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. 2024 Aug;23(8):e14181.
doi: 10.1111/acel.14181. Epub 2024 Apr 30.

Exerkine FNDC5/irisin-enriched exosomes promote proliferation and inhibit ferroptosis of osteoblasts through interaction with Caveolin-1

Lin Tao  1 , Jinpeng Wang  1 , Ke Wang  1 , Qichang Liu  1 , Hongyang Li  1 , Site Xu  1 , Chunjian Gu  1 , Yue Zhu  1
Affiliations

Exerkine FNDC5/irisin-enriched exosomes promote proliferation and inhibit ferroptosis of osteoblasts through interaction with Caveolin-1

Lin Tao et al. Aging Cell. 2024 Aug.

Abstract

Postmenopausal osteoporosis is a prevalent metabolic bone disorder characterized by a decrease in bone mineral density and deterioration of bone microstructure. Despite the high prevalence of this disease, no effective treatment for osteoporosis has been developed. Exercise has long been considered a potent anabolic factor that promotes bone mass via upregulation of myokines secreted by skeletal muscle, exerting long-term osteoprotective effects and few side effects. Irisin was recently identified as a novel myokine that is significantly upregulated by exercise and could increase bone mass. However, the mechanisms underlying exercise-induced muscle-bone crosstalk remain unclear. Here, we identified that polyunsaturated fatty acids (arachidonic acid and docosahexaenoic acid) are increased in skeletal muscles following a 10-week treadmill exercise programme, which then promotes the expression and release of FNDC5/irisin. In osteoblasts, irisin binds directly to Cav1, which recruits and interacts with AMP-activated protein kinase α (AMPKα) to activate the AMPK pathway. Nrf2 is the downstream target of the AMPK pathway and increases the transcription of HMOX1 and Fpn. HMOX1 is involved in regulating the cell cycle and promotes the proliferation of osteoblasts. Moreover, upregulation of Fpn in osteoblasts enhanced iron removal, thereby suppressing ferroptosis in osteoblasts. Additionally, we confirmed that myotube-derived exosomes are involved in the transportation of irisin and enter osteoblasts through caveolae-mediated endocytosis. In conclusion, our findings highlight the crucial role of irisin, present in myotube-derived exosomes, as a crucial regulator of exercise-induced protective effects on bone, which provides novel insights into the mechanisms underlying exercise-dependent treatment of osteoporosis.

Keywords: Caveolin 1; exercise; exosome; ferroptosis; irisin; osteoporosis.

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Conflict of interest statement

The authors are not aware of any affiliations, memberships, funding or financial holdings that might be perceived as affecting the objectivity of this study.

Figures

FIGURE 1
FIGURE 1
Serum levels of irisin were reduced in postmenopausal patients and could be rescued by exercise. (a) Serum levels of irisin in participants in the control (Ctrl) group, osteoporosis (OP) group, and exercise (Exe) group. (b) Representative images of FNDC5 IHC in skeletal muscle sections. (c) Micro‐CT of the distal femur of mice in the three groups. (d) Three‐dimensional reconstructed micro‐CT images of the femur. Upper panels: trabecular bone; lower panels: cortical bone. (e–i) Femoral trabecular bone mass was assessed by micro‐CT, including the trabecular bone mineral density (Tb.BMD) (e), the trabecular thickness (Tb.Th) (f), the ratio of the bone volume to the total volume (BV/TV) (g), the trabecular separation (Tb.Sp) (h) and the number of trabeculae (Tb.N) (I) (n = 5 per group). (j, k) Biomechanical analysis of the femur using a 3‐point bending test, including the yield point (j) and ultimate force (k) (n = 5 per group). (l) Representative Goldner's trichrome–stained sections of femurs in three groups. Left panels: complete bone (scale bar, 3000 μm), middle panels: distal femur (scale bar, 1000 μm), and right panels: femoral shaft (scale bar, 300 μm). OVX, bilaterally ovariectomized; ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3).
FIGURE 2
FIGURE 2
Ten‐week treadmill exercise ameliorates ovariectomy‐induced metabolomic alterations in skeletal muscle. (a,b) Volcano plots of differentially abundant metabolites in skeletal muscles between the Ovx group and Sham group (a) and the treadmill group and Ovx group (b). (c) Clustering heatmap for differentially abundant metabolites among the three groups. The red box with arrows represents polyunsaturated fatty acids (AA, DHA and PA). (d–f) Quantitative values of AA (d), DHA (e), and PA (f) are displayed in the boxplots. (g) Representative images of PGC1α IHC in skeletal muscle sections. Scale bar, 300 μm. (h) Quantification of IHC in (g). (i) Immunoblotting showed the expression of PGC1α and FNDC5 in myotubes treated with AA, DHA and PA for 5 days at 0.01 mM. (j) Quantification of WB in (i). (k, l) Cell viability was analysed with CCK‐8 analysis to determine the effects of AA and DHA on myotubes. (m) Immunoblotting showed the expression of PGC1α and FNDC5 in myotubes treated with AA (12 μM). (n) Quantification of WB in (m). (o) Immunoblotting showed the expression of PGC1α and FNDC5 in myotubes treated with DHA (4 μM). (p) Quantification of WB in (o). (q) ELISA was used to detect concentrations of irisin in the medium of myotubes treated with AA (12 μM) and DHA (4 μM) were measured by ELISAs. AA, arachidonic acid; DHA, docosahexaenoic acid; OVX, bilaterally ovariectomized; PA, palmitic acid; ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3).
FIGURE 3
FIGURE 3
Irisin increased the bone mass of Ovx mice and promoted the differentiation of osteoblasts. (a) Cell viability of osteoblasts treated with different concentrations of conditioned medium (CM) from myotubes. (b) Representative western blot analysis of FNDC5 expression indicated that FNDC5 was successfully overexpressed in myotubes. (c) Quantification of WB in (b). (d) Immunoblotting showed the expression of COL1, RUNX2 and ALP in osteoblasts treated with CM from myotubes transfected with or without FNDC5 plasmid. (e) Quantification of WB in (d). (f) Micro‐CT of the distal femur of mice in the three groups. (g) Representative 3D reconstructed micro‐CT images of the femur. Upper panels: trabecular bone; lower panels: cortical bone. (h–k) Trabecular bone microarchitecture of femurs showing Tb.BMD (h), BV/TV (i), Tb.N (j) and Tb.Sp (k) of three groups (n = 10 per group). (l, m) Biomechanical analysis of the femur using a 3‐point bending test, including the yield point (l) and ultimate force (m) (n = 10 per group). (n) Representative Goldner's trichrome–stained sections of femurs in three groups. First column, complete bone (scale bar, 3000 μm); second column, distal femur (scale bar, 300 μm); third column, femoral shaft (scale bar, 100 μm); fourth column, femoral head (scale bar, 1000 μm). (o) Alizarin red staining of osteoblasts cultured in osteogenic medium (OM) treated with irisin (irisin group) or without irisin (OM group) compared with osteoblasts cultured in basal medium (blank group) was performed on the seventh day of differentiation. Scale bar, 300 μm. (p) ALP staining of osteoblasts of three groups on the 21st day of differentiation. OVX, bilaterally ovariectomized; ARS, Alizarin red staining; ALP, Alkaline phosphatase staining; Scale bar, 300 μm. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3).
FIGURE 4
FIGURE 4
Cav1 interacts with FNDC5 and AMPKα to activate the AMPK pathway. (a) GST pulldown assays of irisin. GST‐irisin was used as bait, while GST served as a negative control (upper panel). Immunoblotting identified the expression of CAV1 in GST‐irisin pulldown products (lower panel). (b, c) Secondary mass spectrometry analysis of GST pulldown products exhibiting specific peptide segments identified as FNDC5. (d) Secondary mass spectrometry analysis of GST pulldown products exhibiting specific peptide segments identified as CAV1. (e) Immunofluorescence staining of femurs from mice in the control group, Ovx group and treadmill group. Sections were stained with antibodies against FNDC5 (green) and CAV1 (red). Nuclei were stained with DAPI (blue). Scale bar, 1000 μm. (f) Colocalization images of FNDC5 (red) and CAV1 (green) in osteoblasts. Nuclei were stained with DAPI (blue). Osteoblasts with red arrows were used for analysis of fluorescence intensity and colocalization by ImageJ. Scale bar, 100 μm. (g) Intensity profile of FNDC5 and Cav1 immunofluorescence. (h) Colocalization images of Cav1 (red) and AMPKα (green) in osteoblasts. Nuclei were stained with DAPI (blue). Osteoblasts with red arrows were used for analysis of fluorescence intensity and colocalization by ImageJ. Scale bar, 100 μm. (i) Intensity profile of FNDC5 and Cav1 immunofluorescence. (j, k) Coimmunoprecipitation (CoIP) of FNDC5 and CAV1 in osteoblasts. (l, m) CoIP of CAV1 and AMPKα in osteoblasts. (n) Osteoblasts were transfected with FNDC5‐siRNA and irisin (1 μg/mL). Immunoblotting showed the expression of AMPKα, FNDC5 and CAV1. (o) Quantification of WB in (n). (p) Osteoblasts were transfected with CAV1‐siRNA and CAV1 plasmid. Immunoblotting showed the expression of AMPKα and CAV1. (q) Quantification of WB in (p). (r) IF staining of femurs to evaluate the expression of AMPKα in femur tissues. OVX, bilaterally ovariectomized; IRI, irisin; ns p > 0.05, *p < 0.05, **p < 0.01 (n = 3).
FIGURE 5
FIGURE 5
Irisin could regulate the cell cycle and promote the proliferation of osteoblasts via the upregulation of HMOX1. (a) Volcano plots of differentially abundant metabolites in serum between the Ovx group and IRI group. (b) Clustering heatmap for differentially abundant metabolites between the Ovx group and IRI group. (c) Column graph showing the fold change of the top 10 increased and decreased metabolites. (d) Violin plots showing the serum level of biliverdin between the Ovx group and IRI group. (e) KEGG pathway enrichment analysis of differentially abundant metabolites between the Ovx group and IRI group. (f, h) Interaction between irisin and HMOX1. (f) Irisin was labelled as (a), and HMOX1 was labelled as (b). (g) Irisin is shown as cyan sticks, and HMOX1 is shown as slate sticks. (h) Irisin is represented as a slate cartoon, HMOX1 is presented as a cyan cartoon, and their binding sites are displayed as a series of pink sticks. (i) Osteoblasts were transfected with Cav1‐siRNA and Cav1 plasmid. Immunoblotting showed the expression of Nrf2, Fpn, Cav1 and HMOX1 in osteoblasts. (j) Quantification of WB in (i). (k) Osteoblasts were transfected with Cav1 plasmid and further treated with C.C. (1 μM) for 2 h. Immunoblotting showed the expression of Nrf2, AMPKα, Fpn, HMOX1 and Cav1 in osteoblasts. (l) Quantification of WB in (k). (m) Osteoblasts were transfected with HMOX1‐siRNA and HMOX1 plasmid. Immunoblotting showed the expression of CycA2, E2F2, HMOX1 and CDK2 in osteoblasts. (n) Quantification of WB in (m). (o) Osteoblasts were transfected with HMOX1‐siRNA and then treated with irisin (0.1 μg/mL) for 5 days. Immunoblotting showed the expression of CycA2, E2F2, HMOX1 and CDK2 in osteoblasts. (p) Quantification of WB in (o). (q–s) The cell cycle distribution of osteoblasts in the three groups was analysed by flow cytometry. (t) Stacked column graph exhibiting cell cycle distribution in (q–s). (u) A CCK‐8 assay was used to analyse the proliferation of osteoblasts in the three groups. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3).
FIGURE 6
FIGURE 6
Irisin inhibited ferroptosis of osteoblasts via upregulation of Fpn. (a) Osteoblasts were first transfected with Fpn‐siRNA. Immunoblotting showed the expression of Fpn, SLC7A11 and GPX4 in osteoblasts. (b) Quantification of WB in (a). (c) RSL3 (1 μM) was added to induce ferroptosis of osteoblasts. Irisin (1 μg/mL) was used to suppress ferroptosis. Immunoblotting showed the expression of SLC7A11 and GPX4 in osteoblasts. (d) Quantification of WB in (c). (e) Representative images of JC‐1 staining in osteoblasts. (f–i) Quantitation of JC‐1 fluorescence by flow cytometry in osteoblasts. (j) Stacked column graph of JC‐1 staining via fluorescence images in (e). (k) Stacked column graph of JC‐1 staining via flow cytometry in (f–i). (l) Representative images of ROS staining in osteoblasts. (m) Quantification of ROS fluorescence images in (l). (n) Quantitation of ROS fluorescence by flow cytometry in osteoblasts. (o) Representative images of lipid ROS staining in osteoblasts with C11‐BODIPY. (p) Representative images of intracellular iron staining in osteoblasts with FerroOrange. (q) Quantification of C11‐BODIPY fluorescence images in (o). (r) Quantification of FerroOrange fluorescence images in (p). (s) The microstructure of mitochondria in osteoblasts was observed by TEM. Red arrows indicate mitochondria. Typical morphological changes in mitochondria are distinctive characteristics of ferroptosis, exhibiting mitochondrial shrinkage with condensed mitochondrial membrane densities and loss of mitochondrial ridges. Scale bar, 2 μm. (t) Quantification of damaged mitochondria percentages in (S). IRI, irisin; 1, Ctrl group; 2, RSL3 group; 3, RSL3 + IRI group; 4, RSL3 + IRI + si‐Fpn group; ns p > 0.05, *p < 0.05, **p < 0.01 (n = 3).
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