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Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.

Gray RD, Lucas CD, Mackellar A, Li F, Hiersemenzel K, Haslett C, Davidson DJ, Rossi AG - J Inflamm (Lond) (2013)

Bottom Line: Inhibition of novel and atypical PKC had no effect.Conventional PKCs have a prominent role in NET formation.Furthermore PKCβ is the major isoform implicated in NET formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, Scotland, UK. r.d.gray@ed.ac.uk.

ABSTRACT

Background: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.

Methods: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.

Results: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001).

Conclusions: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

No MeSH data available.


Related in: MedlinePlus

Effect of isozyme specific PKC inhibitors on NET formation: Cells were plated as described and either pre-treated with at increasing concentrations of Gö 6976 (conventional PKC inhibitor), Rottlerin (atypical PKC inhibitor), PKCζ psuedosubstrate (novel PKC inhibitor), or control media, before stimulation with PMA at 10 nM for 4 h. A) Pre-treatment with Gö 6976 significantly reduced NET formation at 100 nM (P<0.05) and 1 μM (p<0.001). B) Pre-treatment with Rottlerin had no effect on NET formation. C) Pre-treatment with PKCζ psuedosubstrate had no effect on NET formation. Data show mean +/− SEM for n=3 independent experiments. * indicates p<0.05, *** indicates p<0.001.
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Figure 3: Effect of isozyme specific PKC inhibitors on NET formation: Cells were plated as described and either pre-treated with at increasing concentrations of Gö 6976 (conventional PKC inhibitor), Rottlerin (atypical PKC inhibitor), PKCζ psuedosubstrate (novel PKC inhibitor), or control media, before stimulation with PMA at 10 nM for 4 h. A) Pre-treatment with Gö 6976 significantly reduced NET formation at 100 nM (P<0.05) and 1 μM (p<0.001). B) Pre-treatment with Rottlerin had no effect on NET formation. C) Pre-treatment with PKCζ psuedosubstrate had no effect on NET formation. Data show mean +/− SEM for n=3 independent experiments. * indicates p<0.05, *** indicates p<0.001.

Mentions: Ro-31-8220 is an inhibitor of multiple PKC isoforms, therefore in order to investigate PKC isoform specificity, PKC isoform classes were targeted with specific inhibitors; Gö 6976 (Conventional), rottlerin (Novel), PKCζ psuedosubstrate (Atypical) (Figure 3A, B, C). Figure 3A demonstrates that Gö 6976 significantly inhibited NET formation at 100 nM (p<0.05) and 1 μM (p<0.001). In contrast, Rottlerin and PKCζ psuedosubstrate had no significant effect on NET formation (Figure 3B and C). These data were confirmed microscopically and demonstrate a key role for conventional PKC in NET formation.


Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.

Gray RD, Lucas CD, Mackellar A, Li F, Hiersemenzel K, Haslett C, Davidson DJ, Rossi AG - J Inflamm (Lond) (2013)

Effect of isozyme specific PKC inhibitors on NET formation: Cells were plated as described and either pre-treated with at increasing concentrations of Gö 6976 (conventional PKC inhibitor), Rottlerin (atypical PKC inhibitor), PKCζ psuedosubstrate (novel PKC inhibitor), or control media, before stimulation with PMA at 10 nM for 4 h. A) Pre-treatment with Gö 6976 significantly reduced NET formation at 100 nM (P<0.05) and 1 μM (p<0.001). B) Pre-treatment with Rottlerin had no effect on NET formation. C) Pre-treatment with PKCζ psuedosubstrate had no effect on NET formation. Data show mean +/− SEM for n=3 independent experiments. * indicates p<0.05, *** indicates p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643828&req=5

Figure 3: Effect of isozyme specific PKC inhibitors on NET formation: Cells were plated as described and either pre-treated with at increasing concentrations of Gö 6976 (conventional PKC inhibitor), Rottlerin (atypical PKC inhibitor), PKCζ psuedosubstrate (novel PKC inhibitor), or control media, before stimulation with PMA at 10 nM for 4 h. A) Pre-treatment with Gö 6976 significantly reduced NET formation at 100 nM (P<0.05) and 1 μM (p<0.001). B) Pre-treatment with Rottlerin had no effect on NET formation. C) Pre-treatment with PKCζ psuedosubstrate had no effect on NET formation. Data show mean +/− SEM for n=3 independent experiments. * indicates p<0.05, *** indicates p<0.001.
Mentions: Ro-31-8220 is an inhibitor of multiple PKC isoforms, therefore in order to investigate PKC isoform specificity, PKC isoform classes were targeted with specific inhibitors; Gö 6976 (Conventional), rottlerin (Novel), PKCζ psuedosubstrate (Atypical) (Figure 3A, B, C). Figure 3A demonstrates that Gö 6976 significantly inhibited NET formation at 100 nM (p<0.05) and 1 μM (p<0.001). In contrast, Rottlerin and PKCζ psuedosubstrate had no significant effect on NET formation (Figure 3B and C). These data were confirmed microscopically and demonstrate a key role for conventional PKC in NET formation.

Bottom Line: Inhibition of novel and atypical PKC had no effect.Conventional PKCs have a prominent role in NET formation.Furthermore PKCβ is the major isoform implicated in NET formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, Scotland, UK. r.d.gray@ed.ac.uk.

ABSTRACT

Background: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.

Methods: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.

Results: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001).

Conclusions: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

No MeSH data available.


Related in: MedlinePlus