Limits...
Acid sphingomyelinase activity triggers microparticle release from glial cells.

Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E, Matteoli M, Verderio C - EMBO J. (2009)

Bottom Line: ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice.We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release.Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: CNR Institute of Neuroscience and Department of Medical Pharmacology, University of Milano, Milano, Italy.

ABSTRACT
We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Show MeSH

Related in: MedlinePlus

Src kinase-dependent phosphorylation of p38 MAPK mediates P2X7-induced A-SMase activity. (A) Time course of p38 MAPK phosphorylation in astrocytes exposed to 100 μM BzATP for 2, 15 and 30 min. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to ribophorin from three independent experiments. (B) Western blot analysis of cortical astrocytes exposed for 2 min to 100 μM BzATP in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the kinase inhibitor genistein (10 μM). Astrocytes were also exposed to BzATP in the absence of extracellular Na+ and Ca2+ and high extracellular K+ ions to inhibit ions influx. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to the astrocyte marker ribophorin. (C) Time course analysis of Yo-PRO-1 uptake in astrocytes exposed to 100 μM BzATP in the absence or in the presence of the inhibitors of p38 MAPK phosphorylation pathway. Yo-PRO-1 uptake is sensitive to the inhibition p38 MAPK phosphorylation (n=3; P<0.01, ANOVA analysis, Dunnett's method). (D) A-SMase activity triggered by 100 μM BzATP treatment in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the src-kinase inhibitor PP2 (10 μM) (n=3; asterisks: P<0.01 versus control) normalised as described in Figure 3. (E, F) A-SMase exposure onto the PM induced by 100 μM BzATP treatment in the presence (E)/absence (F) of the p38 MAPK inhibitor SB-203580 (400 nM) measured by FACS in intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated versus negative controls. The results shown are from one experiment representative of three reproducible ones. The RFI values are determined as described in Figure 3. (G) Quantitative analysis of MPs pelleted at 10 000 g from the supernatants of FM1-43-labelled astrocytes exposed to 100 μM BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. FM1-43-labelled MPs pelleted at 10 000 g from the supernatants of astrocytes exposed to BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. (H) ELISA for IL-1β on supernatant conditioned for 30 min by 100 μM BzATP-stimulated astrocytes in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2664656&req=5

f5: Src kinase-dependent phosphorylation of p38 MAPK mediates P2X7-induced A-SMase activity. (A) Time course of p38 MAPK phosphorylation in astrocytes exposed to 100 μM BzATP for 2, 15 and 30 min. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to ribophorin from three independent experiments. (B) Western blot analysis of cortical astrocytes exposed for 2 min to 100 μM BzATP in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the kinase inhibitor genistein (10 μM). Astrocytes were also exposed to BzATP in the absence of extracellular Na+ and Ca2+ and high extracellular K+ ions to inhibit ions influx. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to the astrocyte marker ribophorin. (C) Time course analysis of Yo-PRO-1 uptake in astrocytes exposed to 100 μM BzATP in the absence or in the presence of the inhibitors of p38 MAPK phosphorylation pathway. Yo-PRO-1 uptake is sensitive to the inhibition p38 MAPK phosphorylation (n=3; P<0.01, ANOVA analysis, Dunnett's method). (D) A-SMase activity triggered by 100 μM BzATP treatment in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the src-kinase inhibitor PP2 (10 μM) (n=3; asterisks: P<0.01 versus control) normalised as described in Figure 3. (E, F) A-SMase exposure onto the PM induced by 100 μM BzATP treatment in the presence (E)/absence (F) of the p38 MAPK inhibitor SB-203580 (400 nM) measured by FACS in intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated versus negative controls. The results shown are from one experiment representative of three reproducible ones. The RFI values are determined as described in Figure 3. (G) Quantitative analysis of MPs pelleted at 10 000 g from the supernatants of FM1-43-labelled astrocytes exposed to 100 μM BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. FM1-43-labelled MPs pelleted at 10 000 g from the supernatants of astrocytes exposed to BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. (H) ELISA for IL-1β on supernatant conditioned for 30 min by 100 μM BzATP-stimulated astrocytes in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway.

Mentions: P2X7R is a ligand-gated ion channel, which induces the opening of large membrane pores, recently identified as pannexin-1 hemichannels (Pelegrin and Surprenant, 2006, 2007; Locovei et al, 2007). Independently of the molecular entity of the pore, several pieces of evidence indicate that P2X7-induced pore formation requires activation of p38 MAPK cascade (Suzuki et al, 2004). We observed by western blotting that a brief BzATP exposure induces a prompt p38 MAPK phosphorylation in microglia (not shown) and cortical astrocytes (Figure 5A). p38 phosphorylation peaked 2–5 min after receptor stimulation and then slowly declined, being still above control levels 30 min after agonist addition. BzATP-induced p38 phosphorylation was prevented by the p38 specific inhibitor SB-203580 (400 nM, Figure 5B), by the kinase inhibitor genistein (50 μM, Figure 5B), and the src-kinase inhibitor PP2 (10 μM, not shown), indicating that p38 phosphorylation is mediated by an src-kinase family member, as suggested previously (Suzuki et al, 2004). BzATP-induced p38 activation was independent of the channel activity of the receptor, as it efficiently occurred also in the absence of extracellular Na+ and Ca2+ and in the presence of high extracellular K+ ions (Figure 5B). Consistent with the involvement of an src-family protein kinase in P2X7-mediated p38 activation, exposure of astrocytes to BzATP in the presence of either PP2, genistein as well as SB203580 failed to induce pore opening, as assayed by YO-PRO-1 uptake (Figure 5C).


Acid sphingomyelinase activity triggers microparticle release from glial cells.

Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E, Matteoli M, Verderio C - EMBO J. (2009)

Src kinase-dependent phosphorylation of p38 MAPK mediates P2X7-induced A-SMase activity. (A) Time course of p38 MAPK phosphorylation in astrocytes exposed to 100 μM BzATP for 2, 15 and 30 min. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to ribophorin from three independent experiments. (B) Western blot analysis of cortical astrocytes exposed for 2 min to 100 μM BzATP in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the kinase inhibitor genistein (10 μM). Astrocytes were also exposed to BzATP in the absence of extracellular Na+ and Ca2+ and high extracellular K+ ions to inhibit ions influx. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to the astrocyte marker ribophorin. (C) Time course analysis of Yo-PRO-1 uptake in astrocytes exposed to 100 μM BzATP in the absence or in the presence of the inhibitors of p38 MAPK phosphorylation pathway. Yo-PRO-1 uptake is sensitive to the inhibition p38 MAPK phosphorylation (n=3; P<0.01, ANOVA analysis, Dunnett's method). (D) A-SMase activity triggered by 100 μM BzATP treatment in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the src-kinase inhibitor PP2 (10 μM) (n=3; asterisks: P<0.01 versus control) normalised as described in Figure 3. (E, F) A-SMase exposure onto the PM induced by 100 μM BzATP treatment in the presence (E)/absence (F) of the p38 MAPK inhibitor SB-203580 (400 nM) measured by FACS in intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated versus negative controls. The results shown are from one experiment representative of three reproducible ones. The RFI values are determined as described in Figure 3. (G) Quantitative analysis of MPs pelleted at 10 000 g from the supernatants of FM1-43-labelled astrocytes exposed to 100 μM BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. FM1-43-labelled MPs pelleted at 10 000 g from the supernatants of astrocytes exposed to BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. (H) ELISA for IL-1β on supernatant conditioned for 30 min by 100 μM BzATP-stimulated astrocytes in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664656&req=5

f5: Src kinase-dependent phosphorylation of p38 MAPK mediates P2X7-induced A-SMase activity. (A) Time course of p38 MAPK phosphorylation in astrocytes exposed to 100 μM BzATP for 2, 15 and 30 min. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to ribophorin from three independent experiments. (B) Western blot analysis of cortical astrocytes exposed for 2 min to 100 μM BzATP in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the kinase inhibitor genistein (10 μM). Astrocytes were also exposed to BzATP in the absence of extracellular Na+ and Ca2+ and high extracellular K+ ions to inhibit ions influx. Right panel shows the quantitative analysis of P-p38 immunoreactivity normalised to the astrocyte marker ribophorin. (C) Time course analysis of Yo-PRO-1 uptake in astrocytes exposed to 100 μM BzATP in the absence or in the presence of the inhibitors of p38 MAPK phosphorylation pathway. Yo-PRO-1 uptake is sensitive to the inhibition p38 MAPK phosphorylation (n=3; P<0.01, ANOVA analysis, Dunnett's method). (D) A-SMase activity triggered by 100 μM BzATP treatment in the presence/absence of the p38 MAPK inhibitor SB-203580 (400 nM) or the src-kinase inhibitor PP2 (10 μM) (n=3; asterisks: P<0.01 versus control) normalised as described in Figure 3. (E, F) A-SMase exposure onto the PM induced by 100 μM BzATP treatment in the presence (E)/absence (F) of the p38 MAPK inhibitor SB-203580 (400 nM) measured by FACS in intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated versus negative controls. The results shown are from one experiment representative of three reproducible ones. The RFI values are determined as described in Figure 3. (G) Quantitative analysis of MPs pelleted at 10 000 g from the supernatants of FM1-43-labelled astrocytes exposed to 100 μM BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. FM1-43-labelled MPs pelleted at 10 000 g from the supernatants of astrocytes exposed to BzATP in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway. (H) ELISA for IL-1β on supernatant conditioned for 30 min by 100 μM BzATP-stimulated astrocytes in the presence/absence of inhibitors of p38 MAPK phosphorylation pathway.
Mentions: P2X7R is a ligand-gated ion channel, which induces the opening of large membrane pores, recently identified as pannexin-1 hemichannels (Pelegrin and Surprenant, 2006, 2007; Locovei et al, 2007). Independently of the molecular entity of the pore, several pieces of evidence indicate that P2X7-induced pore formation requires activation of p38 MAPK cascade (Suzuki et al, 2004). We observed by western blotting that a brief BzATP exposure induces a prompt p38 MAPK phosphorylation in microglia (not shown) and cortical astrocytes (Figure 5A). p38 phosphorylation peaked 2–5 min after receptor stimulation and then slowly declined, being still above control levels 30 min after agonist addition. BzATP-induced p38 phosphorylation was prevented by the p38 specific inhibitor SB-203580 (400 nM, Figure 5B), by the kinase inhibitor genistein (50 μM, Figure 5B), and the src-kinase inhibitor PP2 (10 μM, not shown), indicating that p38 phosphorylation is mediated by an src-kinase family member, as suggested previously (Suzuki et al, 2004). BzATP-induced p38 activation was independent of the channel activity of the receptor, as it efficiently occurred also in the absence of extracellular Na+ and Ca2+ and in the presence of high extracellular K+ ions (Figure 5B). Consistent with the involvement of an src-family protein kinase in P2X7-mediated p38 activation, exposure of astrocytes to BzATP in the presence of either PP2, genistein as well as SB203580 failed to induce pore opening, as assayed by YO-PRO-1 uptake (Figure 5C).

Bottom Line: ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice.We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release.Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: CNR Institute of Neuroscience and Department of Medical Pharmacology, University of Milano, Milano, Italy.

ABSTRACT
We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Show MeSH
Related in: MedlinePlus