Splicing factor YBX1 regulates bone marrow stromal cell fate during aging

Abstract

Senescence and altered differentiation potential of bone marrow stromal cells (BMSCs) lead to age-related bone loss. As an important posttranscriptional regulatory pathway, alternative splicing (AS) regulates the diversity of gene expression and has been linked to induction of cellular senescence. However, the role of splicing factors in BMSCs during aging remains poorly defined. Herein, we found that the expression of the splicing factor Y-box binding protein 1 (YBX1) in BMSCs decreased with aging in mice and humans. YBX1 deficiency resulted in mis-splicing in genes linked to BMSC osteogenic differentiation and senescence, such as Fn1, Nrp2, Sirt2, Sp7, and Spp1, thus contributing to BMSC senescence and differentiation shift during aging. Deletion of Ybx1 in BMSCs accelerated bone loss in mice, while its overexpression stimulated bone formation. Finally, we identified a small compound, sciadopitysin, which attenuated the degradation of YBX1 and bone loss in old mice. Our study demonstrated that YBX1 governs cell fate of BMSCs via fine control of RNA splicing and provides a potential therapeutic target for age-related osteoporosis.

Keywords: Y-box binding protein 1; aging; alternative splicing; bone marrow stromal cells.

Figure 1. Dysregulated pre‐mRNA alternative splicing and altered gene expression patterns in BMSCs during aging

1. A

   Schematic diagram of the isolation and culture of BMSCs from 2‐ and 24‐month‐old mice. BMSCs, bone marrow stromal cells.

2. B

   Representative images of β‐Gal staining (left panel) and quantification of β‐Gal‐positive cells (right panel) of BMSCs isolated from 2‐ and 24‐month‐old mice. Scale bar: 100 μm. β‐Gal, beta‐galactosidase.

3. C

   Representative images of Alizarin Red staining at 21 days of osteogenic induction (left panel) and quantification of calcification (right panel) of BMSCs isolated from 2‐month‐old and 24‐month‐old mice by detecting the amount of Alizarin Red extracted from the matrix.

4. D

   Schematic diagram of the experimental process of alternative splicing analysis.

5. E

   Gene ontology analysis of differentially expressed genes between BMSCs isolated from 2‐ and 24‐month‐old mice. ΔPSI, percentage spliced in.

6. F

   Heat map of differentially expressed genes between BMSCs isolated from 2‐ and 24‐month‐old mice.

7. G

   Histogram of the differential splicing events between BMSCs isolated from 2‐ and 24‐month‐old mice. AS, alternative splicing.

8. H

   Venn diagrams of overlapping genes between differentially expressed genes, RNA‐binding proteins, and splicing factor datasets between BMSCs isolated from 2‐ and 24‐month‐old mice.

9. I

   Enrichment network representing the top 10 enriched terms of differentially expressed RNA splicing factors between BMSCs isolated from 2‐ and 24‐month‐old mice. Enriched terms with high similarity were clustered and rendered as a network, while each node represents an enriched term and is colored according to its cluster. The node size indicates the number of enriched genes, and the line thickness indicates the similarity score shared by two enriched terms.

10. J

    The list of differentially expressed RNA splicing factors between BMSCs isolated from 2‐ and 24‐month‐old mice whose functions are clustered in mRNA splicing and regulation of RNA splicing.

Data information: In (B) and (C), n = 5 in each group from three independent experiments. In (E) and (F), n = 3 in each group. Data are shown as the mean ± SEM. **P < 0.01; ***P < 0.001; Student's t‐test.

Figure 2. Splicing factor YBX1 regulated fate decision and senescence of BMSCs and showed decreased expression during aging

1. A, B

   Representative images (A) of immunofluorescence staining and quantification (B) of YBX1 (green) in primary BMSCs.

2. C, D

   Representative immunohistochemical staining images of YBX1 (red) and LEPR (green) (C) and quantification of YBX1⁺ LEPR⁺ cell numbers (D) in femoral bone marrow. MP, metaphysis; T Ar, Tissue area. Arrows point to YBX1⁺ LEPR⁺ cells.

3. E

   Age‐associated changes in YBX1 levels in human BMSCs from 30 males (left panel) and 30 females (right panel).

4. F, G

   Representative images of Alizarin Red staining (F) and quantification of calcification (G) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction.

5. H, I

   Representative images of Oil Red O staining (H) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix (I) in BMSCs after 10 days of adipogenic induction.

6. J, K

   Representative images of β‐Gal staining (J) and quantification (K) of β‐Gal‐positive cells among BMSCs.

7. L, M

   Relative mRNA levels of osteogenic differentiation related genes (L), adipogenic differentiation and senescence related genes (M) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA.

8. N

   Heat map of differentially expressed genes between BMSCs transfected with adenovirus driven control and Ybx1 shRNA.

Data information: In (B), (D), (G), (I), and (K), n = 5 in each group from three independent experiments. In (L) and (M), n = 3 in each group from three independent experiments. In (N), n = 3 in each group. Data are shown as the mean ± SEM. Scale bar: 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; Student's t‐test for (B), (D), (L), (M) and one‐way ANOVA for (G), (I), (K).

Figure 3. Depletion of YBX1 in BMSCs results in accelerated bone loss and bone marrow fat accumulation

1. A

   qRT‐PCR analysis of the levels of Ybx1 in BMSCs isolated from Ybx1 ^Prx1‐CKO mice and Ybx1 ^flox/flox mice.

2. B–F

   Representative μCT images, Scale bar: 1 mm. (B) and quantitative μCT analysis of trabecular bone microarchitecture (C–F) in distal femora from 3‐ and 12‐month‐old male Ybx1 ^Prx1‐CKO mice and Ybx1 ^flox/flox mice (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).

3. G

   Representative images of H&E staining (Hematoxylin–Eosin Staining) in distal femora. Scale bar: 100 μm.

4. H

   Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar).

5. I

   Representative images of osteocalcin (OCN) immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. Scale bar: 50 μm.

6. J

   Quantification of osteocalcin positive cells on the bone surface. Number of OCN⁺ cells per bone perimeter (N. Ocn⁺/B.Pm).

7. K

   Representative images of TRAP staining in distal femora. Scale bar: 100 μm.

8. L

   Quantification of TRAP positive cells on the bone surface. Number of TRAP⁺ cells per bone perimeter (N. Trap⁺/B.Pm).

9. M

   Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm.

10. N

    Quantification of the bone formation rate (BFR) based on calcein double labeling.

11. O

    Quantification of the mineral apposition rate (MAR) based on calcein double labeling.

Data information: n = 6, biological replicates in each group. Data are shown as the mean ± SEM. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; Student's t‐test for (A), one‐way ANOVA for (C), (D), (E), (F), (H), (J), (L), (N), and (O).

Figure 4. Overexpression of Ybx1 attenuated fat accumulation and promoted bone formation in aged mice

1. A

   Representative images of Alizarin Red staining (left panel) and quantification of calcification (right panel) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with the control or Ybx1 plasmid at 21 days of osteogenic induction.

2. B

   Representative images (left panel) and quantification (right panel) of Oil Red O staining in BMSCs transfected with the control or Ybx1 plasmid at 10 days of adipogenic induction.

3. C

   Representative images of β‐Gal staining (left panel) and quantification (right panel) of β‐Gal‐positive cells in BMSCs transfected with the control or Ybx1 plasmid.

4. D

   qRT‐PCR analysis of the expression of Ybx1 in BMSCs from mice with rAAV8‐GFP‐Ybx1 or rAAV8‐GFP transfection.

5. E, F

   Representative μCT images, Scale bar: 1 mm. (E) and quantitative μCT analysis of trabecular bone microarchitecture (F) in distal femora from 24‐month‐old mice with rAAV8‐GFP‐Ybx1 or rAAV8‐GFP transfection (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).

6. G

   Representative images of H&E staining in distal femora.

7. H

   Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar).

8. I

   Representative images of osteocalcin immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells.

9. J

   Quantification of osteocalcin positive cells in bone surface. Number of OCN⁺ cells per bone perimeter (N. Ocn⁺/B.Pm).

10. K

    Representative images of TRAP staining in distal femora.

11. L

    Quantification of TRAP positive cells on the bone surface. Number of TRAP⁺ cells per bone perimeter (N. Trap⁺/B.Pm).

12. M

    Representative images of calcein double labeling of trabecular bone.

13. N

    Quantification of the bone formation rate (BFR) and mineral apposition rate (MAR) based on calcein double labeling.

Data information: n = 6, biological replicates in each group. Data are shown as the mean ± SEM. Scale bar: 50 μm. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; Student's t‐test.

Figure 5. YBX1 regulated the fate of BMSCs through regulating the splicing of pre‐mRNAs critical for differentiation and senescence

1. A

   Venn diagrams of overlapping genes targeted by YBX1 and that showed alternative splicing upon YBX1 deletion. CLIP, ultraviolet cross‐linking immunoprecipitation.

2. B

   CLIP‐seq read coverage across Fn1, Nrp2, Sp7, Sirt2, and Spp1 from BMSCs. The dotted line regions indicate the location of the exon skipping. ES, Exon Skipping.

3. C

   Semiquantitative PCR showing the different isoforms of Fn1, Nrp2, Sp7, and Sirt2 from BMSCs of Ybx1 ^Prx1‐CKO mice and Ybx1 ^flox/flox mice.

4. D–G

   Representative images of Alizarin Red staining (D and F) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (E and G) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1.

5. H, I

   Representative images (H) and quantification of Oil Red O staining (I) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction.

6. J, K

   Representative images of β‐Gal staining (J) and quantification (K) of β‐Gal‐positive cells in BMSCs transfected with different isoforms of Sirt2.

7. L–S

   BMSCs were isolated from Ybx1 ^Prx1‐CKO mice and Ybx1 ^flox/flox mice, among them, the BMSCs from Ybx1 ^flox/flox mice were transfected with the blank control, BMSCs from Ybx1 ^Prx1‐CKO mice were transfected with the blank control or different isoforms of the target genes. (L–O) Representative images of Alizarin Red staining (L and N) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (M and O). (P, Q) Representative images (P) and quantification of Oil Red O staining (Q) in BMSCs after 10 days of adipogenic induction. (R, S) Representative images of β‐Gal staining (R) and quantification (S) of β‐Gal‐positive cells among BMSCs.

Data information: In (C), n = 2 in each group from three independent experiments. In (E), (G), (I), (K), (M), (O), (Q), and (S), n = 5 in each group from three independent experiments. Data are shown as the mean ± SEM. Scale bar: 100 μm. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; one‐way ANOVA. Source data are available online for this figure.

Figure 6. Sciadopitysin bound to and inhibited ubiquitin degradation of YBX1

1. A

   The homology modeling structure of mouse YBX1 and nine top‐ranked candidates.

2. B

   Cell proliferation rate assessed using a CCK8 assay after administration of different compounds.

3. C

   qRT‐PCR analysis of Ybx1 expression in BMSCs after administration of different compounds.

4. D

   Representative images of Alizarin Red staining (up panel), Oil Red O staining (middle panel, scale bar: 100 μm) and β‐Gal staining (bottom panel, scale bar: 100 μm) in BMSCs treated with different compounds.

5. E–G

   Quantification of calcium mineralization based on Alizarin Red staining (E), quantification of Oil Red O based on Oil Red O staining (F), and quantification of β‐Gal‐positive cells based on β‐Gal staining in BMSCs treated with different compounds (G).

6. H

   The molecular structure of sciadopitysin and a model of the interaction between sciadopitysin and mouse YBX1.

7. I

   Western blotting analysis of YBX1 in sciadopitysin pretreated BMSCs after cycloheximide (CHX) treatment.

8. J

   Western blotting analysis of YBX1 related ubiquitination in sciadopitysin pretreated BMSCs administered with MG132.

9. K

   Co‐IP of His‐FBXO33 with HA‐YBX1 and a series of mutant HA‐YBX1 proteins following transfection into BMSCs.

10. L

    Co‐IP of FBXO33 withYBX1 with or without the administration of sciadopitysin.

11. M

    Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin.

12. N

    Semiquantitative PCR showed the isoforms of Fn1, Nrp2 and Sirt2 in cultured BMSCs isolated from 2‐ or 24‐month‐old mice then treated with or without sciadopitysin.

Data information: In (B), (C), (E), (F), and (G), n = 5 in each group from three independent experiments. (I–N) is representative of three independent experiments. Data are shown as the mean ± SEM. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; one‐way ANOVA. Source data are available online for this figure.

Figure 7. Sciadopitysin treatment alleviated aging‐related bone loss in mice

1. A

   Schematic of the time of oral treatment of sciadopitysin in mice.

2. B–F

   Representative μCT images, Scale bar: 1 mm. (B) and quantitative analysis of trabecular bone microarchitecture (C–F) in distal femora of 25‐month‐old mice administered with sciadopitysin or vehicle (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).

3. G, H

   Representative images (G) and quantification (H) of osteocalcin (OCN)‐positive cells in distal femora of 25‐month‐old mice administered with sciadopitysin or vehicle. Number of Ocn⁺ cells per bone perimeter (N. Ocn⁺/B.Pm). Arrows point to osteocalcin positive cells. Scale bar: 50 μm.

4. I, J

   Representative images (I) of H&E staining in distal femora and quantification (J) of the number of adipocytes related to the tissue area (right panel, N. adipocytes/T.Ar) in distal femora of 25‐month‐old mice administered with sciadopitysin or vehicle. Scale bar: 100 μm.

5. K, L

   Representative images (K) and quantification (L) of TRAP‐positive cells in distal femora of 25‐month‐old mice administered with sciadopitysin or vehicle. Number of TRAP⁺ cells per bone perimeter (N. Trap⁺/B.Pm). Scale bar: 50 μm.

6. M

   Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm.

7. N, O

   Quantification of the bone formation rate (BFR, N) and mineral apposition rate (MAR, O) based on calcein double labeling.

8. P

   RNA binding protein YBX1 regulates a cluster of genes including Fn1, Nrp2, Spp1, Sirt2 and Sp7 as a splicing factor in the nucleus, which further stimulates osteogenic differentiation and restrains the senescence of BMSCs. The decreased expression level of YBX1 during aging contributes to the debility of BMSCs, including increased senescence and reduced osteogenesis. Sciadopitysin can delay the ubiquitination degradation of YBX1 by preventing YBX1 from binding to ubiquitin ligase FBXO33 (model based on data from previous figures).

Data information: n = 8, biological replicates in each group. Data are shown as the mean ± SEM. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; Student's t‐test.