Research ArticleImmunology

Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells

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Science Signaling  16 Aug 2016:
Vol. 9, Issue 441, pp. ra82
DOI: 10.1126/scisignal.aae0453
  • Fig. 1 Characterization of RTK- and GPCR-driven PIP3 responses in BMDMs: Roles of class I PI3Ks.

    (A to D) Serum-starved BMDMs (1.2 × 106 cells) from wild-type (WT) mice were stimulated with 30 nM C5a (A) or M-CSF (30 ng/ml) (C) for the indicated times, whereas BMDMs (1.2 × 106) from WT (closed bars), β-KO (open bars), δ-KI (dark gray bars), or γ-KO (light gray bars) mice were serum-starved overnight before being stimulated for 30 s with either 30 nM C5a (B) or M-CSF (30 ng/ml) (D) or vehicle control (B and D). Where indicated (hatched bars, D), the p110α-selective inhibitor BYL719 (1 μM final) was added to WT BMDMs 5 min before the cells were stimulated. (E) BMDMs (1.2 × 106 cells) from WT or δ-KI mice were serum-starved and incubated with the indicated concentrations of TGX221 (WT, black diamonds; δ-KI, black squares) or 1 μM IC87114 (WT, gray diamonds; δ-KI, gray squares) for 5 min before being stimulated for 30 s with M-CSF (30 ng/ml). As a control, WT BMDMs were also left unstimulated (gray triangle). (F) BMDMs (1.2 × 106 cells) from WT (black diamonds) or δ-KI (black squares) mice were serum-starved and incubated with vehicle (WT) or 1 μM IC87114 (δ-KI) and the indicated concentrations of BYL719 for 5 min before being stimulated for 30 s with M-CSF (30 ng/ml). As a control, WT BMDMs were also left unstimulated (gray triangle). (A to F) Reactions were quenched, lipids were extracted, and the amounts of PIP3 and PI were quantitated by mass spectrometry (MS) as described in Materials and Methods. Data are means ± SEM of at least three experiments, each performed in duplicate [except for the WT control in (E), where n = 1], and are expressed as the ratio of the abundance of PIP3 to that of PI (PIP3/PI) to account for any variations in cell input. The following statistical analyses were performed. (B) For the comparisons shown: NS, not significant; *P < 0.05, **P < 0.01, by one-way analysis of variance (ANOVA) with Holm-Sidak post hoc test. (E) For M-CSF + 0 nM TGX221 versus M-CSF + IC87114, P = 0.0156 (WT), P = 0.7417 (δ-KI); for M-CSF + 0 nM TGX221 versus M-CSF + TGX221, P < 0.05 (WT), P < 0.05 (δ-KI), by ratio paired t test with Holm-Sidak correction for multiple comparisons and independent t test. (F) For comparison to the 0 μM BYL719 control, *P < 0.05, **P < 0.01, and ***P < 0.005 by repeated-measures ANOVA with Holm-Sidak post hoc test, with Holm-Sidak correction for multiple comparisons.

  • Fig. 2 Synergistic PIP3 production by BMDMs in response to costimulation of RTKs and GPCRs is dependent on p110β and requires binding to Gβγ proteins.

    (A) Left: BMDMs (1.2 × 106 cells) from WT mice were serum-starved overnight before being stimulated for 30 s with 30 nM C5a or M-CSF (30 ng/ml) alone or in combination, as indicated. PIP3 was quantified by MS as described for Fig. 1. Data are means ± SEM of at least 10 experiments, each performed in duplicate, and are expressed as a percentage of the response to M-CSF. Right: Ratios for costimulation (the PIP3 response to simultaneous addition of C5a and M-CSF) divided by additive (the sum of the individual PIP3 responses to C5a and M-CSF) for the same experiments were calculated as described in Materials and Methods. (B) Left: WT (closed bars), β-KO (open bars), δ-KI (dark gray bars), and γ-KO (light gray bars) BMDMs (1.2 × 106) were left unstimulated (control) or were stimulated with the indicated agonists, and PIP3/PI ratios were quantitated as described in (A). Data are means ± SEM of at least three experiments, each performed in duplicate, and are expressed as a percentage of the PIP3 abundance of WT macrophages in response to M-CSF. Data for the unstimulated and singly stimulated cells include data from the experiments shown in (A) and Fig. 1 (B and D). Right: Ratios (costimulation/additive) for the same experiments were calculated as described in (A). (C) Left: BMDMs from WT (closed bars), β-RBD (hatched bars), β-Gβγ (striped bars), and β-KO (open bars) mice were prepared and treated as described in (A). Data are means ± SEM of at least three experiments, each performed in duplicate, and are expressed as a percentage of the response of WT cells to M-CSF. Right: Ratios (costimulation/additive) for the same experiments were calculated as described in (A). *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.0001 by ratio paired t test (A, right) with Holm-Sidak correction for multiple comparisons (B and C, right).

  • Fig. 3 PIP3 generation in BMNs in response to the GPCR agonists fMLP and C5a requires p110γ but not p110β.

    (A and B) WT BMNs (0.5 × 106) were stimulated in suspension with 10 μM fMLP (A) or 100 nM C5a (B) for the indicated times. The reactions were quenched, and lipids were extracted, derivatized, and quantitated by MS as described earlier. Data are means ± SEM of the PIP3/PI ratio of at least three experiments, each performed in at least duplicate. (C and D) BMNs (0.5 × 106) from WT, β-KO, and γ-KO mice were preincubated with 40 nM TGX221 (hatched bars) or 0.05% DMSO (vehicle, closed bars) before being stimulated for 10 s with fMLP (C) or C5a (D). Reactions were then stopped, and lipids were extracted and quantitated as described earlier. The PIP3/PI ratio in unstimulated WT BMNs (WT-CON) is also shown. Data are means ± SEM of the PIP3/PI ratio of at least three experiments, each performed in at least duplicate. NS, not significant. ***P < 0.005 by one-way ANOVA with Holm-Sidak post hoc test comparisons as indicated (C and D).

  • Fig. 4 Adhesion, spreading, and ROS production in BMNs in response to immune complexes and adhesive proteins are p110β-dependent responses.

    (A) BMNs from WT, β-Gβγ, β-RBD, and β-KO mice were incubated for 10 min with either DMSO or 40 nM TGX221 before being applied to glass coverslips coated with poly-RGD+. Cells were incubated at 37°C for 20 min, and adherent cells were fixed in 3.7% paraformaldehyde, washed, and mounted on glass microslides before being visualized with a Nikon Ti-E Live Cell Imager inverted microscope with a 20× objective lens. Images of a 1:15 field of view of adherent spread (S) and nonspread (NS) cells are representative of three experiments performed in duplicate (except for cells from β-KO mice, for which n = 2). (B) WT BMNs (0.5 × 106) were preincubated with horseradish peroxidase (HRP) and luminol before being added to a 96-well plate coated with IgG-BSA (triangles), FGN (diamonds), or poly-RGD+ (circles), or to a plate containing IgG-opsonized SRBCs (squares). ROS generation was measured in triplicate for each condition by chemiluminescence and recorded with a Berthold MicroLumat Plus luminometer and are expressed as relative light units (RLUs) per second. Data are from one experiment and are representative of at least three experiments. (C and D) BMNs from the indicated mice were pretreated for 10 min with either DMSO or 40 nM TGX221 before being incubated on duplicate coverslips coated with IgG-BSA, FGN, or poly-RGD+, as indicated, and were processed as described for (A). Images were analyzed for cell adhesion and spreading with ImageJ software (Fiji), selecting for size and brightness as described in Materials and Methods. Data are means ± SEM of the number of adherent cells per field of view (C) and of the percentage of spread cells (D) from three experiments performed in duplicate (except for cells from β-KO mice, for which n = 2). (E) BMNs (0.5 × 106) from WT, β-RBD, β-Gβγ, and β-KO mice were preincubated with HRP/luminol in the presence of 40 nM TGX221 (hatched bars) or DMSO (0.05%, closed bars) before being added to wells precoated with IgG-BSA, FGN, or poly-RGD+, as indicated. ROS was then measured as described earlier. Data are means ± SEM for accumulated light emission (RLU) over a 20-min recording of at least three experiments performed in at least duplicate and are expressed as a percentage of the ROS generated in DMSO-treated WT BMNs. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 by t test with Holm-Sidak corrections for multiple comparisons.

  • Fig. 5 The p110β-dependent generation of PIP3 in BMNs in response to adhesion to poly-RGD+ requires both an intact RBD and binding to Gβγ proteins.

    BMNs from WT, β-RBD, β-Gβγ, and β-KO mice were preincubated with 40 nM TGX221 or vehicle control before being added to glass coverslips coated with poly-RGD+ or were left in suspension. Where indicated, WT BMNs were additionally preincubated with 100 nM wortmannin (wort). Cells were allowed to adhere and spread for 10 min at 37°C and then harvested for PIP3 measurement by MS as described earlier. Data are means ± SEM of PIP3/PI ratios from three experiments (except for data from wortmannin-treated cells, for which n = 2), each performed in duplicate. N.D., not determined. *P < 0.05 by one-sample t test with Holm-Sidak correction for multiple comparisons.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/441/ra82/DC1

    Methods

    Fig. S1. Targeting of Pik3cbKK526,527DD.

    Fig. S2. Confirmation of the correct targeting of Pik3cbKK526,527DD.

    Fig. S3. p110β quantities in BMDMs and BMNs are unaffected by the introduction of the RBD- and Gβγ-insensitive mutations.

    Fig. S4. Dose responses of GPCR (C5a) and RTK (M-CSF) agonists on PIP3 production by BMDMs.

    Fig. S5. ROS generation in BMNs in response to the phagocytosis of IgG-SRBCs, but not soluble GPCR agonists, requires p110β activity.

    Fig. S6. ROS generation in BMNs in response to adhesion to poly-RGD+, but not immune complexes, is independent of BLT1 activation.

    Reference (70)

  • Supplementary Materials for:

    Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells

    Daniel M. Houslay, Karen E. Anderson, Tamara Chessa, Suhasini Kulkarni, Ralph Fritsch, Julian Downward, Jonathan M. Backer, Len R. Stephens,* Phillip T. Hawkins*

    *Corresponding author. Email: phillip.hawkins{at}babraham.ac.uk (P.T.H.); len.stephens{at}babraham.ac.uk (L.R.S.)

    This PDF file includes:

    • Methods
    • Fig. S1. Targeting of Pik3cbKK526,527DD.
    • Fig. S2. Confirmation of the correct targeting of Pik3cbKK526,527DD.
    • Fig. S3. p110β quantities in BMDMs and BMNs are unaffected by the introduction of the RBD- and Gβγ-insensitive mutations.
    • Fig. S4. Dose responses of GPCR (C5a) and RTK (M-CSF) agonists on PIP3 production by BMDMs.
    • Fig. S5. ROS generation in BMNs in response to the phagocytosis of IgG-SRBCs, but not soluble GPCR agonists, requires p110β activity.
    • Fig. S6. ROS generation in BMNs in response to adhesion to poly-RGD+, but not immune complexes, is independent of BLT1 activation.
    • Reference (70)

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    Citation: D. M. Houslay, K. E. Anderson, T. Chessa, S. Kulkarni, R. Fritsch, J. Downward, J. M. Backer, L. R. Stephens, P. T. Hawkins, Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells. Sci. Signal. 9, ra82 (2016).

    © 2016 American Association for the Advancement of Science

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