spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 25 November 2008
doi: 10.1242/jcs.032763

Journal of Cell Science 121, 4124-4133 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Papakonstanti, E. A.
Right arrow Articles by Vanhaesebroeck, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Papakonstanti, E. A.
Right arrow Articles by Vanhaesebroeck, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages

Evangelia A. Papakonstanti1,2,*, Olivier Zwaenepoel2, Antonio Bilancio1,{ddagger}, Emily Burns1, Gemma E. Nock1, Benjamin Houseman3, Kevan Shokat3, Anne J. Ridley4 and Bart Vanhaesebroeck1,§

1 Centre for Cell Signalling, Institute of Cancer, Queen Mary, University of London, Charterhouse Square, London EC1M 6BQ, UK
2 Department of Biochemistry, School of Medicine, University of Crete, Vassilika Vouton, GR-71110 Heraklion, Greece
3 Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA
4 Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 1. Effect of pharmacological or genetic inactivation of class IA PI3K isoforms on CSF1-induced phosphorylation of Akt. Upper panel, WT BMMs were pre-treated for 1 hour with PW12 (0.5 µM), TGX155 (0.5 µM) or IC87114 (5 µM), followed by incubation with CSF1 (30 ng/ml) for 10 minutes and analysis of phosphorylation of Akt (on T308 and S473) by western blotting of total cell lysates (80 µg/lane). Graph represents the mean ± s.e.m. of a representative experiment performed in triplicate (**P<0.01). Lower panel, BMMs with heterozygotic inactivation of p110{alpha} or p110{delta} were stimulated with 30 ng/ml CSF1 for the indicated time points, followed by analysis as described above. Graphs represent the mean ± s.e.m. of three separate experiments (*P<0.05).

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 2. Effect of pharmacological or genetic inactivation of class IA PI3K isoforms on the activation of small GTPases and PTEN. (A,B) Equal volumes of cell lysates of the indicated BMMs were subjected to pull-down assay with GTP-PBD, followed by detection of precipitated Rac1 by western blotting. Total cell lysates were resolved and immunoblotted for Rac1. Graphs represent the mean ± s.e.m. of three experiments. *P<0.05; **P<0.01 when compared with cells treated with vehicle only for each time point or with WT cells. (C,D) Equal volumes of cell lysates of the indicated BMMs were subjected to pull-down assay with GST-RBD, followed by western blot detection of precipitated RhoA. Total cell lysates were resolved on the same SDS-PAGE gel and immunoblotted for RhoA. Graphs for BMMs represent the mean ± s.e.m. of three experiments. *P<0.05; **P<0.01, compared with cells treated with vehicle only for each time point or with WT cells. (E) BMMs were pre-treated for 1 h with PW12 (0.5 µM), TGX155 (0.5 µM) or IC87114 (5 µM), followed by assay of PTEN lipid phosphatase activity as described (left panel). One representative experiment done in triplicate is shown (*P<0.05). Right panels show the effect of genetic inactivation of p110{delta} or p110{alpha} on PTEN lipid phosphatase activity. PTEN was immunoprecipitated from the respective BMM lysates followed by determination of its phosphatase activity towards synthetic PIP3 by ELISA (Echelon). One representative experiment done in triplicate is shown (*P<0.05).

 

Figure 3
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 3. Effect of genetic inactivation of p110{alpha} or p110{delta} on basal and CSF1-induced morphological changes in BMMs. (A) CSF1-starved or CSF1-stimulated cells were stained for F-actin. Scale bar: 30 µm. (B-D) Measurement of elongation ratio (ratio of the longest to the shortest cell axes, B), total area of adhesion (C) and ruffling (D). The results shown are mean ± s.e.m. of >=180 cells from each population. ***P<0.0001; **P<0.05. Comparison labelled 1 was with unstimulated WT; 2 with unstimulated {alpha}D933A/WT; and 3 with unstimulated {delta}D910A/WT cells.

 

Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Effect of genetic inactivation of p110{alpha} or p110{delta} on chemotaxis of BMMs. (A) Chemotaxis of BMMs exposed to a gradient of CSF1 in Dunn chambers, monitored for 16 hours by time-lapse microscopy. The cell tracks from three separate experiments were merged into a single file for analysis. Circular histograms (upper panels) show the proportion of cells migrating into each of 20 segments of the angular trajectory plot (measured when each cell migrated past a horizon of 30 µm from its starting point with the source of CSF1 at the top). Arrows indicate significant mean directionality of the cell population. The green shaded areas mark the 95% confidence intervals of statistical significance. Similar chemotaxis plots were obtained for horizon limits of 50, 80, 100 µm, although the cell numbers varied (not shown). Vector plots (lower panel) show the end point of the cells with the starting point of each cell at the intersection between x and y axes and with the source of CSF1 at the top of each plot. (B) Summary of the effect of genetic inactivation of p110{alpha} or p110{delta} on the activation of Rac1 and RhoA, and on chemotaxis. (C) Effect of pharmacological inactivation of PI3K isoforms on chemotaxis in BMMs and BAC1.2F5 cells. Cells on the lower surface of the top chamber were stained and counted from at least six randomly chosen frames. *P<0.05 compared with DMSO-treated cells.

 

Figure 5
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 5. Effect of pharmacological or genetic inactivation of class IA PI3K isoforms on DNA synthesis in BAC1.2F5 cells and BMMs. (A) Effect of the p110{delta} inhibitor IC87114 or the pan-PI3K inhibitor LY294002 on DNA synthesis induced in BAC1.2F5 and WT BMM cells by increasing doses of CSF1. (B) Effect of pharmacological inactivation of p110{alpha}, p110β or p110{delta} on DNA synthesis in BMMs and BAC1.2F5 cells induced by CSF1 (30 ng/ml). [3H]Thymidine incorporation of inhibitor-treated cells was expressed relative to that of cells treated with vehicle (DMSO) only (set as 100%). *P<0.05, compared with DMSO-treated cells. (C) Effect of homozygous deletion of p110{alpha} on DNA synthesis in BMMs. The inset shows the expression level of p110{alpha} in Cre– and Cre+ BMMs in a representative experiment. (D) Summary of impact of p110 isoform neutralisation on CSF1-induced DNA synthesis in macrophages.

 

Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Effect of pharmacological inactivation of class IA PI3K isoforms on CSF1-induced phosphorylation of Akt in macrophage cell lines. Cells were pre-treated for 1 hour with PW12 (0.5 µM), TGX155 (0.5 µM) or IC87114 (5 µM), followed by incubation with CSF1 (30 ng/ml) for 10 minutes and analysis of phosphorylation of Akt (on T308 and S473) by western blotting of total cell lysates (80 µg/lane). Graph represents the mean ± s.e.m. of two experiments performed in duplicate (*P<0.05; **P<0.01; ***P<0.001).

 

Figure 7
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 7. (A) Expression levels of class IA PI3K isoforms in BAC1.2F5 cells and BMMs (WT or expressing inactive p110{alpha} or p110{delta}) were determined by immunoblotting of total cell lysates (80 µg per lane). (B) Selective recruitment of p110{delta} to the CSF1R in BMMs but not in BAC1.2F5 cells. WT BMMs or BAC1.2F5 cells were incubated with CSF1 (30 ng/ml) for the indicated times, followed by lysis and immunoprecipitation of p110 isoforms. The co-precipitated CSF1R was detected by western blotting. Equal loading was assessed by reprobing the membrane with antibodies against p85. TCL, total cell lysate (100 µg). For BMMs, a representative experiment out of three is shown. For BAC1.2F5 cells, the results of two independent, representative experiments are shown.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008