Limits...
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

Extracellular Ca2+ regulates TG2 secretion. (A) P2X7R-mediated TG2 export at different [Ca2+]ex. P2X7R cells expressing TG2 were stimulated with BzATP for 10 min in medium containing 0.9 or 2.2 mM Ca2+ or in Ca2+-free medium, and chased for 30 min in respective media without BzATP. Conditioned media were analyzed by western blotting for TG2 and flotillin-2. (B) TG2 catalytic activity is not required for P2X7R-mediated export. P2X7R cells expressing TG2 or the TG2 C277S mutant were stimulated with BzATP in medium containing 0.9 or 2.2 mM Ca2+ and TG2 export was assessed as above. (C,D) [Ca2+]ex regulates P2X7R activity. P2X7R or parental cells were stimulated with BzATP, as indicated, in PSS containing YO-PRO1 and different concentrations of Ca2+. To determine YO-PRO1 uptake by cells after BzATP application, changes in well-specific fluorescence (λex, 480-10 nm; λem, 520-10 nm) were monitored over time. A representative experiment of dye uptake in Ca2+-free PSS is shown as mean±s.e.m. of two wells (C). In D, the initial rates of YO-PRO1 uptake at different [Ca2+]ex in response to 300 μM BzATP are given (mean±s.e.m.; n=2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696497&req=5

JCS175968F5: Extracellular Ca2+ regulates TG2 secretion. (A) P2X7R-mediated TG2 export at different [Ca2+]ex. P2X7R cells expressing TG2 were stimulated with BzATP for 10 min in medium containing 0.9 or 2.2 mM Ca2+ or in Ca2+-free medium, and chased for 30 min in respective media without BzATP. Conditioned media were analyzed by western blotting for TG2 and flotillin-2. (B) TG2 catalytic activity is not required for P2X7R-mediated export. P2X7R cells expressing TG2 or the TG2 C277S mutant were stimulated with BzATP in medium containing 0.9 or 2.2 mM Ca2+ and TG2 export was assessed as above. (C,D) [Ca2+]ex regulates P2X7R activity. P2X7R or parental cells were stimulated with BzATP, as indicated, in PSS containing YO-PRO1 and different concentrations of Ca2+. To determine YO-PRO1 uptake by cells after BzATP application, changes in well-specific fluorescence (λex, 480-10 nm; λem, 520-10 nm) were monitored over time. A representative experiment of dye uptake in Ca2+-free PSS is shown as mean±s.e.m. of two wells (C). In D, the initial rates of YO-PRO1 uptake at different [Ca2+]ex in response to 300 μM BzATP are given (mean±s.e.m.; n=2).

Mentions: TG2 secretion was effectively stimulated by P2X7R activation in medium that contains 0.9 mM Ca2+, which is similar to the free ionized extracellular Ca2+ concentration estimated at 1.1–1.3 mM (Riccardi and Kemp, 2012), but surprisingly not in medium containing high Ca2+ (Figs 1D and 5A). BzATP treatment of cells in the absence of Ca2+ led to enhanced TG2 secretion during stimulation only (Fig. 5A), indicating that TG2 export was faster but not sustained. In contrast, flotillin-2 release occurring at 0.9 mM Ca2+ was greatly reduced when cells were stimulated with agonist at either 0 or 2.2 mM Ca2+ (Fig. 5A). This shows that TG2 and flotillin-2 secretion is differentially affected by the extracellular Ca2+ concentration ([Ca2+]ex) and hence, that the underlying mechanisms are distinct. As microvesicle shedding is a Ca2+-dependent process, TG2 release in Ca2+-free medium supports a vesicle-independent mode of release, in line with previous data (Fig. 4).Fig. 5.


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

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

Extracellular Ca2+ regulates TG2 secretion. (A) P2X7R-mediated TG2 export at different [Ca2+]ex. P2X7R cells expressing TG2 were stimulated with BzATP for 10 min in medium containing 0.9 or 2.2 mM Ca2+ or in Ca2+-free medium, and chased for 30 min in respective media without BzATP. Conditioned media were analyzed by western blotting for TG2 and flotillin-2. (B) TG2 catalytic activity is not required for P2X7R-mediated export. P2X7R cells expressing TG2 or the TG2 C277S mutant were stimulated with BzATP in medium containing 0.9 or 2.2 mM Ca2+ and TG2 export was assessed as above. (C,D) [Ca2+]ex regulates P2X7R activity. P2X7R or parental cells were stimulated with BzATP, as indicated, in PSS containing YO-PRO1 and different concentrations of Ca2+. To determine YO-PRO1 uptake by cells after BzATP application, changes in well-specific fluorescence (λex, 480-10 nm; λem, 520-10 nm) were monitored over time. A representative experiment of dye uptake in Ca2+-free PSS is shown as mean±s.e.m. of two wells (C). In D, the initial rates of YO-PRO1 uptake at different [Ca2+]ex in response to 300 μM BzATP are given (mean±s.e.m.; n=2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696497&req=5

JCS175968F5: Extracellular Ca2+ regulates TG2 secretion. (A) P2X7R-mediated TG2 export at different [Ca2+]ex. P2X7R cells expressing TG2 were stimulated with BzATP for 10 min in medium containing 0.9 or 2.2 mM Ca2+ or in Ca2+-free medium, and chased for 30 min in respective media without BzATP. Conditioned media were analyzed by western blotting for TG2 and flotillin-2. (B) TG2 catalytic activity is not required for P2X7R-mediated export. P2X7R cells expressing TG2 or the TG2 C277S mutant were stimulated with BzATP in medium containing 0.9 or 2.2 mM Ca2+ and TG2 export was assessed as above. (C,D) [Ca2+]ex regulates P2X7R activity. P2X7R or parental cells were stimulated with BzATP, as indicated, in PSS containing YO-PRO1 and different concentrations of Ca2+. To determine YO-PRO1 uptake by cells after BzATP application, changes in well-specific fluorescence (λex, 480-10 nm; λem, 520-10 nm) were monitored over time. A representative experiment of dye uptake in Ca2+-free PSS is shown as mean±s.e.m. of two wells (C). In D, the initial rates of YO-PRO1 uptake at different [Ca2+]ex in response to 300 μM BzATP are given (mean±s.e.m.; n=2).
Mentions: TG2 secretion was effectively stimulated by P2X7R activation in medium that contains 0.9 mM Ca2+, which is similar to the free ionized extracellular Ca2+ concentration estimated at 1.1–1.3 mM (Riccardi and Kemp, 2012), but surprisingly not in medium containing high Ca2+ (Figs 1D and 5A). BzATP treatment of cells in the absence of Ca2+ led to enhanced TG2 secretion during stimulation only (Fig. 5A), indicating that TG2 export was faster but not sustained. In contrast, flotillin-2 release occurring at 0.9 mM Ca2+ was greatly reduced when cells were stimulated with agonist at either 0 or 2.2 mM Ca2+ (Fig. 5A). This shows that TG2 and flotillin-2 secretion is differentially affected by the extracellular Ca2+ concentration ([Ca2+]ex) and hence, that the underlying mechanisms are distinct. As microvesicle shedding is a Ca2+-dependent process, TG2 release in Ca2+-free medium supports a vesicle-independent mode of release, in line with previous data (Fig. 4).Fig. 5.

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