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Roscovitine-induced apoptosis in neutrophils and neutrophil progenitors is regulated by the Bcl-2-family members Bim, Puma, Noxa and Mcl-1.

Gautam S, Kirschnek S, Wiesmeier M, Vier J, Häcker G - PLoS ONE (2013)

Bottom Line: The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss.In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa.In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center, Freiburg, Freiburg, Germany.

ABSTRACT
Neutrophil granulocyte (neutrophil) apoptosis plays a key role in determining inflammation in infectious and non-infectious settings. Recent work has shown that inhibitors of cyclin-dependent kinases (cdk) such as roscovitine can potently induce neutrophil apoptosis and reduce inflammation. Using a conditional Hoxb8-expression system we tested the participation of Bcl-2-family proteins to roscovitine-induced apoptosis in mouse neutrophils and in neutrophil progenitor cells. Bcl-2 strongly protected against roscovitine-induced apoptosis in neutrophils. The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss. The only substantial change in protein levels observed was the loss of Mcl-1, which was not transcriptional and was inhibited by proteasome blockade. In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa. The pattern of protein expression and Mcl-1-regulation in progenitor cells was very similar to the one observed in differentiated neutrophils. In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.

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Role of caspases and BH3-only proteins in apoptosis in neutrophil progenitor cells.(A) PI-staining of wt progenitor cells treated with roscovitine (25 µM) alone or in the presence of the caspase-inhibitor Q-VD-OPh (50 µM) for 12 h. (B) PI staining of wt, Bim-/-, Noxa-/-, Bim/Noxa-/-, Bim/Puma-/- and Bcl-2-transgenic progenitors treated with roscovitine (25 µM) for the indicated times. Bar diagram represents mean/SEM of three independent experiments. Statistical significance is calculated using the Student’s t-test (ns, non-significant; *, p<0.05; ** p< 0.01; *** p<0.001).
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pone-0079352-g005: Role of caspases and BH3-only proteins in apoptosis in neutrophil progenitor cells.(A) PI-staining of wt progenitor cells treated with roscovitine (25 µM) alone or in the presence of the caspase-inhibitor Q-VD-OPh (50 µM) for 12 h. (B) PI staining of wt, Bim-/-, Noxa-/-, Bim/Noxa-/-, Bim/Puma-/- and Bcl-2-transgenic progenitors treated with roscovitine (25 µM) for the indicated times. Bar diagram represents mean/SEM of three independent experiments. Statistical significance is calculated using the Student’s t-test (ns, non-significant; *, p<0.05; ** p< 0.01; *** p<0.001).

Mentions: These cells were slightly less sensitive to roscovitine than differentiated cells, with about 60 % dying in the first 8 h but 90 % cell death at 12 h, which was inhibited by caspase-inhibition (Figure 5A, B). Bcl-2 again protected almost completely against roscovitine-induced apoptosis (Figure 5B). Surprisingly, the loss of Bim failed to protect these cells against roscovitine-induced apoptosis. When both Bim and Puma were absent, some protection was seen. A very strong effect was however seen when cells lacking Noxa were exposed to roscovitine, while additional loss of Bim did not further reduce sensitivity (Figure 5B).


Roscovitine-induced apoptosis in neutrophils and neutrophil progenitors is regulated by the Bcl-2-family members Bim, Puma, Noxa and Mcl-1.

Gautam S, Kirschnek S, Wiesmeier M, Vier J, Häcker G - PLoS ONE (2013)

Role of caspases and BH3-only proteins in apoptosis in neutrophil progenitor cells.(A) PI-staining of wt progenitor cells treated with roscovitine (25 µM) alone or in the presence of the caspase-inhibitor Q-VD-OPh (50 µM) for 12 h. (B) PI staining of wt, Bim-/-, Noxa-/-, Bim/Noxa-/-, Bim/Puma-/- and Bcl-2-transgenic progenitors treated with roscovitine (25 µM) for the indicated times. Bar diagram represents mean/SEM of three independent experiments. Statistical significance is calculated using the Student’s t-test (ns, non-significant; *, p<0.05; ** p< 0.01; *** p<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815126&req=5

pone-0079352-g005: Role of caspases and BH3-only proteins in apoptosis in neutrophil progenitor cells.(A) PI-staining of wt progenitor cells treated with roscovitine (25 µM) alone or in the presence of the caspase-inhibitor Q-VD-OPh (50 µM) for 12 h. (B) PI staining of wt, Bim-/-, Noxa-/-, Bim/Noxa-/-, Bim/Puma-/- and Bcl-2-transgenic progenitors treated with roscovitine (25 µM) for the indicated times. Bar diagram represents mean/SEM of three independent experiments. Statistical significance is calculated using the Student’s t-test (ns, non-significant; *, p<0.05; ** p< 0.01; *** p<0.001).
Mentions: These cells were slightly less sensitive to roscovitine than differentiated cells, with about 60 % dying in the first 8 h but 90 % cell death at 12 h, which was inhibited by caspase-inhibition (Figure 5A, B). Bcl-2 again protected almost completely against roscovitine-induced apoptosis (Figure 5B). Surprisingly, the loss of Bim failed to protect these cells against roscovitine-induced apoptosis. When both Bim and Puma were absent, some protection was seen. A very strong effect was however seen when cells lacking Noxa were exposed to roscovitine, while additional loss of Bim did not further reduce sensitivity (Figure 5B).

Bottom Line: The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss.In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa.In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University Medical Center, Freiburg, Freiburg, Germany.

ABSTRACT
Neutrophil granulocyte (neutrophil) apoptosis plays a key role in determining inflammation in infectious and non-infectious settings. Recent work has shown that inhibitors of cyclin-dependent kinases (cdk) such as roscovitine can potently induce neutrophil apoptosis and reduce inflammation. Using a conditional Hoxb8-expression system we tested the participation of Bcl-2-family proteins to roscovitine-induced apoptosis in mouse neutrophils and in neutrophil progenitor cells. Bcl-2 strongly protected against roscovitine-induced apoptosis in neutrophils. The isolated loss of either Bim or noxa provided significant, partial protection while protection through combined loss of Bim and noxa or Bim and Puma was only slightly greater than this individual loss. The only substantial change in protein levels observed was the loss of Mcl-1, which was not transcriptional and was inhibited by proteasome blockade. In progenitor cells there was no protection by the loss of Bim alone but substantial protection by the loss of both Bim and Puma; surprisingly, strongest protection was seen by the isolated loss of noxa. The pattern of protein expression and Mcl-1-regulation in progenitor cells was very similar to the one observed in differentiated neutrophils. In addition, roscovitine strongly inhibited proliferation in progenitor cells, associated with an accumulation of cells in G2/M-phase.

Show MeSH
Related in: MedlinePlus