Adiponectin regulates bone marrow mesenchymal stem cell niche through a unique signal transduction pathway: an approach for treating bone disease in diabetes

Abstract

Adiponectin (APN) is an adipocyte-secreted adipokine that exerts well-characterized antidiabetic properties. Patients with type 2 diabetes (T2D) are characterized by reduced APN levels in circulation and impaired stem cell and progenitor cell mobilization from the bone marrow for tissue repair and remodeling. In this study, we found that APN regulates the mobilization and recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to participate in tissue repair and regeneration. APN facilitated BMSCs migrating from the bone marrow into the circulation to regenerate bone by regulating stromal cell-derived factor (SDF)-1 in a mouse bone defect model. More importantly, we found that systemic APN infusion ameliorated diabetic mobilopathy of BMSCs, lowered glucose concentration, and promoted bone regeneration in diet-induced obesity mice. In vitro studies allowed us to identify Smad1/5/8 as a novel signaling mediator of APN receptor (AdipoR)-1 in BMSCs and osteoblasts. APN stimulation of MC3T3-E1 osteoblastic cells led to Smad1/5/8 phosphorylation and nuclear localization and increased SDF-1 mRNA expression. Although APN-mediated phosphorylation of Smad1/5/8 occurred independently from adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1, it correlated with the disassembly of protein kinase casein kinase 2 and AdipoR1 in immunoprecipitation experiments. Taken together, this study identified APN as a regulator of BMSCs migration in response to bone injury. Therefore, our findings suggest APN signaling could be a potential therapeutic target to improve bone regeneration and homeostasis, especially in obese and T2D patients.

Keywords: Adiponectin; Bone regeneration; Cell mobilization; Mesenchymal stem cells.

Figure 1. APN regulates the SDF-1/CXCR4 axis affecting BMSCs and osteoblasts

(A, B) qRT-PCR analysis of the relative mRNA expression levels of CXCR4 in C3H10T1/2 cells and WT-BMSCs, with or without APN (10 µg/ml) treatment (n = 3). (C) qRT-PCR analysis of the relative SDF-1 mRNA expression in MC3T3-E1 cells co-cultured with C3H10T1/2 cells with or without APN treatment (n =3). (D) WT femur-derived bone segments without bone marrow (control, devitalized bone on the left; and live bone on the right) were cultured with WT-BMSCs for 7 days. IHC with an anti-nestin antibody was performed to detect nestin⁺ BMSCs (cells in red). Red line represents the bone edge. Scale bars, 1 mm. (E) IHC staining for nestin+ cell detection in WT mice femur sections. Black arrow, nestin⁺ cell; BM, bone marrow; Scale bar, 50µm. Data are shown as mean ± SD. *p < 0.05; **p < 0 .01.

Figure 2. APN deficiency leads to increased adipocytes and nestin⁺ cells in bone marrow and increases SDF-1 level in bone marrow and peripheral blood

(A, B) qRT-PCR analysis of the relative (A) MMP9 and (B) CXCR4 mRNA expression levels in WT and APN−/− BMSCs (n = 3). (C) qRT-PCR analysis of the relative SDF-1 mRNA expression levels in calvarial primary osteoblasts of WT and APN^−/− mice. (D) H&E and immunohistochemical staining (AEC) of WT and APN^−/− bone marrow. Sections show increased number of adipocytes and nestin⁺ cells in APN^−/− mice bone marrow. Scale bars, HE 200µm, IHC 50µm. (E) FACS analysis on nestin⁺ cells in bone marrow and peripheral blood from WT and APN^−/− mice. (n=4~6 per group). (F) Immunohistochemistry of APN^−/− and WT mice femur sections. The SDF-1 staining was analyzed by immunohistochemical staining (AEC) kit. Scale bars, 50 µm. (G) The CCK-8 analysis of WT-BMSCs proliferation at different doses of APN (0, 1, 3, or 10 µg/ml). Data are shown as mean ± the SD. *p < 0.05; **p < 0 .01; ***p < 0.001; NS, no significant difference.

Figure 3. APN promotes BMSC migration and bone regeneration

(A) Transwell assays demonstrate APN promotes BMSC migration in vitro. (n = 3). (B) qRT-PCR analysis of the relative mRNA expression levels of MMP9 in WT-BMSCs with or without APN (10 µg/ml) treatment. (n = 3). (C) Serum SDF-1 levels in WT, APN^−/−, and WT+APN mice at day 0, 3, 7, and 14 post-surgery. (n= 4~5 mice per group per time point.) (D) Relative SDF-1 mRNA expression levels in bone marrow cells of WT, APN^−/−, and WT+APN mice at day 0, 3, 7, and 14 post-surgery. (n= 5 mice per group per time point) (E) FACS analysis on nestin⁺ cells in bone marrow or peripheral blood from WT and APN-treated WT mice at day 1 post-surgery. (n=4~6 per group) (F) H&E staining and (G) percentage of newly formed bone in calvarial bone defects from WT, APN^−/−, and WT+APN mice at 14 days post-surgery. (Scale bars, 500µm. CB=calvarial bone, NB= new bone, SS=silk scaffold) Error bars represent the SD. *p < 0.05; **p < 0.01; ***p < 0.001; NS, no significant difference.

Figure 4. APN lowers blood glucose level and improves osteogenesis in DIO mice

(A, B) Fasting blood glucose concentration of (A) DIO and (B) APN-treated DIO mice before and after surgery. (C, D) Relative mRNA expression levels of (C) SDF-1 and (D) CXCR4 in WT, DIO, and DIO+APN mice bone marrow cells 14 days post-surgery. (n = 4 mice per group). (E) H&E staining and (F) percentage of newly formed bone in calvarial defects from DIO and DIO+APN mice. (n=4 mice per group, scale bars, 500µm; CB=calvarial bone; NB=new bone; SS=silk scaffold). Data are shown as the mean ± SD, *, p < 0.05; NS, no significant difference.

Figure 5. APN activates the Smad signaling pathway

(A) Western blot analysis of pSmad1/5/8 in MC3T3-E1 cells after APN (0.3 µg/ml) incubation for 0, 5, 15, 30, 60, and 120 minutes. (B) pSmad1/5/8 level in MC3T3-E1 cells following 30 min of 0.5 µM LDN-193189 pretreatment followed by 30 min of APN (0.3 µg/ml) stimulation. (C) qRT-PCR analysis of the relative SDF-1 mRNA expression level in MC3T3-E1 cells and MC3T3-E1 cells + BMP inhibitor LDN193189 with or without APN (10 µg/ml) treatment (n =3) for 24 hours. (D) Western blot analysis of pSmad1/5/8 and APPL1 in MC3T3-E1 cells transfected with APPL1 or Scramble siRNA for 48 hours. (E) Immunofluorescence in MC3T3-E1 cells showing the pSmad1/5/8 nuclear translocation with or without 30 min of APN stimulation. Scale bars, 2µm. Data are shown as mean ± SD. *p < 0.05; **p < 0 .01; ***p < 0.001.

Figure 6. Smad1/5/8 as a novel intracellular partner of AdipoR1 signaling

(A, B) Co-immunoprecipitation experiments with MC3T3-E1 cells extracts to analyze interactions of AdipoR1, CK2β and Smad1/5/8. Cells were treated with or without APN (0.3 µg/ml) for 30 min. (A) Protein immunoprecipitated by the anti-Smad1/5/8 antibody was subjected to IB analysis to detect levels of Smad1/5/8, AdipoR1 and CK2β. (B) Protein immunoprecipitated by the anti-CK2β antibody was analyzed by IB analysis to detect levels of Smad1/5/8, AdipoR1, and CK2β. IB, immunoblotting; IP, immunoprecipitation; Input, whole protein lysis as positive control; mIgG: mouse IgG as negative control. (C, D) Effects of CK2 inhibitor TBB in Smad1/5/8 phosphorylation induced by APN or BMP-2. (C) MC3T3-E1 cells were stimulated with CK2 inhibitor TBB, APN, or pretreated with TBB followed by APN. (D) MC3T3-E1 cells were stimulated with TBB, BMP-2, or pretreated with TBB followed by BMP-2. pSmad1/5/8 level was determined by Western blot analysis. Data are shown as mean ± SD. *p < 0.05; **p < 0 .01; NS, no significant difference.

Figure 7. Schematic diagram of APN effects in the BM niche and APN-induction of SDF-1 expression

(A) APN regulates a SDF-1 chemotactic gradient for BMSCs migration from the bone marrow to the circulation by increasing SDF-1 concentration in circulation and decreasing SDF-1 expression in bone marrow after injury. Bone marrow-derived MSCs express CXCR4 receptor and follow the favorable gradient to repair the bone defect. MMP9 expression is increased and facilitates recruitment of MSC to the circulation. (B) Simplified representation of AdipoR1 signal transduction pathway in osteoblastic cells. AdipoR1, CK2, and Smad1/5/8 form an inactive complex in the absence of APN. APN treatment disassembles the complex and releases Smad1/5/8 for phosphorylation and nuclear translocation. Once at the nucleus, pSmad1/5/8 activates SDF-1 expression.