Targeting miR-337 mitigates disuse-induced bone loss

Abstract

Disuse-induced bone loss occurs in long-term bed-ridden patients and in astronauts during spaceflight. The underlying mechanisms are poorly understood. In a rodent model of disuse-induced bone loss (called hindlimb unloading (HU)), we observed that decreased numbers of leptin receptor (LepR) positive mesenchymal stem cells (MSCs) in adult bone marrow, contribute to bone loss. MicroRNA-337-3p (miR-337) was upregulated in MSCs upon HU and inhibited MSC proliferation by directly targeting IRS-1 to suppress the PI3kinase-Akt-mTOR pathway. Piezo1 was the upstream receptor for sensing mechanical stress and regulated miR-337 through the Hippo-YAP signaling pathway. Remarkably, the knockout of miR-337 significantly attenuated HU-induced, but not ovariectomy-induced, bone loss by increasing MSC proliferation and osteogenesis. Finally, the transplantation of miR-337^-/- MSCs into wild-type HU mice was sufficient to mitigate bone loss. These findings reveal the cellular and molecular mechanisms underlying disuse-induced bone loss and highlight a feasible therapeutic strategy to prevent disuse- or microgravity-induced bone loss on Earth and during spaceflight.

Fig. 1. The MSC frequency was markedly reduced in BM of HU rats.

a Schematic of HU models and WB controls. Representative micro-CT images (b) and quantification of three-dimensional microstructural parameters (c) of femurs from HU rats and WB controls. d Relative serum Ocn and CTX1 levels in WB and HU rats. The data are presented as the fold changes relative to those of the WB samples. e Representative images of tibia sections from rats subjected to HU for 7 days and stained with LepR. f Quantification of LepR⁺ cells. The data are presented as percentages of positively stained cells to total cells (DAPI-stained nuclei) *P < 0.05, **P < 0.01, and ***P < 0.001 compared with WB (one-way ANOVA with Dunnett’s post hoc test), n = 6 per group. The data are presented as the means ± SEM. g Flow cytometry analysis showing the percentage of CD45/CD31^–LepR⁺ cells in the BM of WB rats and rats suspended for 7 days. The data are presented as the means ± SD, n = 6 per group, and are representative of three independent experiments. h Representative image of CFU clones formed by the BM cells derived from rats subjected to unloading for 7 days. The number of clones larger than 40 cells was calculated.

Fig. 2. The number of proliferating LepR⁺ cells was decreased in the BM of unloaded rats.

a Representative images of mIHC of tibia sections from WB controls and rats suspended for 7 days. Scale bars, (upper panel) 600 μm; (lower panel) 50 μm. b Schematic of the LepR⁺ cell niches. c Quantification of the 4 subsets of LepR⁺ cells in the 3 niches (< 50 μm), n = 6 per group.

Fig. 3. CMS-activated quiescent MSCs by the miR-337-PI3K-Akt-mTOR pathway.

Flow cytometry analysis showing the percentage of EdU⁺ MSCs treated with the indicated CMS elongations (a) or 10% CMS (b) for the indicated time after serum-starvation for 8 h. c Schematic of the different MSC treatments groups used for the RNA-seq analysis. d Flow cytometry analysis of the percentage of EdU⁺ MSCs starved for 12 h and then stimulated with 10% CMS for another 24 h and treated with DMSO, LY294002, or rapamycin. Antagonistic agents were added at the time of the CMS treatment. The baseline (black dotted line) indicates the percentage of EdU⁺ MSCs before CMS treatment. e Rapamycin administration decreased the bone volume of WB rats as indicated by the micro-CT analysis of tibia sections from rats treated for 28 days. f Quantification of three-dimensional microstructural parameters from micro-CT images. g Heatmap of differentially expressed miRNAs that specifically target the PI3K-Akt pathway in MSCs treated with 10% CMS or FBS for 24 h after 8 h of serum starvation. h Quantitative RT-PCR was used to measure the expression of miR-337 in de novo isolated LepR⁺ cells from rats suspended for 7 days as well as WB controls. i Representative Western blot images showing the activation of the PI3K-Akt-mTOR pathways. The cells were transfected with the miR-337 mimic or inhibitor before they were subjected to 10% CMS-stimulated proliferation. j Quantification of the relative protein levels was normalized to the intensity of GAPDH in 3 independent experiments. k Flow cytometry analysis showing the percentage of EdU⁺ cells after being transfection with the miR-337 mimic or inhibitor, followed by 10% CMS stimulation for 24 h. l Representative images of CFU-F and ALP staining. MSCs were transfected with the miR-337 mimic or inhibitor as indicated for 24 h before the CFU-F and osteogenic assays were performed. m Quantification of CFU and ALP staining intensities shown in (l). n Flow cytometry analysis revealed that the fluorescent intensity of the GFP-IRS1 reporter was regulated by miR-337. o Schematic of the in vivo reporter assay. p Flow cytometry analysis showing the fluorescence intensity of GFP in CD45^–/CD31^–/LepR⁺ cells in rats suspended for 7 days after an intramedullary injection of the AAV-GFP-IRS1 reporter. Statistical significance was assessed by Student’s t-test of the results from 3 independent experiments. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001. The data are presented as means ± SD. The miR-337 expression levels were normalized to those of U6.

Fig. 4. miR-337 regulates the PI3K-Akt signaling pathway through IRS-1.

a Flow cytometry analysis showing the percentage of EdU⁺ rMSCs after transfection with the miR-337 inhibitor or IRS-1 si-RNA as indicated, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. b Representative Western blot images showing the activation of the PI3K-Akt-mTOR pathway. rMSCs were transfected with the miR-337 inhibitor or IRS-1 si-RNA as indicated before they were subjected to 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. c Flow cytometry analysis showing the percentage of EdU⁺ rMSCs after infection with LV-nc or LV-IRS-1 and subsequent transfection with the miR-337 mimic as indicated, followed by 10% CMS stimulation for 24 h after 8 h of serum starvation. d Representative Western blot image showing the activation of the PI3K-Akt-mTOR pathway. rMSCs were infected with LV-nc or LV-IRS-1 and transfected with the miR-337 mimic as indicated sequentially, followed by 10% CMS stimulation for 24 h after 8 h of serum starvation. e, f Flow cytometry analysis showing the percentage of EdU⁺ con-rMSCs or mut-rMSCs after transfection with the miR-337 inhibitor or mimic as indicated, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. g Quantitative RT-PCR was used to measure the expression of miR-337 in hMSCs treated as indicated for 24 h after they were serum-starved for 8 h. h, j Flow cytometry analysis showing the percentage of EdU⁺ hMSCs after transfection with the miR-337 inhibitor or mimic as indicated, followed by treatment as indicated for 24 h after being serum-starved for 8 h. i, k Representative Western blot images showing the activation of the PI3K-Akt-mTOR pathway in hMSCs. The cells were transfected with the miR-337 inhibitor or mimic before being treated as indicated for 24 h after being serum-starved for 8 h. Statistical significance was assessed by Student’s t-test of the results from 3 independent experiments. n = 3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001. The data are means ± SD. The miR-337 expression levels were normalized to those of U6.

Fig. 5. MSCs sense stress stimulation through the Piezo1-miR-337-PI3K-Akt axis.

a Representative Western blot images showing the Piezo1 protein level at 72 h after siRNA transfection. b Quantitative RT-PCR analysis of the expression of miR-337 in rMSCs transfected with the Piezo1 si-RNA followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. c Flow cytometry analysis showing the percentage of EdU⁺ rMSCs after transfection with the Piezo1 si-RNA, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. d Representative Western blot images showing the activation of the PI3K-Akt-mTOR pathway. rMSCs were transfected with the Piezo1 si-RNA before they were subjected to 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. e Quantitative RT-PCR was used to measure the expression of miR-337 in rMSCs treated with DMSO or Yoda1 and transfected with the miR-337 mimic as indicated simultaneously, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. f Flow cytometry analysis showing the percentage of EdU⁺ rMSCs after treatment with DMSO or Yoda1 and transfection with the miR-337 mimic as indicated simultaneously, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. g Representative Western blot images showing the activation of the PI3K-Akt-mTOR pathway. rMSCs were treated with DMSO or Yoda1 and simultaneously transfected with the miR-337 mimic as indicated, followed by treatment with 0% CMS or 10% CMS for 24 h after 8 h of serum starvation. h Schematic illustration of the TEAD binding site in the wild-type and mutant miR-337 promoters. The TEAD binding motif in the promoter of miR-337 was predicted using the JASPAR database. i Fluorescence activity in 293 T cells expressing the miR-337 wild-type or mutant promoter with TEAD pcDNA3.1. j Representative Western blot images showing the YAP protein level at 72 h after siRNA transfection. k Quantitative RT-PCR analysis of the expression of miR-337 in rMSCs treated with DMSO or Yoda1 and transfected with the YAP si-RNA simultaneously for 24 h after 8 h of serum starvation. l Quantitative RT-PCR analysis of the expression of miR-337 in rMSCs treated with the indicated medium for 24 h after 8 h of serum starvation. Statistical significance was assessed by Student’s t-test of the results from 3 independent experiments. n = 3 per group. *P < 0.05, **P < 0.01, and ***P < 0.001. The data are presented as means ± SD. The miR-337 expression levels were normalized to those of U6.

Fig. 6. Knockout of miR-337 reversed mechanical unloading-induced bone loss.

a Representative images of micro-CT and bone histomorphometry. WT and miR-337^–/– rats were suspended by the tail for 28 days before the femurs were used for micro-CT scanning. n = 6 per group. b Quantification of the three-dimensional microstructural parameters from micro-CT scans of femurs. c Representative images showing the flow cytometry analysis of CD45/CD31^–LepR⁺ cells in the BM of WT and miR-337^–/– rats suspended for 7 days. d An EdU incorporation assay was used to determine the percentage of proliferative LepR⁺ cells in the BM of WT- or KO-rats after being suspended for 7 days. e CFU frequencies of BM cells isolated from WT- or KO-rats after being suspended for 7 days. Colonies larger than 40 cells were counted. f The number of subsets of LepR⁺ cells counted in 3 BM niches (< 50 μm) from mIHC images. g Heatmap of 21 DEGs enriched in the PI3K-Akt pathway, whose expression changed after 10% CMS in wild-type MSCs, but did not change significantly in KO-MSCs stimulated with FBS or 10% CMS. h Representative images of Western blots showing PI3K-Akt-mTOR pathway activity in WT- and miR-337^–/– MSCs after serum starvation for 12 h. Statistical significance was assessed by Student’s t-test of the results from 3 to 6 independent experiments. All the data are presented as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7. miR-337^–/– MSCs ameliorated the HU-induced decrease in bone formation.

a Flow cytometry analysis of EdU⁺ MSCs treated as indicated for 24 h. b Representative images of the CFU frequency of WT- and miR-337^–/– MSCs in the growth medium either without serum or supplemented with a PI3K inhibitor (LY) or an mTOR inhibitor (Rapa). c The number of colonies containing more than 40 cells was quantified. d Representative images of ALP staining and ARS staining of WT- and miR-337^–/– MSCs treated as indicated. e Quantification of the staining intensity in three independent experiments. f Relative expression of Runx2 and Col1a1 in WT- and miR-337^–/– MSCs treated as indicated for 7 days. The data are normalized to the level of GAPDH. g Schematic of the in vivo transplantation assays. h Representative micro-CT images of femurs from WT rats suspended for 28 days. i Quantification of three-dimensional microstructural parameters from micro-CT scans of femurs. The baselines (black dotted lines) indicate the parameters of WT rats under WB conditions. j Flow cytometry analysis of transplanted GFP⁺ cells in the BM of WT rats suspended for 7 days. k Representative images of EdU⁺GFP⁺ cells in the BM of WT rats suspended for 7 days. l Flow cytometry analysis of endogenous CD45/CD31^–LepR⁺ cells in the BM of WT rats transplanted with the indicated cells before 7 days of HU. The statistical significance of the results shown in (a–f and j–l) was assessed by Student’s t-test of 3 to 6 independent experiments. The data are represented as the means ± SDs. The statistical significance of the results from the experiments shown in i was assessed by one-way ANOVA with Dunnett’s post hoc test. n = 6 per group. The data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 8. Schematic diagram illustrating the regulation of mesenchymal stem cell proliferation by mechanical stress via miR-337 expression.

HU results in the loss of mechanical stress around quiescent LepR⁺ mesenchymal stem cells, leading to the closure of the Piezo1 channel. Through the YAP-TEAD pathway, this closure increases the expression of miR-337, which inhibits the PI3K-Akt-mTOR signaling pathway. Consequently, the number of activated LepR⁺ MSCs in adult BM decreases, contributing to bone loss.