Abstract
Skeletal growth relies on both biosynthetic and catabolic processes1,2. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis3. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates4. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34–beclin-1. Autophagy is completely suppressed in growth plates from Fgf18−/− embryos, while Fgf18+/− heterozygous and Fgfr4−/− mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18+/− and Fgfr4−/− phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.
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Acknowledgements
We thank A. Ballabio, G. Karsenty, A. Auricchio, M. Sandri and G. Diez Roux for critical reading of the manuscript. We thank L. Staiano and C. Lanzara for technical support. We thank A. Carissimo for statistical analysis. We thank N. Mizushima, D. Ornitz and J. Seavitt for sharing mouse lines used in this work. This work was supported by grants from the Italian Telethon Foundation (TCP12008), Marie Curie European Reintegration Grant (PCIG13-GA-2013-618805), STAR/Banco di Napoli and the Italian Ministry of Research (FIRB RBFR13LH4X) to C.S.
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Contributions
L.C. performed the majority of the experiments involving mice. A.F. performed the majority of the in vitro experiments. R.B. and M.S. performed immunoprecipitation experiments. R.V. provided essential reagents and helped with the collagen experiments. S.M. performed the high-content screening experiments. E.P. performed TEM analysis. E.N. performed Tat–beclin-1 injections and tissue harvesting. A.R. provided critical suggestions and protocols. R.P. supervised the TEM. D.L.M. supervised the high-content screening experiments. M.A.D.M. provided critical suggestions, protocols and reagents. C.S. conceived, designed and supervised the study and wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Autophagy flux increases during early post-natal bone development.
a, Representative images of comparative TEM of P0 and P6 wild-type growth-plate chondrocytes showing increased AV number at P6. Arrow indicates AVs. Bar graphs show number and size of AVs within 5.3 μm field of view. Values represent mean ± s.e.m. Student’s t-test, **P < 0.005. N = 16 (P0); 18 (P6). b, Western blot analysis of LC3I/II of femoral growth plates from mice at the indicated ages. Mice were injected with Leupeptin where indicated (Leup; 40 mg kg−1; 6 h before being killed). β-Actin was used as a loading control. Bar graphs show quantification of LC3II protein relative to β-actin. Mean ± s.e.m. Student’s t-test, *P < 0.05. n = 3 per group. c, Western blot analysis of ATG7, LC3 and SQSTM1 proteins in Atg7fl/fl, Col2a1-Cre; Atg7fl/fl and Prx1-Cre; Atg7fl/fl growth-plate lysates. Histone 3 (H3) was used as loading control. Results are representative of 3 independent experiments. d–f, Western blot analysis of ATG7 and LC3 proteins isolated from different tissues isolated from mice with indicated genotypes. GAPDH and β-actin were used as loading control. Bar graph shows quantification (±s.e.m.) of ATG7 and LC3II proteins relative to β-actin in different tissues. n = 2 (d), 3 (e), 4 (f) mice per genotype.
Extended Data Figure 2 Analysis of bone growth in mice lacking chondrocyte autophagy.
a–d, Alcian blue/alizarin red skeletal staining of Atg7fl/fl, Col2a1-Cre; Atg7fl/fl and Prx1-Cre; Atg7fl/fl mice at P0 (a), P9 (b), P30 (c) and P120 (d). Details of femur and tibia magnifications. In c and d only male mice were analysed. Graphs show femur and tibia lengths from mice with indicated genotypes. Values represent mean ± s.e.m. Student’s t-test, *P < 0.05, **P < 0.005, ***P < 0.0005. n = 3 (P0), 3 (P9), 4 (P30), 4 (P120) mice per genotype. Scale bar, 2 mm.
Extended Data Figure 3 Autophagy does not regulate chondrocyte proliferation, differentiation and apoptosis.
a, b, Haematoxylin and eosin (H&E) staining of femoral sections of P6 (a) and P9 (b) Atg7fl/fl and Prx1-Cre; Atg7fl/fl mice showing a reduced femoral length starting from P9 in Prx1-Cre; Atg7fl/fl mice compared with controls (black arrowheads). White arrows show normal differentiation of the secondary ossification centre in Prx1-Cre; Atg7fl/fl compared with control. The data are representative of three independent experiments. Scale bar, 2 mm. c–e, Representative images of H&E staining of hypertrophic chondrocytes (c), bromodeoxyuridine (BrdU) staining (d) and TUNEL assay (e) in P6 Atg7fl/fl and Prx1-Cre; Atg7fl/fl growth plates. Arrows indicate TUNEL-positive cells. Graph shows quantification of BrdU index in femoral and tibial growth plates from Atg7fl/fl and Prx1-Cre; Atg7fl/fl mice. Values represent ± s.e.m. n = 5 mice per genotype. Scale bar, 100 μm. c, e, Data are representative of 3 independent experiments.
Extended Data Figure 4 Lack of autophagy leads to PC2 accumulation in the ER of growth-plate chondrocytes.
a, Total levels of glycosaminoglycans (GAGs) in femoral and tibia growth plates of P5 and P9 mice with indicated genotypes. Values were normalized to total DNA and expressed as percentage relative to control (Atg7fl/fl) mice. Values represent mean ± s.e.m. n = 3 (P5); n = 5 (P9) mice per genotype. b, Extracellular Col2 staining in chondroitinase ABC-treated growth-plate femoral sections isolated from Atg7fl/fl and Prx1-Cre; Atg7fl/fl mice. The data are representative of two independent experiments. Scale bar, 500 μm. c, Representative images of confocal analysis of PC2, calreticulin (CRT) Sec31, VapA, P115, GM130, giantin and LAMP1 proteins in Prx1-Cre; Atg7fl/fl growth-plate chondrocytes at P6. Insets show high magnification of boxed areas. Scale bar, 10 μm. Quantification (±s.e.m.) of PC2 co-localization with indicated organelle markers (Mander’s coefficient, ImageJ plug-in). At least 2 sections per mouse containing 400 cells were analysed. n = 3 mice per group. d, ER distention in Atg7fl/fl; Prx1-Cre growth-plate chondrocytes compared with control chondrocytes (Atg7fl/fl). Arrows indicate ER. Insets show a high magnification of selected areas. The data are representative of two independent experiments. Scale bar, 500 nm.
Extended Data Figure 5 Autophagy regulates PC2 secretion.
a, Western blot analysis of ATG7 and LC3II levels in control (scrambled) and Atg7 siRNA-treated RCS chondrocytes. GAPDH was used as a loading control. KD, knockdown; ns, non-silencing. b, Western blot analysis of LC3II levels in RCS chondrocytes treated with Spautin-1 at the indicated concentrations for 24 h. β-Actin was used as a loading control. c, Co-localization of GFP–LC3 with PC2 in primary chondrocytes isolated from GFP–LC3 transgenic mice and treated with BafA1 (4 h, 200 nM). Values are expressed as percentage ± s.d. relative to total GFP area. N = 500 cells; n = 2 independent preparations. d, e, Confocal analysis of ATG12 (d), ATG16L (e) (green) and mCherry–PC2 (red) in RCS chondrocytes. The data are representative of 3 independent experiments. Insets show a high magnification of selected areas. Blue, DAPI. Scale bar, 10 μm. f, Immunofluorescence staining of PC2 (blue), HSP47 (red) and GFP–LC3 (green) in RCS chondrocytes, showing that HSP47 does not co-localize with PC2 in AVs. The data are representative of 2 independent experiments. Insets show higher magnifications and single colour channels of the boxed area. Scale bar, 5 μm.
Extended Data Figure 6 Role of autophagy during PC2 secretion.
a, Immunofluorescence staining of HSP47 chaperone (red) in Atg7fl/fl and Atg7fl/fl; Prx1-Cre chondrocytes, showing altered HSP47 distribution in Prx1-Cre; Atg7fl/fl chondrocytes. The data are representative of 3 independent experiments. Insets show a higher magnification of the boxed area. Scale bar, 10 μm. Blue, DAPI. b, Co-localization of PC2 with HSP47 in growth-plate chondrocytes of mice with indicated genotypes. The data are representative of 3 independent experiments. Scale bar, 20 μm. c, Altered HSP47 and PC2 trafficking in Spautin-1-treated chondrocytes. HSP47 and PC2 immunostaining in control (vehicle) or Spautin-1-treated RCS chondrocytes. Synchronized PC2 secretion was obtained after incubating chondrocytes for 3 h at 40 °C to block PC2 in the ER, and then shifting the temperature to 32 °C (ER block release) for 10 min. The data are representative of 2 independent experiments. Scale bar, 10 μm. d, Proposed model of autophagy function in chondrocytes. Autophagy in chondrocytes prevents PC2 aggregation and maintains ER homeostasis during the process of PC2 secretion. e, Confocal analysis of GFP–LAMP1 (green) and mCherry–PC2 (red) in vehicle- and Spautin-1-treated chondrocytes at the indicated time points (min) after the ER block release. The insets show a high magnification of the selected area. Scale bar, 5 μm. f, Quantification of GFP–LAMP1/mCherry–PC2 co-localization. Values represent mean ± s.d. from three independent experiments. N = 30. ANOVA, P = 4.91 × 10−5; Tukey’s post-hoc test, ***P < 0.0005. g, h, Confocal analysis of RCS chondrocytes treated with tannic acid (0.5% final concentration in the medium) for 1 h, showing that PC2 vesicles (red) at the periphery do not co-localize with LC3 (g) or with LAMP1 (h) (green). The data are representative of 2 independent experiments. Scale bar, 10 μm.
Extended Data Figure 7 FGF18 induces autophagy in chondrocytes.
a, Representative images of high-content imaging analysis of primary chondrocytes isolated from GFP–LC3 transgenic mice and treated with vehicle or FGF18 (25 ng ml−1 for 24 h). BafA1 was used where indicated (4 h, 200 nM). Scale bar, 50 μm. b, Quantification of green vesicles (AVs) in cells treated with the indicated factors for 24 h. Vesicles were counted in at least 1,000 cells per treatment. Values represent mean values ± s.d. of n = 3 independent experiments. Statistical analysis was performed using repeated-measures ANOVA, P = 0.0001; Tukey’s post-hoc test, **P < 0.005. c, Western blot analysis of primary chondrocytes isolated from wild-type mice treated as indicated (FGF18, 25 ng ml−1, 24 h). Where indicated, BafA1 was added (200 nM, 4 h). Bar graphs represent mean values ± s.e.m. of n = 3 independent experiments. Student’s t-test, ***P < 0.0005. d, Representative images of immunofluorescence staining of RCS chondrocytes expressing the tandem fluorescent-tagged LC3 (mRFP–eGFP–LC3) protein. Graphs show increased number of autolysosomes (AL) and of total vesicles (AV+AL) in FGF18- (25 ng ml−1 for 24 h) compared with vehicle-treated RCS chondrocytes. Bar graph represent mean values ± s.d. N = 10 cells per experiment were analysed from 3 independent experiments. Student’s t-test, *P < 0.05, ***P < 0.0005. BafA1 (4 h at 200 nM) was used as a control to inhibit AV–lysosome fusion. Scale bar, 10 μm. e, Representative images of confocal analysis of GFP–LC3 puncta (autophagosomes) in femoral growth plates from P0 and P6 GFP-LC3tg/+; Fgf18+/+ and GFP-LC3tg/+; Fgf18+/− mice. Scale bar, 20 μm. Bar graph shows quantification of the data in P6 mice. Values are mean ± s.e.m. n = 5 mice per group. Two sections per mouse containing at least 400 nuclei were analysed. Student’s t-test, *P < 0.05. f, Western blot analysis of Fgf18+/+ and Fgf18+/− growth plate lysates. Mice were injected with leupeptin where indicated (Leup; 40 mg kg−1, 6 h before being killed). β-Actin was used as loading control. Bar graph shows quantification of LC3II protein in vehicle and leupeptin-injected mice. Values represent the mean values (±s.e.m.) relative to β-actin. n = 5 mice per genotype. ANOVA, P = 7.17 × 10−6; Tukey’s post-hoc test, *P < 0.05, ***P < 0.0005. g, Western blot analysis of SQSTM1 protein in three Fgf18+/+ and three Fgf18+/− growth plate lysates at P30. β-Actin was used as a loading control. Bar graph shows quantification of SQSTM1 protein relative to β-actin. Values represent mean ± s.e.m. n = 3 mice per group. Student’s t-test, *P < 0.05.
Extended Data Figure 8 FGF18 regulates autophagy via FGFR4 and JNK1/2 signalling.
a, Representative images of immunofluorescence analysis of LC3 positive vesicles in RCS chondrocytes treated with siRNA for Fgfr1, Fgfr2, Fgfr3 and Fgfr4 and then stimulated with FGF18 for 2 h. BafA1 was added (200 nM, 3 h). Values represent mean values ± s.e.m. of n = 3 independent experiments (N = 40 cells per treatment were analysed). Student’s t-test, ***P < 0.0005. NS, not significant. Scale bar, 10 μm. b, Immunoprecipitation of FGFR3 or of FGFR4 from RCS chondrocytes stably expressing FGFR3 or FGFR4, respectively, followed by western blotting with phosphotyrosine antibody (pY). Cells were untreated (−) or treated (+) with FGF18 (100 ng ml−1, 20 min). c, Confocal analysis of FGFR3 and FGFR4 in growth-plate chondrocytes isolated from P6 mice. No signal was detected when sections were incubated with secondary antibody alone (Neg. CTR). The data are representative of two independent experiments. Scale bar, 20 μm. d, Western blot analysis of LC3I/II, phospo-JNK1/2, JNK1/2, phospo-ERK1/2, ERK1/2, phospo-P38 MAPK and P38 MAPK in growth plates isolated from three Fgf18+/+ and three Fgf18+/− mice at P6. β-Actin was used as a loading control. The bar graph shows quantification of LC3II relative to β-actin and of phosphorylated proteins relative to the corresponding total proteins. Values are mean ± s.e.m. from n = 3 mice per genotype. Student’s t-test, *P < 0.05, ***P < 0.0005. e, Western blot analysis of three Fgf18+/+ and three Fgf18+/− growth-plate lysates showing no differences in the phosphorylation levels of the proteins analysed. Bar graph shows quantification of the ratio of phosphorylated to total protein (values represent mean ± s.e.m.; n = 3).
Extended Data Figure 9 FGF18 regulates autophagy through beclin-1 complex activation.
a, Western blot analysis of BCL2 phosphorylation at Ser 70, p-BCL2 (S70) and of human influenza hemagglutinin (HA) in RCS chondrocytes expressing human BCL2–HA. Where indicated, chondrocytes were treated with FGF18 (2h, 25 ng ml−1) and with JNK inhibitor (JNK inh; 4 h, 50 μM). b, Immunoprecipitation assays testing physical interactions between endogenous beclin-1, BCL2 and VPS34 in untreated and FGF18-treated (2 h, 25 ng ml−1) RCS chondrocytes. Cell lysates were immunoprecipitated with a beclin-1-specific antibody or control immunoglobulin G (IgG), followed by probing with antibodies specific for beclin-1, BCL2 or VPS34. c, Representative images of membrane-associated PtdIns(3)K assay in situ. RCS chondrocytes were transfected with GFP–2•FYVE and then treated with or without FGF18 (2 h, 25 ng ml−1) and with JNK inhibitors (4 h, 50 μM) where indicated. Graph shows quantitative analysis of cells with GFP–2•FYVE dots. Values are expressed as mean (±s.e.m.) of n = 3 independent experiments. N = 65 (vehicle), 237 (FGF18), 203 (FGF18 + JNK inhibitor) cells. ANOVA, P = 1.12 × 10−6; Tukey’s post-hoc test, ***P < 0.0005. Scale bar, 10 μm. d, Measure of PtdIns(3)K activity associated with beclin-1, expressed as fold change relative to control cells (vehicle treated). Graph shows mean ± s.e.m. Student’s t-test, *P < 0.05. e, PC2 (red) and GFP–LC3 (green) confocal analysis of resting chondrocytes in P6 GFP-LC3tg/+; Fgf18+/− mice showing autophagosomes containing PC2 (arrows). The data are representative of 2 independent experiments. The inset shows a high magnification and single channels of the boxed areas. Scale bar, 10 μm. f, Coomassie blue staining of femoral growth-plate collagen isolated from Fgf18+/+, Fgf18+/− and Fgf18+/− mice injected with Tat–beclin-1. M, marker. g, Total collagen concentration in femoral and tibia growth plates of Fgfr4+/+, Fgfr4−/− and Fgfr4−/− mice treated with Tat–beclin-1 at P9. Values (mean ± s.e.m.) were normalized to DNA and expressed as percentage relative to control mice. n = 5 mice per group. ANOVA, P = 0.0004; Tukey’s post-hoc test, **P < 0.005, ***P < 0.0005. h, i, Femoral lengths of Fgfr4+/+, Fgfr4−/− and Fgfr4−/− mice treated with Tat–beclin-1 at P9 (h) and P15 (i). Values (mean ± s.e.m.) were expressed as percentage relative to littermate control mice. n = 5 mice per group (h) and n = 4 mice per group (i). ANOVA, P = 8.8 × 10−10 (h), P = 5.83 × 10−5 (i); Tukey’s post-hoc test, ***P < 0.0005.
Extended Data Figure 10 Proposed model of FGF18-dependent regulation of autophagy in chondrocytes during post-natal bone growth.
During early post-natal bone growth, FGF18 induces the activation of FGFR4 and of JNK kinase, which phosphorylates BCL2 and activates the VPS34-beclin-1 autophagy complex. This process induces autophagy, which maintains PC2 homeostasis by preventing accumulation of PC2 in the ER. Chondrocyte autophagy appears to be dispensable when low levels of PC2 secretion are needed (for example, during prenatal bone growth).
Supplementary information
Supplementary Figure 1
This file contains the uncroped gels with size marker indications for Figures 1c, 1e, 3a, 3b, 3d, 3e, 3f and Extended Data Figures 1b, 1d, 1e, 1f, 5a, 5b, 7c, 7f, 7g, 8b, 8d, 8e, 9a, 9b. (PDF 2088 kb)
PC2 is an autophagy substrate
GFP–LC3 and mCherry–PC2 co-transfected Rx chondrocytes were synchronized at 40 °C then ER block released at 32 °C. Frames were taken roughly every 20s for 45 min after ER block release. Arrow indicates the mCherry–PC2 molecules that are progressively engulfed by forming GFP–LC3 vesicles. At least 5 separate cells were recorded in 3 independent experiments. (AVI 24689 kb)
Neither autophagosomes nor lysosomes directly mediate PC2 exocytosis
GFP-LC3 and mCherry–PC2 co-transfected Rx chondrocytes were synchronized at 40 °C then ER block released at 32 °C. TIRF imaging was performed with a critical angle of 65° and an evanescent field of 137 nm, for 15 min after 32 °C release, for a 15 min duration. Squares enclose points where mCherry–PC2 puncta colocalize with GFP but no secretion event occurs. Arrows represent secretion events of mCherry–PC2. No fusion events of co-labeled GFP/mCherry occur in any observed cell. At least 12 separate cells were recorded in 3 independent experiments for each GFP construct. (AVI 23492 kb)
Neither autophagosomes nor lysosomes directly mediate PC2 exocytosis
GFP–LAMP1 and mCherry–PC2 co-transfected Rx chondrocytes were synchronized at 40 °C then ER block released at 32 °C. TIRF imaging was performed with a critical angle of 65° and an evanescent field of 137 nm, for 15 min after 32 °C release, for a 15 min duration. Squares enclose points where mCherry–PC2 puncta colocalize with GFP but no secretion event occurs. Arrows represent secretion events of mCherry–PC2. No fusion events of co-labeled GFP/mCherry occur in any observed cell. At least 12 separate cells were recorded in 3 independent experiments for each GFP construct. (AVI 29766 kb)
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Cinque, L., Forrester, A., Bartolomeo, R. et al. FGF signalling regulates bone growth through autophagy. Nature 528, 272–275 (2015). https://doi.org/10.1038/nature16063
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DOI: https://doi.org/10.1038/nature16063
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