histogram represents the total cell number of HMEC receiving dye (calcein) per monocyte (mean ±SD, n=3; **P-values <0.01 vs control). B. VER155008 does not inhibit the HSP70 release by M-CSF-stimulated monocytes for 12 h (dosed by ELISA; mean ± SD, n=4; *P-values <0.05 vs control). C. VER155008 antagonizes the blocking effect of rhHSP70 on GJIC between HMEC (gap-FRAP analysis). Histogram shows the constant k measured for the coupled cells after 1 h of cell treatment (mean ± SD, n=4; *P-values<0.05 vs control). D. Effects of the endothelial Cx43 knockdown on the GJIC coupling between HMEC and HSP70 depleted monocytes. Cultured HMEC and monocytes were transfected respectively with siRNA Cx43 (HMEC) and siRNA HSP70 (monocytes) or control siRNA, 48h prior to various analysis. Insert is representative western blot showing the specific depletion of Cx43 in HMEC. Transfected monocytes (donors), stimulated overnight with M-CSF, were pre-loaded with calcein and DiL-C18 before to be plated. Microphotographs of monocytes in contact with transfected HMEC (receivers) after 3 h of culture (representative of 6 experiments; Bar 100 μm). Histogram represents the mean cell number of neighboring HMEC receiving dye (calcein) per monocyte (mean ±SD; n=50 labeled monocytes examined; n=3). E. Effects of the endothelial Cx43 knockdown and released HSP70 on the transendothelial migration of monocytes. Control or transfected HMEC monolayers grown on Transwells were kept in FCS-free conditions overnight. Control or transfected monocytes (3×105) stimulated overnight by M-CSF, were labeled with phycoerythrin-conjugated anti-CD14+ before to be added into the wells. Cells migrating through the endothelial layers were counted (after 3 h). Data are percentage of total applied monocytes counted by flow cytometry (mean ± SD; n=5).
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Figure 7: The endothelial Cx43 expression is required for the transendothelial migration of monocytesA. The adenosine-derived inhibitor of HSP70, VER155008 (10 μM) favors the establishment of GJIC between monocytes (M-CSF stimulated) and HMEC within 3 h. Phase-contrast microphotographs are representative of 4 experiments. Bar 100 μm. Right, histogram represents the total cell number of HMEC receiving dye (calcein) per monocyte (mean ±SD, n=3; **P-values <0.01 vs control). B. VER155008 does not inhibit the HSP70 release by M-CSF-stimulated monocytes for 12 h (dosed by ELISA; mean ± SD, n=4; *P-values <0.05 vs control). C. VER155008 antagonizes the blocking effect of rhHSP70 on GJIC between HMEC (gap-FRAP analysis). Histogram shows the constant k measured for the coupled cells after 1 h of cell treatment (mean ± SD, n=4; *P-values<0.05 vs control). D. Effects of the endothelial Cx43 knockdown on the GJIC coupling between HMEC and HSP70 depleted monocytes. Cultured HMEC and monocytes were transfected respectively with siRNA Cx43 (HMEC) and siRNA HSP70 (monocytes) or control siRNA, 48h prior to various analysis. Insert is representative western blot showing the specific depletion of Cx43 in HMEC. Transfected monocytes (donors), stimulated overnight with M-CSF, were pre-loaded with calcein and DiL-C18 before to be plated. Microphotographs of monocytes in contact with transfected HMEC (receivers) after 3 h of culture (representative of 6 experiments; Bar 100 μm). Histogram represents the mean cell number of neighboring HMEC receiving dye (calcein) per monocyte (mean ±SD; n=50 labeled monocytes examined; n=3). E. Effects of the endothelial Cx43 knockdown and released HSP70 on the transendothelial migration of monocytes. Control or transfected HMEC monolayers grown on Transwells were kept in FCS-free conditions overnight. Control or transfected monocytes (3×105) stimulated overnight by M-CSF, were labeled with phycoerythrin-conjugated anti-CD14+ before to be added into the wells. Cells migrating through the endothelial layers were counted (after 3 h). Data are percentage of total applied monocytes counted by flow cytometry (mean ± SD; n=5).

Mentions: Dye coupling was also seen with the non-transfected monocytes pretreated by the adenosine derived inhibitor of HSP70, VER155008 (Fig. 7A), i.e. VER155008-treated cells established functional gap junctions with HMEC in response to M-CSF. VER155008 treatment did not change the high level of HSP70 protein expressed in cells stimulated by M-CSF (Fig. 7B). Treatment of HMEC alone by VER155008 improved the blocking action of rhHSP70 on GJIC (Fig. 7C).


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 endothelial Cx43 expression is required for the transendothelial migration of monocytesA. The adenosine-derived inhibitor of HSP70, VER155008 (10 μM) favors the establishment of GJIC between monocytes (M-CSF stimulated) and HMEC within 3 h. Phase-contrast microphotographs are representative of 4 experiments. Bar 100 μm. Right, histogram represents the total cell number of HMEC receiving dye (calcein) per monocyte (mean ±SD, n=3; **P-values <0.01 vs control). B. VER155008 does not inhibit the HSP70 release by M-CSF-stimulated monocytes for 12 h (dosed by ELISA; mean ± SD, n=4; *P-values <0.05 vs control). C. VER155008 antagonizes the blocking effect of rhHSP70 on GJIC between HMEC (gap-FRAP analysis). Histogram shows the constant k measured for the coupled cells after 1 h of cell treatment (mean ± SD, n=4; *P-values<0.05 vs control). D. Effects of the endothelial Cx43 knockdown on the GJIC coupling between HMEC and HSP70 depleted monocytes. Cultured HMEC and monocytes were transfected respectively with siRNA Cx43 (HMEC) and siRNA HSP70 (monocytes) or control siRNA, 48h prior to various analysis. Insert is representative western blot showing the specific depletion of Cx43 in HMEC. Transfected monocytes (donors), stimulated overnight with M-CSF, were pre-loaded with calcein and DiL-C18 before to be plated. Microphotographs of monocytes in contact with transfected HMEC (receivers) after 3 h of culture (representative of 6 experiments; Bar 100 μm). Histogram represents the mean cell number of neighboring HMEC receiving dye (calcein) per monocyte (mean ±SD; n=50 labeled monocytes examined; n=3). E. Effects of the endothelial Cx43 knockdown and released HSP70 on the transendothelial migration of monocytes. Control or transfected HMEC monolayers grown on Transwells were kept in FCS-free conditions overnight. Control or transfected monocytes (3×105) stimulated overnight by M-CSF, were labeled with phycoerythrin-conjugated anti-CD14+ before to be added into the wells. Cells migrating through the endothelial layers were counted (after 3 h). Data are percentage of total applied monocytes counted by flow cytometry (mean ± SD; n=5).
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Related In: Results  -  Collection

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Figure 7: The endothelial Cx43 expression is required for the transendothelial migration of monocytesA. The adenosine-derived inhibitor of HSP70, VER155008 (10 μM) favors the establishment of GJIC between monocytes (M-CSF stimulated) and HMEC within 3 h. Phase-contrast microphotographs are representative of 4 experiments. Bar 100 μm. Right, histogram represents the total cell number of HMEC receiving dye (calcein) per monocyte (mean ±SD, n=3; **P-values <0.01 vs control). B. VER155008 does not inhibit the HSP70 release by M-CSF-stimulated monocytes for 12 h (dosed by ELISA; mean ± SD, n=4; *P-values <0.05 vs control). C. VER155008 antagonizes the blocking effect of rhHSP70 on GJIC between HMEC (gap-FRAP analysis). Histogram shows the constant k measured for the coupled cells after 1 h of cell treatment (mean ± SD, n=4; *P-values<0.05 vs control). D. Effects of the endothelial Cx43 knockdown on the GJIC coupling between HMEC and HSP70 depleted monocytes. Cultured HMEC and monocytes were transfected respectively with siRNA Cx43 (HMEC) and siRNA HSP70 (monocytes) or control siRNA, 48h prior to various analysis. Insert is representative western blot showing the specific depletion of Cx43 in HMEC. Transfected monocytes (donors), stimulated overnight with M-CSF, were pre-loaded with calcein and DiL-C18 before to be plated. Microphotographs of monocytes in contact with transfected HMEC (receivers) after 3 h of culture (representative of 6 experiments; Bar 100 μm). Histogram represents the mean cell number of neighboring HMEC receiving dye (calcein) per monocyte (mean ±SD; n=50 labeled monocytes examined; n=3). E. Effects of the endothelial Cx43 knockdown and released HSP70 on the transendothelial migration of monocytes. Control or transfected HMEC monolayers grown on Transwells were kept in FCS-free conditions overnight. Control or transfected monocytes (3×105) stimulated overnight by M-CSF, were labeled with phycoerythrin-conjugated anti-CD14+ before to be added into the wells. Cells migrating through the endothelial layers were counted (after 3 h). Data are percentage of total applied monocytes counted by flow cytometry (mean ± SD; n=5).
Mentions: Dye coupling was also seen with the non-transfected monocytes pretreated by the adenosine derived inhibitor of HSP70, VER155008 (Fig. 7A), i.e. VER155008-treated cells established functional gap junctions with HMEC in response to M-CSF. VER155008 treatment did not change the high level of HSP70 protein expressed in cells stimulated by M-CSF (Fig. 7B). Treatment of HMEC alone by VER155008 improved the blocking action of rhHSP70 on GJIC (Fig. 7C).

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