Oxylipin-PPARγ-initiated adipocyte senescence propagates secondary senescence in the bone marrow

Abstract

The chronic use of glucocorticoids decreases bone mass and quality and increases bone-marrow adiposity, but the underlying mechanisms remain unclear. Here, we show that bone-marrow adipocyte (BMAd) lineage cells in adult mice undergo rapid cellular senescence upon glucocorticoid treatment. The senescent BMAds acquire a senescence-associated secretory phenotype, which spreads senescence in bone and bone marrow. Mechanistically, glucocorticoids increase the synthesis of oxylipins, such as 15d-PGJ2, for peroxisome proliferator-activated receptor gamma (PPARγ) activation. PPARγ stimulates the expression of key senescence genes and also promotes oxylipin synthesis in BMAds, forming a positive feedback loop. Transplanting senescent BMAds into the bone marrow of healthy mice is sufficient to induce the secondary spread of senescent cells and bone-loss phenotypes, whereas transplanting BMAds harboring a p16INK4a deletion did not show such effects. Thus, glucocorticoid treatment induces a lipid metabolic circuit that robustly triggers the senescence of BMAd lineage cells that, in turn, act as the mediators of glucocorticoid-induced bone deterioration.

Keywords: INK-family proteins; PPARγ; bone marrow adipocytes; bone marrow adiposity; cellular senescence; glucocorticoids; osteoporosis; oxylipins; prostaglandins; senescence-associated secretory phenotype.

Figure 1. Glucocorticoid treatment induces rapid senescence of BMAds in mice.

Three-month-old p16^tdTom mice were treated with methylprednisolone (MPS) at 10 mg/m²/day or vehicle by daily intraperitoneal injection for different time periods as indicated. (A-B) Representative images of tdTom⁺ cells from proximal and distal femur in (A) and analysis of cell number per mm² tissue area (N. tdTom⁺ cells) in (B). (C) Schematic diagram illustrating the experimental procedure. Femoral bone and bone marrow tissue was collected from mice and digested, and the isolated cells were subjected to flow cytometry analysis. (D-E) Representative flow cytometry images of tdTom-expressing cells of the femoral bone in (D) and the percentages of tdTom⁺ cells out of total bone/bone marrow cells in (E). (F-H) Immunofluorescence staining of perilipin in femoral bone sections. Representative images are shown in (F) Quantification of the percentage perilipin⁺ cells in total tdTom positive cell population (% perilipin⁺ cells/total tdTom⁺ cells) and the percentage of tdTom⁺ cells in total perilipin positive cell population (% tdTom⁺ cells/total perilipin⁺ cells) are shown in (G) and (H), respectively. (I-J) Three-month-old C57BL/6 mice were treated with MPS or vehicle for 2 weeks. (I) Fluorescent in situ hybridization followed by immunofluorescence staining was performed to identify SADS-positive senescent cells (in red) and perilipin⁺ adipocytes (in green), respectively. (J) Double-immunofluorescence staining of γH2AX and perilipin in femoral bone sections. (K-M) Three-month-old C57BL/6J mice were treated with MPS at 10 mg/m²/day or vehicle for different time periods as indicated. Double-immunofluorescence staining of γH2AX and perilipin in femoral bone sections. Representative images are shown in (K) Quantification of the number of perilipin-positive cells per mm² tissue area (N. Perilipin⁺ cells) and the number of γH2AX-positive cells per mm² tissue area (N. γH2AX⁺ cells) are shown in (L) and (M), respectively. n=5 mice. Data are represented as mean ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s t-tests for 2 groups and one-way ANOVA for 3 or more groups.

Figure 2. Senescent vascular cells and osteoblasts accumulate in murine bone/bone marrow in a late phase of glucocorticoid treatment.

Three-month-old p16^tdTom mice were treated with MPS at 10 mg/m²/day or vehicle for different time periods as indicated. (A-D) Immunofluorescence staining of Endomucin (Emcn) (A) or Osteocalcin (Ocn) (C) in femoral bone sections. Yellow arrow heads, cells are tdTom- and Emcn-double positive in (A) or tdTom- and Ocn-double positive in (C). BM, bone marrow; BS, bone surface. Quantified numbers of Emcn- and tdTom-double positive cells per mm² tissue area (N. Emcn⁺tdTom⁺ cells) and Ocn- and tdTom-double positive cells per mm² tissue area (N. Ocn⁺tdTom⁺ cells) are shown in (B) and (D), respectively. (E-F) Bone/bone marrow cells were isolated from femoral bone for flow cytometry analysis. Representative images are shown in (E). Percentages of tdTom-expressing cells in total CD144⁺ vascular cell population are shown in (F). Three-month-old C57BL/6J mice were treated with MPS at 10 mg/m²/day or vehicle for different time periods as indicated. (G-J) Double-immunofluorescence staining of γH2AX and perilipin in femoral bone sections. Representative images are shown in (G). Quantification of the number of perilipin-positive cells per mm² tissue area (N. Perilipin⁺ cells), the number of γH2AX-positive cells per mm² tissue area (N. γH2AX⁺ cells), and the percentage of γH2AX⁺ cells in total perilipin-positive cell population (% γH2AX⁺ cells/total BMAds) in (H), (I), and (J), respectively. (K-N) Representative images of SA-βGal and Oil red staining of femoral bone sections are shown in (K). Quantification of the numbers of Oil Red⁺ cells per mm² tissue area, SA-βGal⁺ cells per mm² tissue area, and the percentage of SA-βGal⁺ cells in total Oil Red⁺ BMAds are shown in (L), (M), (N), respectively. n=5 mice, Data are represented as mean ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 as determined by two-tailed Student’s t-tests for 2 groups and one-way ANOVA for 3 groups.

Figure 3. Glucocorticoids directly activate a cellular senescence program in cultured BMAd lineage cells.

(A-F) Bone marrow mesenchymal stromal cells (BMSCs) were isolated from 3-month-old p16-3MR mice and differentiated into bone marrow adipocytes/preadipocytes with adipogenesis medium for 10 days (A). The differentiated adipocytes/preadipocytes were then stimulated with vehicle, dexamethasone (DEX) or DEX + Ganciclovir (GCV) for 3 days. Cells were fixed and subjected to perilipin immunostaining (B), Cdkn2a (p16 encoding gene) mRNA measurement by qRT-PCR (C), SA-βGal staining (D), γH2AX immunostaining (E), and Lamin-B1 immunostaining (F), respectively. (G-H) BMAds were isolated from 3-month-old p16-3MR mice as shown in (G) and were treated with vehicle, DEX or DEX + GCV for 3 days. The cells were then subjected to RFP staining with representative pictures in (H). (I-M) BMSCs were isolated from C57BL/6 mice and differentiated into bone marrow adipocytes/preadipocytes with adipogenesis medium for 10 days (I). The differentiated adipocytes/preadipocytes were stimulated with vehicle or DEX and subjected for bulk RNA-Seq. Principal-component analysis (PCA) of the DEG expression profile of sequenced adipocyte samples was shown in (J). Venn map analyzing the number of DEGs overlap with previously identified aging and senescence ASIGs genes (K). Top enriched pathways of ASIGs genes significantly upregulated or downregulated in DEX-treated cells relative to vehicle-treated cells based on RNA-seq data (L). Heatmap showing increased expression of INK family genes in DEX-treated cells relative to vehicle-treated cells in (M). In vitro experiments were repeated 3 times. Data are represented as mean ±s.e.m. ***p<0.001 as determined by two-tailed Student’s t-tests.

Figure 4. Glucocorticoids induce senescence of BMAd lineage cells by activating prostaglandin pathway.

(A) Heatmap of RNA-seq data showing expression changes encoding oxylipin synthesis genes in DEX-treated relative to vehicle-treated adipocytes/preadipocytes. (B) Schematic diagram showing the procedure of the conditioned medium (CM) preparation from cultured BMAds. (C) The concentrations of prostaglandins (PGJ2, PGD2, PGE2) in CM were measured by ELISA. (D) BMAds were isolated and treated with Vehicle, DEX, or 15d-PGJ2 for 3 days. mRNA levels of Cdkn2a, Cdkn1a, and Cdkn1b in the cells were measured by qRT-PCR analysis. (E) Adipocytes/preadipocytes differentiated from BMSCs were treated with Vehicle, DEX, or 15d-PGJ2 for 3 days. SA-βGal staining was performed. (F) Adipocytes/preadipocytes differentiated from BMSCs were treated with Vehicle, DEX, or DEX plus celecoxib for 3 days. mRNA levels of Cdkn2a, Cdkn1a, and Cdkn1b in the cells were measured by qRT-PCR analysis. In vitro experiments were repeated 3 times. Data are represented as mean ±s.e.m. **p<0.01, ***p<0.001 as determined by two-tailed Student’s t-tests for 2 groups and one-way ANOVA for 3 groups.

Figure 5. An oxylipins-PPARγ positive feedback loop mediates glucocorticoid-induced BMAd senescence.

(A-E) Adipocytes/preadipocytes differentiated from murine BMSCs were stimulated with different treatments as indicated. mRNA levels of the indicated genes were measured by qRT-PCR analysis. (F-G) Adipocytes/preadipocytes differentiated from murine bone marrow MSCs were treated with different reagents as indicated and subjected to SA-βGal staining. (H) Heatmap of RNA-seq data showing the changes in INK family genes in cells with the indicated treatments. (I-J) qRT- PCR analysis of Ptgds and Ptgis levels in BMAds with the indicated treatments. Experiments were repeated 3 times. Data are represented as mean ±s.e.m. *p< 0.05, **p<0.01, ***p<0.001, as determined by one-way ANOVA.

Figure 6. Senescent BMAds acquire a SASP that spreads senescence to bone marrow vascular cells and osteoblasts.

(A) Heatmap of RNA-seq data showing SASP gene expression in adipocytes/preadipocytes treated with vehicle, DEX or DEX+GW9662. (B-C) BMAds were isolated from 3-month-old p16-3MR mice, cultured in alpha-MEM for 1 day, and stimulated with vehicle, DEX, or DEX+GCV for 3 days. The secretion of cytokines in the CMs were measured (B). Heatmap of differentially expressed cytokines in CM was shown in (C). (D) A schematic diagram illustrating the procedure of the CM-based senescence assays. (E-F) Calvarium osteoblasts (OB) or HUVECs (EC) were cultured with CM prepared from (D). SA-βGal staining of the cells in (E). Western blot analysis of p16 protein expression in (F). (G) Schematic diagram showing the experimental design for transplantation. (H-L) Representative micro-CT images in (H) and quantitative analysis of trabecular bone volume (BV/TV) (I), trabecular thickness (Tb. Th) (J), trabecular number (Tb. N) (K), and trabecular separation (Tb. Sp) (L). n=5 mice. Data are represented as mean ±s.e.m. ***p<0.001, as determined by two-tailed Student’s t-tests for 2 groups.

Figure 7. Targeting senescent BMAds rescues glucocorticoid-induced bone loss.

(A-G) Three-month-old AdipoQ-Cre::p16 flox/flox (p16 cKO) mice and p16 flox/flox (WT) mice were injected with daily MPS treatment at 10 mg/m²/day or vehicle for 2 weeks. Representative images of SA-βGal staining and Oil red staining in femoral bone tissue sections (A). Quantification of the number of SA-βGal-expressing BMAds per mm² tissue area (N. βGal⁺ BMAds) in (B). Representative micro-CT images of p16 cKO or WT littermates treated with vehicle or DEX are shown in (C) with quantitative analysis of trabecular bone volume (BV/TV) (D), trabecular thickness (Tb. Th) (E), trabecular number (Tb. N) (F), and trabecular separation (Tb. Sp) (G). (H) Schematic diagram showing the experimental design for transplantation. (I-M) Representative micro-CT images of WT mice transplanted with BMAds from p16 cKO or WT littermates treated with vehicle or DEX are shown in (I) with quantitative analysis of BV/TV (J), Tb. Th (K), Tb. N (L), and Tb. Sp (M). (N) Diagram model illustrating the mechanisms of glucocorticoids induced BMAds senescence in bone and bone marrow. n=5 mice. Data are represented as mean ±s.e.m. *p< 0.05, **p<0.01, ***p<0.001 as determined by two-way ANOVA.