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P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Adamczyk M, Griffiths R, Dewitt S, Knäuper V, Aeschlimann D - J. Cell. Sci. (2015)

Bottom Line: Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement.A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity.These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

View Article: PubMed Central - PubMed

Affiliation: Matrix Biology & Tissue Repair Research Unit and Arthritis Research UK Biomechanics and Bioengineering Center of Excellence, College of Biomedical and Life Sciences, Cardiff University, Cardiff CF14 4XY, UK AdamczykM@Cardiff.ac.uk AeschlimannDP@Cardiff.ac.uk.

No MeSH data available.


Related in: MedlinePlus

TG2 export is independent of K+ efflux and membrane depolarization. (A) Calmidazolium (calm) blocks flotillin-2 but not TG2 release. TG2-transfected P2X7R cells were pre-treated for 10 min and then stimulated with BzATP in medium containing 1 µM calmidazolium or vehicle. Cells were chased in agonist-free medium, and conditioned media analyzed by western blotting for TG2 and flotillin-2. (B) Calmidazolium does not affect P2X7R-dependent ‘membrane pore’ formation. P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor A740003 or vehicle for 10 min prior to stimulation with 100 µM BzATP in the presence of respective inhibitors or carrier in PSS containing YO-PRO1 and 0.9 mM Ca2+. Dye uptake was monitored over time. Results are shown as mean±s.e.m. of two wells, and are representative of three independent experiments. (C) Calmidazolium ameliorates the large rise in [Ca2+]i. Fluo-4-AM-loaded P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor or vehicle for 20 min prior to stimulation with 100 μM BzATP in the presence of inhibitors or carrier. Fluorescence change (λex, 485-12 nm; λem, 520-10 nm) relative to control in response to agonist treatment was monitored (mean±s.e.m. of eight replicate wells).
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JCS175968F6: TG2 export is independent of K+ efflux and membrane depolarization. (A) Calmidazolium (calm) blocks flotillin-2 but not TG2 release. TG2-transfected P2X7R cells were pre-treated for 10 min and then stimulated with BzATP in medium containing 1 µM calmidazolium or vehicle. Cells were chased in agonist-free medium, and conditioned media analyzed by western blotting for TG2 and flotillin-2. (B) Calmidazolium does not affect P2X7R-dependent ‘membrane pore’ formation. P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor A740003 or vehicle for 10 min prior to stimulation with 100 µM BzATP in the presence of respective inhibitors or carrier in PSS containing YO-PRO1 and 0.9 mM Ca2+. Dye uptake was monitored over time. Results are shown as mean±s.e.m. of two wells, and are representative of three independent experiments. (C) Calmidazolium ameliorates the large rise in [Ca2+]i. Fluo-4-AM-loaded P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor or vehicle for 20 min prior to stimulation with 100 μM BzATP in the presence of inhibitors or carrier. Fluorescence change (λex, 485-12 nm; λem, 520-10 nm) relative to control in response to agonist treatment was monitored (mean±s.e.m. of eight replicate wells).

Mentions: To assess the contribution of the initial ion flux on TG2 secretion, calmidazolium was employed. This compound is an inhibitor with broad selectivity for voltage-gated fast-acting Na+/K+ and L-type Ca2+ channels that also inhibits the initial ATP-evoked ion flux through P2X7R without affecting the downstream membrane pore formation (Virginio et al., 1997). Calmidazolium has an extracellular mode of action on P2X7R. BzATP-induced TG2 export in P2X7R cells was unaffected by the presence of calmidazolium but flotillin-2 secretion was blocked (Fig. 6A). The inhibitor had no effect on pore formation activity of P2X7R (Fig. 6B) but substantially reduced the rise in [Ca2+]i mediated by P2X7R activation (Fig. 6C). This indicates that TG2 secretion is linked to P2X7R-dependent pore formation but not the initial ion flux and associated membrane depolarization.Fig. 6.


P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Adamczyk M, Griffiths R, Dewitt S, Knäuper V, Aeschlimann D - J. Cell. Sci. (2015)

TG2 export is independent of K+ efflux and membrane depolarization. (A) Calmidazolium (calm) blocks flotillin-2 but not TG2 release. TG2-transfected P2X7R cells were pre-treated for 10 min and then stimulated with BzATP in medium containing 1 µM calmidazolium or vehicle. Cells were chased in agonist-free medium, and conditioned media analyzed by western blotting for TG2 and flotillin-2. (B) Calmidazolium does not affect P2X7R-dependent ‘membrane pore’ formation. P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor A740003 or vehicle for 10 min prior to stimulation with 100 µM BzATP in the presence of respective inhibitors or carrier in PSS containing YO-PRO1 and 0.9 mM Ca2+. Dye uptake was monitored over time. Results are shown as mean±s.e.m. of two wells, and are representative of three independent experiments. (C) Calmidazolium ameliorates the large rise in [Ca2+]i. Fluo-4-AM-loaded P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor or vehicle for 20 min prior to stimulation with 100 μM BzATP in the presence of inhibitors or carrier. Fluorescence change (λex, 485-12 nm; λem, 520-10 nm) relative to control in response to agonist treatment was monitored (mean±s.e.m. of eight replicate wells).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696497&req=5

JCS175968F6: TG2 export is independent of K+ efflux and membrane depolarization. (A) Calmidazolium (calm) blocks flotillin-2 but not TG2 release. TG2-transfected P2X7R cells were pre-treated for 10 min and then stimulated with BzATP in medium containing 1 µM calmidazolium or vehicle. Cells were chased in agonist-free medium, and conditioned media analyzed by western blotting for TG2 and flotillin-2. (B) Calmidazolium does not affect P2X7R-dependent ‘membrane pore’ formation. P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor A740003 or vehicle for 10 min prior to stimulation with 100 µM BzATP in the presence of respective inhibitors or carrier in PSS containing YO-PRO1 and 0.9 mM Ca2+. Dye uptake was monitored over time. Results are shown as mean±s.e.m. of two wells, and are representative of three independent experiments. (C) Calmidazolium ameliorates the large rise in [Ca2+]i. Fluo-4-AM-loaded P2X7R cells were pre-treated with calmidazolium, P2X7R inhibitor or vehicle for 20 min prior to stimulation with 100 μM BzATP in the presence of inhibitors or carrier. Fluorescence change (λex, 485-12 nm; λem, 520-10 nm) relative to control in response to agonist treatment was monitored (mean±s.e.m. of eight replicate wells).
Mentions: To assess the contribution of the initial ion flux on TG2 secretion, calmidazolium was employed. This compound is an inhibitor with broad selectivity for voltage-gated fast-acting Na+/K+ and L-type Ca2+ channels that also inhibits the initial ATP-evoked ion flux through P2X7R without affecting the downstream membrane pore formation (Virginio et al., 1997). Calmidazolium has an extracellular mode of action on P2X7R. BzATP-induced TG2 export in P2X7R cells was unaffected by the presence of calmidazolium but flotillin-2 secretion was blocked (Fig. 6A). The inhibitor had no effect on pore formation activity of P2X7R (Fig. 6B) but substantially reduced the rise in [Ca2+]i mediated by P2X7R activation (Fig. 6C). This indicates that TG2 secretion is linked to P2X7R-dependent pore formation but not the initial ion flux and associated membrane depolarization.Fig. 6.

Bottom Line: Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement.A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity.These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

View Article: PubMed Central - PubMed

Affiliation: Matrix Biology & Tissue Repair Research Unit and Arthritis Research UK Biomechanics and Bioengineering Center of Excellence, College of Biomedical and Life Sciences, Cardiff University, Cardiff CF14 4XY, UK AdamczykM@Cardiff.ac.uk AeschlimannDP@Cardiff.ac.uk.

No MeSH data available.


Related in: MedlinePlus