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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

Extracellular rhHSP70 inhibits the endothelial gap-junction couplingA. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Polygons 2-5 are bleached cells, and polygon 1 is an unbleached control cell used for correction of the artefactual loss of fluorescence. Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. Note the fluorescence recovery follows an exponential time course when the bleached cells are interconnected by open gap-junction channels to unbleached cells (polygons 2-5). The relative permeability of gaps is given by the time constant k. B. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP70 (5 μg/ml). (C) Histogram shows k values measured after rhHSP70 addition for 0, 30, 60 min and 24 h (mean ± SD, n=8; **P<0.01, *P<0.05 vs control [t=0 min]).
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Figure 1: Extracellular rhHSP70 inhibits the endothelial gap-junction couplingA. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Polygons 2-5 are bleached cells, and polygon 1 is an unbleached control cell used for correction of the artefactual loss of fluorescence. Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. Note the fluorescence recovery follows an exponential time course when the bleached cells are interconnected by open gap-junction channels to unbleached cells (polygons 2-5). The relative permeability of gaps is given by the time constant k. B. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP70 (5 μg/ml). (C) Histogram shows k values measured after rhHSP70 addition for 0, 30, 60 min and 24 h (mean ± SD, n=8; **P<0.01, *P<0.05 vs control [t=0 min]).

Mentions: The effects of rhHSP70 were analyzed on the functionality of gap junctions established between HMEC in confluent monolayer by using the gap-FRAP technique [30, 35]. Briefly, HMEC were loaded with a diffusible tracer (calcein/AM), and the fluorescence of investigated cells was suppressed by a laser beam. The recovery of fluorescence in these cells, which results from the intercellular diffusion of calcein from neighboring cells, was recorded to measure the diffusion rate constant k (min−1), an index of gap junction permeability. Fig. 1A shows typical changes in the fluorescence of cells, before and after photobleaching. rhHSP70 was used at 5 μg/ml (Fig. 1B, open circles), a concentration that evoked both maximal spreading of HMEC spheroids (Suppl. Fig. S1) and increase in cell motility (Suppl. Fig. S2). rhHSP70 prevented the fluorescence recovery normally observed in cells exposed to control solution (black circles), demonstrating that rhHSP70 blocked the GJIC between HMEC (Fig. 1B). A time-dependent decrease in k was observed within 1 h (from 0.417 ± 0.100 min−1 in untreated to 0.032 ± 0.014 min−1 in rhHSP70-treated cells; mean ± SD, n=8; Fig. 1C). In comparison, HMEC exposure to rhHSP27 rather increased k (from 0.488 ± 0.207 min−1 in untreated cells to 0.643 ± 0.277 min−1; n=8). Addition of polymyxin B (PMB100 ng/ml) did not prevent the time-dependent decrease in k value observed with rhHSP70 (from 0.445 ± 0.111 min−1 in control to 0.097 ± 0.100 min−1; n=3). Thus, the rhHSP70-induced GJIC inhibition is mainly caused by the rhHSP70 protein itself, rather than by any contaminating endotoxin.


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)

Extracellular rhHSP70 inhibits the endothelial gap-junction couplingA. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Polygons 2-5 are bleached cells, and polygon 1 is an unbleached control cell used for correction of the artefactual loss of fluorescence. Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. Note the fluorescence recovery follows an exponential time course when the bleached cells are interconnected by open gap-junction channels to unbleached cells (polygons 2-5). The relative permeability of gaps is given by the time constant k. B. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP70 (5 μg/ml). (C) Histogram shows k values measured after rhHSP70 addition for 0, 30, 60 min and 24 h (mean ± SD, n=8; **P<0.01, *P<0.05 vs control [t=0 min]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496354&req=5

Figure 1: Extracellular rhHSP70 inhibits the endothelial gap-junction couplingA. FRAP analysis of cell-to-cell communication. Digital images of fluorescence distribution in a HMEC monolayer at three times during a typical gap-FRAP experiment: prebleach, just after bleaching (0 min) and after fluorescence recovery (8 min). Polygons 2-5 are bleached cells, and polygon 1 is an unbleached control cell used for correction of the artefactual loss of fluorescence. Bars 20 μm. Corresponding fluorescence intensities (% of prebleach value) versus time in tested cells. Note the fluorescence recovery follows an exponential time course when the bleached cells are interconnected by open gap-junction channels to unbleached cells (polygons 2-5). The relative permeability of gaps is given by the time constant k. B. Graph represents mean ± SEM of the fluorescence redistribution after photobleaching in coupled HMEC in control (●) or after 60 min (○) with rhHSP70 (5 μg/ml). (C) Histogram shows k values measured after rhHSP70 addition for 0, 30, 60 min and 24 h (mean ± SD, n=8; **P<0.01, *P<0.05 vs control [t=0 min]).
Mentions: The effects of rhHSP70 were analyzed on the functionality of gap junctions established between HMEC in confluent monolayer by using the gap-FRAP technique [30, 35]. Briefly, HMEC were loaded with a diffusible tracer (calcein/AM), and the fluorescence of investigated cells was suppressed by a laser beam. The recovery of fluorescence in these cells, which results from the intercellular diffusion of calcein from neighboring cells, was recorded to measure the diffusion rate constant k (min−1), an index of gap junction permeability. Fig. 1A shows typical changes in the fluorescence of cells, before and after photobleaching. rhHSP70 was used at 5 μg/ml (Fig. 1B, open circles), a concentration that evoked both maximal spreading of HMEC spheroids (Suppl. Fig. S1) and increase in cell motility (Suppl. Fig. S2). rhHSP70 prevented the fluorescence recovery normally observed in cells exposed to control solution (black circles), demonstrating that rhHSP70 blocked the GJIC between HMEC (Fig. 1B). A time-dependent decrease in k was observed within 1 h (from 0.417 ± 0.100 min−1 in untreated to 0.032 ± 0.014 min−1 in rhHSP70-treated cells; mean ± SD, n=8; Fig. 1C). In comparison, HMEC exposure to rhHSP27 rather increased k (from 0.488 ± 0.207 min−1 in untreated cells to 0.643 ± 0.277 min−1; n=8). Addition of polymyxin B (PMB100 ng/ml) did not prevent the time-dependent decrease in k value observed with rhHSP70 (from 0.445 ± 0.111 min−1 in control to 0.097 ± 0.100 min−1; n=3). Thus, the rhHSP70-induced GJIC inhibition is mainly caused by the rhHSP70 protein itself, rather than by any contaminating endotoxin.

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