PI3Kα inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification
- Departament de Ciències Fisiològiques, Universitat de Barcelona, IDIBELL, C/ Feixa Llarga s/n 08907 Hospitalet de Llobregat, Spain
- Department of Cell and Chemical Biology, Leiden University Medical Center, Netherlands
- Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Health Research Institute of Asturias (ISPA), Spain
Figures
Figure 1 with 1 supplement
Delayed initiation of treatment with PI3Kα inhibitor effectively prevents heterotopic ossification.
(A) Heterotopic ossification was induced at P7 through the injection of Adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. The pattern of five administrations of DMSO or BYL719 (25 mg/kg) is indicated by gray dots started at P8 (start day 1), P10 (delayed start day 3), and P14 (delayed start day 7). One group was administered DMSO or BYL719 (25 mg/kg) only at P8, P9, and P10 (first 3 days post-HO-induction), as indicated by gray dots. The final time point, P30, was conserved between experimental groups. (B) Quantification of heterotopic ossifications bone volume (BV) (mm3) of each experimental group. Colored symbols indicate the presence of heterotopic ossifications. Black symbols indicate the absence of heterotopic ossifications. Individual mouse values with group median are shown. *p < 0.05, **p < 0.01, Kruskal–Wallis test with Dunn’s multiple comparisons test. (C) Representative 3D microtomography images of the injected hindlimbs of three different mice for each experimental group. White arrows point to heterotopic ossification.
Figure 1—figure supplement 1
Mice body weight from P8 (1 day post-HO-induction) to P30.
Data are shown as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test.
Figure 2 with 2 supplements
Deficiency of PI3Kα at injury sites is sufficient to partially prevent heterotopic ossification.
(A) Heterotopic ossification (HO) was induced at P7 through the injection of Adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. Either DMSO (vehicle) or BYL719 (25 mg/kg) was injected following the scheme indicated with gray dots, starting at P8 (B). Quantification of HOs bone volume (BV) (mm3) of each experimental group. Colored symbols indicate the presence of HOs. Black symbols indicate the absence of HOs. Individual mouse values with group median are shown. *p < 0.05, **p < 0.01, Kruskal–Wallis test with Dunn’s multiple comparisons test. (C) Quantification of the ratio of bone volume per tissue volume (bone volume/tissue volume, BV/TV) within HOs of each experimental group, including only mice with detected HO. Data shown are of each individual mouse with the group median. **p < 0.01, ***p < 0.001, Kruskal–Wallis test with Dunn’s multiple comparisons test. (D) Representative 3D frontal microtomography images of the injected hindlimbs of mice for each experimental group and a detailed close-up image for each selected mouse. White arrows show HO in the close-up images.
Figure 2—figure supplement 1
Absence of toxic effects of PIk3ca deletion and BYL719 administration.
(A) Mice body weight from P8 (1 day post-HO-induction) to P30 (final time point). Data are shown as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparisons test. (B) Close-up 3D microtomography images of the injected hindlimbs of mice for each experimental group. White arrows show heterotopic ossification. (C) Two-dimensional representation of bone volume (BV) and bone volume/tissue volume (BV/TV) within heterotopic ossifications. Data shown are of each individual mouse with observed heterotopic ossification. Each symbol corresponds to an individual mouse. Colors indicate groups, as detailed in the legend.
Figure 2—figure supplement 2
Muscle sections of mice 23 days after injury stained with Masson’s Trichrome.
Different genotypes and treatments specified in the figure. All images were acquired at ×4 magnification (scale bar 500 µm).
Figure 3 with 1 supplement
Analysis of Acvr1 gene expression by qPCR in bone marrow-derived mesenchymal stem cells (BM-MSCs) from Pik3cafl/fl mice infected with virus expressing wild-type Acvr1 (WT) or Acvr1R206H (RH).
(A) The endogenous expression level of the Acvr1 gene in mock-transfected BM-MSCs is shown as a dotted horizontal line. Data are shown as mean ± SD (n = 12 per group). Unpaired t-test between transfected groups. (B) Gene expression analysis of Pik3caα in BM-MSCs from Pik3cafl/fl mice, infected with virus expressing wild-type Acvr1 (WT) or Acvr1R206H (RH) and/or Cre co-infection. Data are shown as mean ± SD (n = 3 per group). ***p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (C) mRNA expression of canonical (Id1 and Sp7), non-canonical (Ptgs2) target genes, and activin A (Inhba) in BM-MSCs Pik3cafl/fl transfected with Acvr1 (wild type or R206H) with or without Cre recombinase. Cells were NT (not treated) or treated with BYL719 (2 µM) and/or activin A (2 nM). Expression data were normalized to those of control cells which were transfected only with Acvr1 WT without any treatment, shown as a dotted horizontal line. Asterisks (*) refer to the differences between different conditions of Acvr1 RH cells compared to control cells. Hash signs (#) refer to the differences between different conditions of Acvr1 RH cells compared to Acvr1 RH cells without Cre recombinase and treated with activin A. Data are shown as mean ± SD (n = 6 per group). * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.
Figure 3—figure supplement 1
BYL719 reduces the number of FAPs and improves muscle regeneration during the ossification process.
(A) Muscle sections of mice 4, 9, 16, and 23 days after induction of heterotopic ossification (HO). PDGFRA+ cells are indicated in red, wheat germ agglutinin staining is indicated in green, and DAPI staining is indicated in blue. Different times and BYL719 treatment are specified in the figure. (B) Quantification of PDGFRA-positive fibroadipogenic precursors (FAPs) obtained from five images obtained per mice, from 4 mice per group, for a total of 20 quantified images per group. (C) Quantification of myotube diameter estimated from wheat germ agglutinin staining obtained from five images per mice, from 4 mice per group, for a total of 20 quantified images per group. One-way ANOVA with Tukey’s multiple comparisons test. ***p < 0.001.
Figure 4 with 1 supplement
BYL719 does not reduce ACVR1 kinase activity.
(A) A schematic depiction of nano-bioluminescence resonance energy transfer (nanoBRET) target engagement assays. TGF-β receptors–Nanoluciferase fusion proteins are expressed in COS-1 cells and an ATP-like tracer analog in close proximity with the Nanoluciferase donor allows energy transfer from the Nanoluciferase donor to the fluorescent tracer acceptor. An ATP analog molecule (type I inhibitor) will compete with the fluorescent tracer, impairing close proximity donor–acceptor and reducing the nanoBRET ratio. (B) The nanoBRET emission spectra consist of the Nanoluciferase donor (460 nm) and the fluorescent acceptor (610 nm). The nanoBRET ratio is shown as milliBRET units (mBU) by dividing the acceptor emission by the donor emission times 1000. (C) NanoBRET target engagement analyses of ACVRL1, ACVR1, ACVR1R206H, BMPR1A, ACVR1B, TGFBR1, TGFBR2, ACVR2A, ACVR2B, and BMPR2 testing 1 or 10 µM BYL719 with n = 4. As controls, LDN193189 (0,5 µM), SB431542 (10 µM), and ML347 (10 µM) were used. Data are shown as mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. (D) Casein phosphorylation by ACVR1R206H kinase. Phosphorylation was performed in the presence of ACVR1R206H kinase and increasing concentrations of the PI3Kα inhibitor BYL719. Quantification of kinase activity. Data are shown as mean ± SD (n = 4 independent experiments). One-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 4—source data 1
Original files for autoradiographies displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/91779/elife-91779-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing original autoradiographies for Figure 4D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/91779/elife-91779-fig4-data2-v1.zip
Figure 4—figure supplement 1
Quantification of the TGF-β receptor–Nanoluciferase protein expression levels measured by the raw donor emission (excitation at 450–480 nm).
Figure 5 with 1 supplement
Analyses of chondrogenic progenitor specification of parental human MSCs (hMSCs), hMSC-ACVR1WT or hMSC-ACVR1R206H treated with TGF-β1 (10 ng/ml) and activin A (100 ng/ml), with and without BYL719 (10 µM) as indicated (A).
Representative images of Alcian Blue staining of micromass cultures after 3 weeks of chondrogenic differentiation. Scale bar represents 1500 µm. (B) Densitometry analysis of the Alcian Blue staining in (A) (n = 3 per group). (C) RT-qPCR results of SOX9, COL2A1, ACAN, and MMP13 after chondrogenic progenitor specification (n = 4 per condition). (D) Representative images of the alkaline phosphatase (ALP) staining after 1 week of chondrogenic progenitor specification. Scale bar represents 1000 µm. (E) Quantification of the ALP activity (n = 3 per group). (F) RT-qPCR results of Acan, Col2a1, Alpl, and Sox9 after chondrogenic progenitor specification from Acvr1WT or ACVR1R206H MSCs treated with activin A (100 ng/ml), with or without BYL719 (10 µM) in TGFβ1-containing differentiation media (DM) as indicated (n = 3 per condition). Data are shown as mean ± SD. In all figure panels, significance is detailed between the indicated conditions. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.
Figure 5—figure supplement 1
Human MSC-ACVR1R206H are responsive upon activin A (50 ng/ml) stimulation compared to hMSC-ACVR1WT after overnight starvation.
(A) Western blot analysis detecting C-terminal phosphorylated SMAD1/5, total SMAD1, and vinculin. (B) RT-qPCR analysis of the BMP target genes ID1 and ID3 (n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s multiple comparisons test.
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Figure 5—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/91779/elife-91779-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
PDF file containing original western blots for Figure 5—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/91779/elife-91779-fig5-figsupp1-data2-v1.zip
Figure 6 with 1 supplement
BYL 719 inhibits ossification and osteoblast differentiation processes in human ACVR1-R206H MSCs.
(A) Schematic depiction of the experimental setup, involving bulk RNA sequencing of human MSC-ACVR1WT and ACVR1R206H upon overnight starvation, 30-min pre-treatment with or without 1 µM BYL719 and with or without 1 hr activin A (50 ng/ml) or BMP6 (50 ng/ml) stimulation. (B) The top 10 most significant gene ontology (GO) terms using all (up- and downregulated) differentially expressed genes between cells expressing ACVR1-WT and ACVR1-R206H under control conditions. Ossification (GO:0001503) and osteoblast differentiation (GO:0001649) were detected within the top 10 of the differentially regulated biological processes. (C) Table of GO terms ossification and osteoblast differentiation upon GO enrichment analysis in all tested conditions ACVR1WT or ACVR1R206H cells, stimulated with BMP6 or activin A and treated with BYL719. Statistical significance upon GO enrichment analysis, count of total differentially expressed genes (DEGs), and their classification as up- or downregulated DEGs are detailed for each comparison. (D) Gene set enrichment analysis (GSEA) with the groups RH_ActA_BYL vs RH_ActA, showing the enrichment plots of the gene ontology sets ossification (GO: 0001503) and osteoblast differentiation (GO:0001649). (E) A heatmap of the top 40 most relevant genes within the ossification GO geneset derived from the leading-edge subset of the enrichment plot (n = 4 per group). Sample names are detailed as receptor (R206H) ligand (AA, Act A, Activin A)_inhibitor (BYL719, if present)_replicate#.
Figure 6—figure supplement 1
Gene set enrichment analysis (GSEA) of bulk RNA sequencing comparing hMSC-ACVR1R206H cells treated with BYL719 (1 µM) with untreated controls, both stimulated with activin A (50 ng/ml) (n = 4 per group).
(A) Significant downregulated enrichment plots of the KEGG gene set TNF signaling pathway (HSA04668), NF-κB signaling (HSA04064) and GO gene set response to interleukin-6 (GO:0070741). (B) The top 40 most relevant genes from the enrichment plot of the TNF signaling pathway as derived from the leading-edge subset.
Figure 7 with 2 supplements
BYL719 reduces the number of monocytes and macrophages at injury sites.
(A) Heterotopic ossification was induced at P7 through the injection of Adenovirus-Cre (Ad.Cre) and cardiotoxin in the mice hindlimb. Either DMSO (vehicle) or BYL719 (25 mg/kg) were injected following the scheme indicated with gray dots, starting at P8 but with a different duration P9 (2 days post-HO-induction), P11 (4 days post-HO-induction), P16 (9 days post-HO-induction), P23 (16 days post-HO-induction), and P30 (23 days post-HO-induction). (B) The quantification of F4/80-positive monocytes/macrophages per field view from immunohistochemistry staining, with ×20 amplification as shown in the representative images depicted in Figure 8C. Five images were randomly acquired per mice, from 2 mice per group, for a total of 10 quantified images per group. F4/80-positive cells were detected as cells with dark brown staining. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Sidak’s multiple comparisons test, comparing both groups on each day (n = 10). (C) Representative images from the quantified immunohistochemistry staining for F4/80 to detect monocytes/macrophages. Representative images are shown for each final time point at day 2, 4, 9, 16, and 23 post-HO-induction. All images were acquired at ×20 (scale bar 100 µm). (D) Quantification of CEM-positive mast cells per field view from CEM staining, with ×20 amplification as shown in the representative images depicted in Figure 8E. Three images were obtained per mice, from 4 mice per group, for a total of 12 quantified images per group. Mast cells were detected as cells with bright blue staining. Data are shown as mean ± SD. ***p < 0.001, two-way ANOVA with Sidak’s multiple comparisons test, comparing both groups in each day (n = 12). (E) C.E.M. staining was performed to detect mast cells, highlighted with black arrows. Representative images are shown for each final time point at day 2, 4, 9, 16, and 23 post-HO-induction. All the images were obtained at ×20 magnification, with a representative scale bar at 100 µm.
Figure 7—figure supplement 1
Time course of the BYL 719 inhibition of heterotopic ossification.
(A) Quantification of heterotopic ossifications bone volume (mm3) of each experimental group. group for each final time point at day 2, 4, 9, 16, and 23 post-HO-induction. Colored symbols indicate the presence of heterotopic ossifications. Black symbols indicate the absence of heterotopic ossifications. Circles indicate DMSO-treated mice, diamonds indicate BYL719-treated mice, as detailed in the legend. Data shown are of each individual mouse with group median. **p < 0.01, two-way ANOVA with Sidak’s multiple comparisons test. (B) Representative 3D microtomography images of the injected hindlimbs of mice from each experimental group. White arrows show heterotopic ossification.
Figure 7—figure supplement 2
Time course of the inhibition of chondrogenesis and osteogenesis by BYL719.
(A) Representative images of injected hindlimbs stained with Fast Green/Safranin O (FGSO) staining. Representative images are shown for each final time point at day 2, 4, 9, 16, and 23 post-HO-induction. All images were obtained at ×4 (scale bar at 500 µm). (B) Representative images of injected hindlimbs stained with Masson’s Trichrome. Representative images are shown for each final time point at day 2, 4, 9, 16, and 23 post-HO-induction. All images were acquired at ×4 magnification (scale bar at 500 µm).
Figure 8 with 1 supplement
Proliferation assays of THP1 (A), RAW264.7 (B), and HMC-1 (C) cells.
Cells were cultured for 6 days and in control conditions, with BMP6 (2 nM) and/or BYL719 (2 or 10 µM). Areas under the proliferation curves were compared by one-way ANOVA with Dunnett’s multiple comparisons test against the control. (*) refers to significant differences between control and single treatments (BMP6 or BYL719). (#) refers to significant differences between control and combined treatments. Data are shown as mean ± SD (n = 4 per group). ** or p < 0.01, *** or ### p < 0.001. (D) Migration assay of THP1 cells with control conditions, or with FBS or BMP6 as chemotactic agents with or without BYL719 treatment. Data are shown as mean ± SD (n = 4 per group). ***p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. Gene expression assays of THP1 (E), RAW264.7 (F), and HMC-1 (G) cells. Cells were treated for 48 hr with BYL719 (2 µM) and/or BMP6 (2 nM). Data are shown as mean ± SD (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test, significance shown between control group and other groups.
Figure 8—figure supplement 1
Proliferation assays of murine MSCs (A) and C2C12 cells (B).
Cells were cultured for 3 days in control conditions or with BYL719 (2 or 10 µM). Areas under the proliferation curves were compared by one-way ANOVA with Dunnett’s multiple comparisons test against the control. (C) RT-qPCR analysis of the myogenic (MyoD1 and Myh1) or myofibroblast (Acta2) markers after culturing C2C12 cells in myogenic differentiation media for 7 days in control conditions, or with BYL719 (2 or 10 µM) (n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test. (D) Representative images of myotube formation of C2C12 cells in myogenic differentiation media for 7 days in control conditions, or with BYL719 (2 or 10 µM). Scale bar represents 200 μm.
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PI3Kα inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification
eLife 12:RP91779.
https://doi.org/10.7554/eLife.91779.4
