RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms

Abstract

RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs.

Graphical abstract

Figure 1

PI 3-Kinase p110β Is Unable to Interact with RAS (A) p110β does not bind to GTP-loaded RAS proteins. Purified recombinant, GTPγS-loaded HRAS, KRAS, NRAS, and RAB5 were incubated with lysates from COS7 cells expressing FLAG-p110α/p85 or FLAG-p110β/p85, wild-type (WT), or RBD double mutant (DM). (B) Active RAS proteins do not bind to immobilized p110β/p85. Purified recombinant GST-p110α/p85 and GST-p110β/p85 were incubated with lysates from COS7 cells expressing Myc-tagged, constitutively active HRAS, KRAS, NRAS, and RAB5. (C) Active RAS proteins do not stimulate p110β in cells. Lipids were extracted and PIP₃ levels measured from COS7 cells expressing constitutively active mutants of HRAS, KRAS, NRAS, and RAB5, alone or in combination with p110α/p85 or p110β/p85 (n = 3; mean with SD; one-way ANOVA). (D) Type I PI3K RBDs show moderate sequence similarity. Alignment of amino acid sequences of all four type I PI3K RBDs (PIK3CB = p110β, PIK3CD = p110δ, PIK3CA = p110α, PIK3CG = p110γ). Orange represents RAS-binding residues in p110γ, and arrows represent conserved “RAS-binding” residues. (E) Mutation of RBD key residues in p110β. The two point mutations are shown together with hypothetical interactor residues modeled on the RAS-p110γ interaction. (F) Unaltered lipid kinase activity of recombinant p110β-RBD-DM protein. Lipid kinase assay assessing basal activities of purified recombinant p110β/p85 complexes (n = 3; mean with SEM). (G) p110β-RBD-DM protein remains sensitive to Gβγ and phosphotyrosine. A representative lipid kinase assay assessing effect of recombinant Gβγ and a PDGFR-derived phosphotyrosine peptide (pY740) on the activity of purified recombinant p85/p110β-WT and p85/p110β-RBD-DM is shown. (H) Activity of p110β-RBD-DM in living cells is reduced. Lipids were extracted and PIP₃ levels measured from COS7 cells expressing wild-type or RBD mutant p110β/p85. Gβγ, coexpression of Gβ₂ and Gγ₁; Myr, myristoylated p110β (n = 3; mean with SEM; paired t test). See also Figure S1.

Figure S1

PI 3-Kinase p110β Is Unable to Interact with RAS, Related to Figure 1 (A) Active RAS proteins fail to stimulate p110β in cells. Constitutively active mutants of HRAS, KRAS and NRAS were expressed in COS7 cells along with empty vector, p110α/p85 or p110β/p85. Cells were serum-starved and protein lysates were made for western blot analysis. (B) p110β-RBD-DM is functionally compromised in intact cells. Wild-type p110β or p110β-RBD-DM were expressed at low levels in COS7 cells, along with p85. Cells were serum-starved and protein lysates were made for western blot analysis. Gβγ, coexpression of Gβ₂ and Gγ₁; Myr, myristoylated p110β. (C) DIRAS1 and DIRAS2 bind p110β in a GTP-dependent manner. cDNAs encoding all 34 murine members of the RAS subfamily of small GTPases (RFGs) were cloned into pGEX-2T and verified by sequencing. GST-tagged RFGs were expressed in E. coli, purified on glutathione agarose, loaded with GDP/GTPγS in vitro and incubated with lysates from transfected COS7 cells, expressing FLAG-p110β/p85. (D) Non-β isoforms bind to RAS proteins and a number of closely related RFGs. GST-tagged RFGs were purified from E. coli lysates and loaded with GDP/GTPγS in vitro. FLAG-tagged p110α, p110γ and p110δ were expressed in COS7 cells along with their respective regulatory subunits p85 or p101. COS7 cell lysates were incubated with GTPases for 1 hr and bound p110 was detected by western blot for FLAG.

Figure 2

RAC and CDC42 Directly Bind and Active p110β (A) p110β interacts with distinct RAS subfamily GTPases to non-β isoforms. Table summarizes results from GST pull-down assays (Figures S1C and S1D) probing 34 murine RAS subfamily GTPases for GTP-dependent binding of type I PI3K isoforms (−, no binding; +, specific binding; ++, strong binding; +++, very strong binding). (B) DIRAS1/-2 do not stimulate p110β in cells. Constitutively active DIRAS1 or DIRAS2 were coexpressed with FLAG-p110β/p85 in COS7 cells. (C) DIRAS does not stimulate p110β lipid kinase activity in vitro. Shown is a lipid kinase assay assessing the effect of purified recombinant, GTPγS-loaded DIRAS1 on p85/p110β (WT) and p85/p110β-RBD-DM (DM) activity (n = 3; mean with SEM). (D) G2 box sequences of H-/KRAS, DIRAS1/-2 and RAC1/CDC42. Color-coded amino acid sequence alignment of the N termini of indicated small GTPases. A square frame highlights the G2 box sequence, and the variant residue (D33 in RAS) is shown in gray. (E) RHO GTPases directly bind p110β in a GTP-dependent manner. Purified recombinant, GDP/GTPγS-loaded RHO GTPases were incubated with purified recombinant p110β/p85 (50 nM). Top: representative experiment; left: quantification (n = 2; mean with SEM); right: ³[H]-GTP uptake of GTPase preparations (n = 2; mean with SEM). (F) Binding of p110β to RAC1 and CDC42 is isoform specific. Purified recombinant GDP/GTPγS-loaded RAC1 and CDC42 were incubated with lysates from COS7 cells expressing FLAG-tagged p110α/p85, p110β/p85, p110γ/p101, and p110δ/p85. (G) RAC1 and CDC42 directly stimulate p110β lipid kinase activity. Purified recombinant GTPγS-loaded RAC1/CDC42 (1 μM) were added to purified recombinant p110β/p85, and lipid kinase activity was assessed in vitro. pTyr, phosphotyrosine peptide (pY740, 10 μM); Δp85, truncated p85, schematic in Figure 3C (n = 2; mean with SD). (H) RAC1 and CDC42 dose-dependently stimulate p110β lipid kinase activity. Increasing concentrations of purified recombinant GTPγS-loaded RAC1/CDC42 were added to purified recombinant p110β/Δp85, and lipid kinase activity was assessed in vitro (n = 3; mean with SEM). (I) RAC1 and CDC42 activate p110β in cells. Lipids were extracted and PIP₃ levels measured from COS7 cells expressing constitutively active RAC1 or CDC42, alone or in combination with FLAG-p110β/p85. Gβγ, coexpression of Gβ₂ and Gγ₁; Myr, myristoylated p110β (n = 2; mean with SD). Western blots show expression levels of FLAG-p110β and Myc-tagged GTPases. See also Figure S2.

Figure S2

RAC and CDC42 Directly Bind and Active p110β, Related to Figure 2 (A) RAS-D33/DIRAS-I37 are essential residues for isoform-specific PI3K binding. Left: GST-tagged wild-type and D33I mutant HRAS were expressed in E. coli, purified on glutathione agarose beads, loaded with GDP/GTPγS in vitro, and incubated with lysates from transfected COS7 cells expressing FLAG-p110α/p85 or FLAG-p110β/p85. Right: GST-tagged wild-type and I37D mutant DIRAS1 were probed for binding FLAG-p110α/p85 or FLAG-p110β/p85. Bound p110 was identified by western blot for FLAG. (B) DIRAS is unable to bind p110β-RBD-DM protein. GST pulldown assay assessing binding of purified recombinant p110β/p85 complexes to immobilized, GDP-/GTPγS-loaded DIRAS1. Lanes 1/2: p85/p110β wild-type; Lanes 3/4: Δp85/p110β wild-type; Lanes 5/6: p85/p110β-RBD-DM. Δp85 is depicted in Figure 3C. (C) G2 box sequences of RHO GTPases. Color-coded amino acid sequence alignment of the G2 boxes of indicated RHO GTPases, frame highlights RAC1-I33 residue. (D) RAC1 and CDC42 bind p110β in a GTP-dependent manner. Purified recombinant, GST-tagged and GDP/GTPγS-loaded RHO subfamily GTPases were incubated with lysates from COS7 cells expressing FLAG-p110β/p85. (E) RAC-I33 is critical for p110β binding. Purified recombinant wild-type RAC1 and a RAC1-I33D mutant were compared for GTP-dependent binding to purified recombinant p110β/p85 (50 nM). Representative experiment and quantification of two independent experiments are shown (mean and SEM). (F) RHO family GTPases do not bind p110α. 11 selected RHO family GTPases were expressed in E. coli, purified on glutathione agarose beads and loaded with GDP/GTPγS in vitro. HRAS was included as positive control. Pulldown was made from a lysate of transfected COS7 cells expressing FLAG-p110α/p85. (G) RAC and CDC42 activate p110β wild-type but not p110β–RBD-DM in transfected cells. Constitutively active mutants of RAC1 and CDC42 (Myc-tagged) were expressed in COS7 cells along with empty vector, FLAG-p110β/p85 or FLAG-p110β-RBD-DM/p85. (H) RAC1 and CDC42, but not RHOA activate p110β in transfected cells. Constitutively active mutants of RAC1, CDC42 and RHOA (Myc-tagged) were expressed in COS7 cells along with empty vector or FLAG-p110β/p85. Cells were serum-starved, and protein lysates were made for western blot analysis. (I) RAC and CDC42 fail to stimulate p110α lipid kinase activity in vitro. Representative lipid kinase assay assessing the effect of increasing concentrations of purified recombinant GTPγS-loaded RAC1 and CDC42 on the activity of purified recombinant p110α/p85 protein complexes. (J) RAC and CDC42 fail to activate p110α, p110γ and p110δ in transfected cells. Constitutively active mutants of RAC1 and CDC42 (Myc-tagged) were expressed in COS7 cells along with empty vector, FLAG-tagged p110α/p85, p110γ/p101 or p110δ/p85. Cells were serum-starved, and protein lysates were made for western blot analysis.

Figure 3

RAC and CDC42 Are Interactors of the p110β RAS-Binding Domain (A) RAC1 and CDC42 do not bind p110β-RBD-DM in vitro. Purified recombinant p85/p110β (WT) and p85/p110β-RBD-DM (DM) protein complexes, at the indicated concentrations, were incubated with GST-tagged, GDP/GTPγS-loaded RAC1 and CDC42. (B) RAC1 and CDC42 do not stimulate p110β-RBD-DM lipid kinase activity. Purified recombinant GTPγS-loaded RAC1/CDC42 (1 μM) were added to purified recombinant p85/p110β-WT or p85/p110β-RBD-DM in lipid kinase assays (n = 2; mean with SD). Data are part of experiment shown in Figure 2G. (C) The N terminus of p85 is not required for p110β binding to RAC1/CDC42. Purified recombinant p110β/p85 protein complexes were incubated with GST-tagged, GDP/GTPγS-loaded RAC1, CDC42 and RAB5. Lanes 1/2: p85/p110β-WT; lanes 3/4: Δp85/p110β-WT; lanes 5/6: p85/p110β-RBD-DM. Δp85 is detailed in the schematic. (D) Single RBD point mutations disrupt p110β binding to RAC1/CDC42. Amino acid alignment of type I PI3K RBDs with secondary structure elements of p110β (H, helix; E, β sheet) and color coding to illustrate effect of residue mutation on p110β binding to RAC1/CDC42. Green, unaltered binding; red, reduced/abolished binding; blue, unstable protein; yellow, p110β-RBD-DM residues; black, not mutated (Figures S3A and S3B for pull-down assays and table). (E) Thermodynamic characterization of the RAC1/CDC42-p110β interaction by ITC. Binding of purified recombinant GTPγS-loaded RAC1 and CDC42 to purified recombinant p110β/Δ85 in solution was studied. Top: differential power over time; bottom: thermal energy (H) over molar ratio. (F) Table summarizes results from ITC experiments. Numbers represent K_d values determined in independent experiments. n.b., no binding. See also Figure S3.

Figure S3

RAC and CDC42 Are Interactors of the p110β RAS-Binding Domain, Related to Figure 3 (A) Single point mutations across the p110β RBD disrupt binding to RAC and CDC42. 43 single point mutations of 37 RBD residues were introduced by site-directed mutagenesis. Mutants were expressed in COS7 cells, along with p85, and lysates were incubated with GST-tagged, GTPγS-loaded RAC1 and CDC42. Bound p110β was detected by western blot for FLAG. Mutants with reduced protein expression levels were deemed unstable and excluded from experiments. (B) Table summarizing results from GST pulldown studies shown in (A). Point mutations made are listed along with their impact on binding to RAC1 and CDC42 (+, no effect on binding; (+), reduced binding; −, complete or near complete loss of binding; unst., unstable protein). (C) Panel of representative ITC experiments investigating the thermodynamics of the interaction between indicated GTPγS-loaded small GTPases and recombinant p110/p85 complexes in solution. Top: the differential power recorded directly over time; bottom: thermal energy (H) over molar ratio.

Figure S4

RAC/CDC42 Binding to p110β Regulates PI3K Activity In Vivo, Related to Figure 4 (A) Targeting strategy to replace exon 6 of the murine Pik3cb gene, encoding the p110β catalytic subunit. An FRT-flanked neomycin selection cassette was inserted into murine genomic DNA provided by a BAC clone, immediately downstream of Pik3cb exon 6, using Red/ET recombination technology (Genebridges). Selection cassette and flanking arms of genomic DNA were sub-cloned and mutations (★) introduced by site-directed mutagenesis. Arrows indicate priming sites for ES cell screening and genotyping. The neomycin selection cassette was later removed by crosses of heterozygous p110β-RBD-DM mice with FLPe mice. (B) ES cell screening and genotyping. Homologous recombination in ES cells was confirmed by long genomic PCR bridging the entire targeting region (top, targeted allele 10kb, wild-type allele 8.5kb). After FLP recombination, MEFs (middle) and mice (bottom) were genotyped using primer pairs flanking the remaining FRT adjacent to exon 6. (C) Genomic sequencing confirms presence of mutations. Genomic DNA from heterozygous and homozygous p110β-RBD-DM mice was isolated and exon 6 was PCR amplified and sequenced using standard techniques. (D) Normal expression of type I PI3K subunits in p110β-RBD-DM MEFs. Wild-type and homozygous p110β-RBD-DM MEFs were maintained in 0%, 1% and 10% FCS, respectively. Lysates were made for western blot analysis. (E) Undisturbed stoichiometry of type I PI3K subunits in p110β-RBD-DM MEFs. p85 protein was immunoprecipitated from whole-cell lysates made from wild-type and p110β-RBD-DM MEFs. The coprecipitation of p110α and p110β along with p85 was assessed by western blot. (F) Deletion of p110α and p110β in conditional knockout MEFs. Immortalized p110α^lox/lox;CreER^+/− and p110β^lox/lox;CreER^+/− MEFs were treated with 4-hydroxytamoxifen (1 μM) for 3 consecutive days, prior to use in experiments.

Figure 4

RAC/CDC42 Binding to p110β Regulates PI3K Activity In Vivo (A) Reduced numbers of homozygous p110β-RBD-DM mice. Offspring from HET × HET crosses was genotyped at 2 weeks of age (n = 389; p < 0.02, chi-square analysis). (B) Newborn p110β-RBD-DM pups are smaller. Newborn pups from HET × HET crosses were collected on the morning of birth, weighed, and genotyped (n = 31; mean ±SEM; p = 0.011, t test). (C) Adult p110β-RBD-DM mice are smaller than their wild-type littermates. Weights of adult homozygous p110β-RBD-DM mice (12–30 weeks old) were compared to same-cage wild-type littermates (n = 39; mean ±SEM; paired t test). (D) Reduced proliferation of p110β-RBD-DM MEFs. Early passage primary MEFs were grown in culture following a modified 3T3 protocol (n = 2 per genotype). Population doubling rate was calculated as PDR = $\log (n\left(day3\right)/n\left(seeded\right))/log2$ (mean ±SEM; p < 0.001, two-way ANOVA). (E) Accumulation of p110β-RBD-DM cells in G1. Cell-cycle profiles of early passage wild-type and homozygous p110β-RBD-DM primary MEFs growing in 1% or 10% FCS (n = 4, means; SEM in the Results section). (F) p110β-RBD-DM MEFs show reduced steady-state phospho-AKT levels. Wild-type and p110β-RBD-DM MEFs were maintained in cell culture medium supplemented with 0%, 1%, or 10% FCS and harvested for western blot analysis. (G) RAC1 and CDC42 cooperatively sustain phospho-AKT levels in wild-type MEFs. Wild-type and p110β-RBD-DM MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting RAC1, CDC42, or both and harvested for western blot analysis 30 hr after transfection. Graph shows phospho-AKT normalized to total AKT (n = 3; mean with SEM, one-way ANOVA). (H) RAC1 and CDC42 activate p110β in vivo. Myc-V12-RAC1 and Myc-V12-CDC42 were nucleofected into immortalized wild-type, p110α-knockout, and p110β-knockout MEFs, along with a kinase-dead AKT reporter construct. The next day, cells were serum starved and harvested for western blot. (I) RAC and CDC42 fail to activate PI3K in p110β-RBD-DM cells. Myc-V12-RAC1, Myc-V12-CDC42, or Myc-V12-HRAS were nucleofected into wild-type and p110β-RBD-DM MEFs as described in (H). See also Figure S4.

Figure 5

RAC Activates p110β Downstream of GPCRs (A) LPA-induced AKT phosphorylation is attenuated in p110β-RBD-DM MEFs. Primary wild-type and homozygous p110β-RBD-DM MEFs were serum starved and stimulated with LPA for 5 min. (B) LPA-induced AKT phosphorylation is more transient in p110β-RBD-DM MEFs. Primary wild-type and p110β-RBD-DM MEFs were serum starved and stimulated with LPA (10 μM) for indicated time periods. (C) LPA-induced AKT phosphorylation is entirely dependent on p110β. Immortalized wild-type, p110α-knockout, p110β-knockout, and p110β-RBD-DM MEFs were serum starved and stimulated with indicated doses of LPA for 5 min. (D) LPA-/S1P-induced AKT phosphorylation requires RAC. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting RAC1, CDC42 or both. Serum-starved cells were stimulated with LPA (10 μM) or S1P (1 μM) for 5 min. (E) LPA-/S1P-induced AKT phosphorylation requires RAC activity. Immortalized wild-type MEFs were serum starved, preincubated with EHT1864 for 30 min, and stimulated with LPA (10 μM) or S1P (1 μM) for 5 min. (F) LPA induces rapid and transient activation of RAC. Serum-starved wild-type and p110β-RBD-DM MEFs were stimulated with LPA (10 μM) for indicated periods of time. RAC⋅GTP and total RAC were measured as described in the Extended Experimental Procedures (n = 3; mean with SEM). See also Figure S5.

Figure S5

RAC Activates p110β Downstream of G Protein-Coupled Receptors, Related to Figure 5 (A) S1P-induced AKT phosphorylation is attenuated in p110β-RBD-DM MEFs. Primary wild-type and homozygous p110β-RBD-DM MEFs were serum-starved and stimulated with sphingosine 1-phosphate (S1P) at indicated doses. Cells were harvested for western blot after 5 min. (B) LPA/S1P-induced phosphorylation of AKT and ERK is sensitive to PTX. Immortalized wild-type MEFs were serum starved overnight and stimulated with EGF, LPA or S1P for 5 min. Pertussis toxin (PTX, 200 ng/ml) was added 16 prior to stimulation where indicated. (C) Normal receptor tyrosine kinase signaling in p110β-RBD-DM MEFs. Primary wild-type and p110β-RBD-DM MEFs were serum-starved and stimulated with EGF, PDGF or insulin at indicated doses. Cells were harvested for western blot after 5 min. (D) Deconvolution of RAC1 and CDC42 siRNA pools. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific individual siRNA oligonucleotides targeting RAC1 or CDC42. 48 hr after transfection, serum-starved cells were stimulated with LPA (10 μM) for 5 min. (E) RAC and CDC42 are not required for AKT phosphorylation induced by receptor tyrosine kinase agonists. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting RAC1 or CDC42. Serum-starved cells were stimulated with EGF (10 ng/ml), PDGF (10 ng/ml) or insulin (5 μg/ml) for 5 min, before they were harvested for western blot. (F) AKT phosphorylation downstream of receptor tyrosine kinases does not require RAC activity. Immortalized wild-type MEFs were serum-starved and incubated with EHT1864 at the indicated doses for 30 min, before they were stimulated with EGF (10 ng/ml), PDGF (10 ng/ml) or insulin (5 μg/ml) for 5 min.

Figure 6

Dock180/Elmo1 Activate RAC Downstream of GPCRs and Upstream of p110β (A) siRNA pools targeting Dbl family RAC-GEFs fail to affect LPA-induced AKT phosphorylation. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting indicated Dbl family RAC-GEFs. A total of 48 hr after transfection, serum-starved cells were stimulated with LPA (10 μM) for 5 min. (B) Dock180 and Elmo1 are essential for LPA-induced AKT phosphorylation. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting indicated Dock family RAC-GEFs. Then 48 hr after transfection, serum-starved cells were stimulated with LPA (10 μM) for 5 min. (C) Dock180 is essential for LPA-induced RAC activation. Immortalized wild-type MEFs, transfected with scrambled duplex or Dock180-specific siRNA pools, were stimulated with LPA (10 μM) or EGF (10 ng/ml) for 20 s and active RAC was quantified (n = 4; mean with SEM; t test; bottom: a representative experiment). (D) LPA-induced RAC activation is PI3K independent. Immortalized MEFs were preincubated with PTX (200 ng/ml, 16 hr) or GDC0941 (10 μM, 1 hr) and stimulated with LPA (10 μM) for the indicated time periods (n = 4; mean with SEM; one-way ANOVA; bottom: representative lysates). (E) Gβγ subunits directly bind to the N terminus of Elmo1. GST-tagged full-length Elmo1 and fragments as shown (schematic) were incubated with lysates from COS7 cells expressing Gβ₂ and Gγ₁. Bound Gβ was detected by western blot. (F) Model of GPCR-induced p110β activation. See text for details. See also Figure S6.

Figure S6

Dock180/Elmo1 Activate RAC Downstream of GPCRs and Upstream of p110β, Related to Figure 6 (A) siRNA pools targeting Dbl family RAC-GEFs fail to affect S1P-induced AKT phosphorylation. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting indicated Dbl family RAC-GEFs. Forty-eight hours after transfection, serum-starved cells were stimulated with S1P (1 μM) for 5 min. (B) Dock180 and Elmo1 are essential for S1P-induced AKT phosphorylation. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting indicated Dock family RAC-GEFs. Forty-eight hours after transfection, serum-starved cells were stimulated with S1P (1 μM) for 5 min. (C) Knockdown of Dock180 or Elmo1 does not affect signaling downstream of receptor tyrosine kinases. Immortalized wild-type MEFs were transfected with scrambled duplex or siRNA pools targeting Dock180 or Elmo1. Cells were serum starved and stimulated with EGF (10 ng/ml), PDGF (10 ng/ml) or insulin (5 μg/ml) for 5 min. (D) Deconvolution of Dock180 siRNA pool. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific individual siRNA oligonucleotides targeting Dock180. Forty-eight hours after transfection, serum-starved cells were stimulated with LPA (10 μM) for 5 min. (E) Deconvolution of Elmo1 siRNA pool. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific single siRNA oligonucleotides targeting Elmo1. Forty-eight hours after transfection, serum-starved cells were stimulated with LPA (10 μM) for 5 min. RNA was extracted in parallel experiments and Elmo1 mRNA levels were compared by qPCR (mean with SEM). (F) Knockdown of Elmo1 but not Elmo2 affects LPA-induced AKT phosphorylation. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting Elmo1 or Elmo2, and serum-starved cells were stimulated with LPA for 5 min. Elmo1/Elmo2 mRNA levels were assessed by qPCR (mean with SEM). (G) Rapid p85 tyrosine phosphorylation upon EGF but not LPA stimulation. Immortalized wild-type MEFs were serum-starved and stimulated with EGF (10 ng/ml) or LPA (10 μM) for 20 and 60 s, before cell lysates were made, and p85 immunoprecipitates were analyzed by western blot as shown.

Figure 7

p110β-RBD-DM Mice Are Protected from Bleomycin-Induced Lung Fibrosis (A) p110β-RBD-DM fibroblasts show reduced migration in gradients of LPA. Migration of wild-type and p110β-RBD-DM MEFs in gradients of LPA and PDGF was assessed in transwell filter assays (n = 3; mean with SD; one-way ANOVA). (B) Dock180, Elmo1, and RAC1 are required for fibroblast migration in gradients of LPA. Immortalized wild-type MEFs were transfected with scrambled duplex or gene-specific siRNA pools targeting Dock180, Elmo1, or RAC1. Migration in gradients of LPA and PDGF was assessed in transwell filter assays and cell numbers were normalized to control conditions (n = 4; mean with SEM; one-way ANOVA). (C) p110β-RBD-DM mice are protected against death from bleomycin-induced lung damage. Wild-type and homozygous p110β-RBD-DM mice were treated with a single intratracheal dose of bleomycin (1.25 U/kg) and observed for 14 days (n = 16 mice per genotype; Mantel-Cox test). (D) p110β-RBD-DM mice are protected against weight loss following bleomycin instillation. Wild-type and p110β-RBD-DM mice received a single intratracheal dose of saline (n = 3 per genotype) or bleomycin (n = 10 per genotype) and weights were taken 14 days later (mean ±SEM; one-way ANOVA). (E) p110β-RBD-DM mice are protected from bleomycin-induced lung fibrosis. Representative lung areas from wild-type and homozygous p110β-RBD-DM mice 14 days after treatment with intratracheal bleomycin (×4 magnification). Top: H&E; middle: IHC for α-SMA; bottom: Sirius red. (F) p110β-RBD-DM mice are protected against loss of transparent lung areas following bleomycin instillation. Lungs were analyzed by H&E 14 days after bleomycin challenge. Multiple nonoverlapping areas of representative sections from each lung were photographed and transparent (white) areas were quantified using Nikon NIS elements software (mean ±SEM; one-way ANOVA; see Figure S7C for raw data). (G) p110β-RBD-DM mice accumulate fewer activated lung fibroblasts following bleomycin instillation. Lungs were analyzed by immunohistochemistry for smooth muscle antigen (α-SMA) 14 days after bleomycin challenge. Multiple nonoverlapping areas of representative sections from each lung were photographed and SMA-positive (brown) areas were quantified using Nikon NIS elements software (mean ±SEM; one-way ANOVA; see Figure S7D for raw data). See also Figure S7.

Figure S7

p110β-RBD-DM Mice Are Protected from Bleomycin-Induced Lung Fibrosis, Related to Figure 7 (A) Fibroblasts migration in gradients of LPA and PDGF requires PI3K activity. Serum-starved immortalized wild-type MEFs were seeded onto fibronectin-coated membrane inserts and placed into 24-well plates containing serum-free medium and the indicated chemoattractants. Where indicated, GDC0941 (5 μM) or PTX (200 ng/ml) were added 30 min and 16 hr prior to seeding, respectively, and were present throughout experiments. After 6 hr, migrated cells were semiautomatically counted and numbers were normalized to control conditions (n = 3; mean with SEM; repeated-measures ANOVA). (B) Constitutively active RAC1 promotes migration of wild-type but not p110β-RBD-DM fibroblasts. Migration of transfected wild-type and p110β-RBD-DM MEFs in starvation medium and gradients of LPA (10 nM) and FCS (1%) were assessed in transwell filter assays (n = 6 from 2 independent experiments; mean with SEM; 1way ANOVA). (C) p110β-RBD-DM mice show less increase in lung weight after bleomycin challenge. Age- and sex-matched wild-type and p110β-RBD-DM mice received saline (n = 3 per genotype) or bleomycin (n = 10 per genotype) via intratracheal instillation and were culled 14 days later (mean ± SEM; t test). (D) Morphometric quantification of transparent lung areas after bleomycin challenge. Wild-type and p110β-RBD-DM mice received a single dose bleomycin via intratracheal instillation. After two weeks, lungs were fixed, sectioned and stained with H&E. As many as possible nonoverlapping areas (A–L), avoiding artifacts from interlobes and major bronchi/vessels, of representative sections from each lung were photographed at low (4×) magnification and transparent (white) areas within each image were quantified using Nikon NIS elements software. (E) Morphometric quantification of α-SMA-positive areas after bleomycin challenge. Wild-type and p110β-RBD-DM mice received a single dose bleomycin via intratracheal instillation. After two weeks, lungs were fixed, sectioned and stained with immunohistochemistry for smooth muscle antigen (α-SMA). As many as possible nonoverlapping areas (A–N), avoiding artifacts from bronchi and blood vessels, of representative sections from each lung were photographed at low (4×) magnification and positive (brown) areas within each image were quantified using Nikon NIS elements software.