Molecular Regulation of Bone Formation
Our group is interested in several areas related to the molecular control of bone formation. We use a variety of genetic, genomic, and transgenic mouse approaches to study the signaling pathways involved and the mechanisms by which they are regulated.
Adiponectin Regulated Bone Metabolism
Adiponectin(APN) is an adipokine playing an important role in regulating energy homeostasis and insulin sensitivity. Our lab 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.
Figure 1. 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. (B) Simplified representation of AdipoR1 signal transduction pathway in osteoblastic cells.
We investigated that the inhibitory effect of adiponectin on osteoclasts was induced by APPL1-mediated down-regulation of Akt1 activity. In addition, overexpression of Akt1 successfully reversed adiponectin-induced inhibition in RANKL-stimulated osteoclast differentiation. Adiponectin is important in maintaining the balance of energy metabolism, inflammatory responses, and bone formation.
Figure 2. Retarded growth of bone explants in adiponectin knock-out mice. A, gross appearance (upper) and radiological analysis (lower) of femoral explants isolated from WT and adiponectin KO mice 4 weeks after explantation is shown. B, three-dimensional microcomputed tomography images are shown. C, hematoxylin and eosin staining sections showing femoral explants 4 weeks after explantation are shown. Tb.B., trabecular bone; Ct.B., cortical bone. D, TRACP staining of bone explants 4 weeks after explantation is shown. Arrows, TRACP+ osteoclasts. Photographs were taken at ×200 magnification using a Nikon Eclipse E600 microscope and Spot Advanced software (Diagnostic Instruments). E, the number of TRACP-positive multinucleated cells were counted, and data are presented as the mean ± S.E. a, p < 0.05 versus WT.
We then determined APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.
Figure 3. Changes in osteogenic and osteoclastic markers after gAPN or vehicle icv infusion in APN-KO and WT mice for 28 days. A: immunohistochemical analysis of special adipose tissue-rich sequence-binding protein 2 (SATB2), osterix (OSX), and osteocalcin (OCN; stained red) in distal femur trabecular bone sections. Scale bars, 20 μm. B: light micrographs of tartrate-resistant acid phosphatase (TRAP) staining (dark purple) from tibia bone sections. Scale bars, 20 μm. C: quantitative RT-PCR analysis of receptor activator of nuclear factor-κB ligand (RANKL) expression in bone marrow stromal cells (BMSC) normalized to β-actin mRNA expression.
The Effects of MiR-335-5p in Bone Formation and Regeneration
Our laboratory has identified and characterized microRNA-335-5P that can promote activation of the Wnt/β-catenin signaling pathway through downregulation of its inhibitor DKK1. Overexpression of MiR-335-5p in Osx-335 transgenic mice resulted in high bone mass formation.
Figure 4. Changes in miRNA expression profiles as indicated by miRNA microarray analysis. (A) Original images of the chips. The chip detects miRNA transcripts listed in Sanger miRBase Release 11.0. (B) Data analysis revealed groups of miRNAs that were down- or upregulated after ascorbic acid treatment, indicating that these miRNAs may be actively involved in the regulation of osteogenic differentiation. (C) miRNA target prediction using a combination of the following computational algorithms: TargetScan, Sloan-Kettering Cancer Center Human MicroRNA Targets Database, and miRBase Targets. (D) Expression levels of some miRNAs predicted to target DKK1 by the aforementioned computer algorithms also were found to be regulated by ascorbic acid treatment, as indicated by miRNA microarray analysis.
We demonstrated constitutive overexpression of miR-335-5p driven by an osterix promoter in the osteoblast lineage induces osteogenic differentiation and bone formation in mice and support the potential application of miR-335-5p–modified BMSCs in craniofacial bone regeneration.
Figure 5. Targeted overexpression of miR-335-5p in osteoblasts lineage resulted in enhanced bone formation in vivo. (A) Femoral tissue sections from 2-week-old and 4-week-old transgenic and WT mice were subjected to H&E staining as well as immunohistochemical staining with OCN and BSP antibodies. Bone histomorphometry was analyzed after H&E staining (n = 4 mice per age group). (B) The µCT analysis was conducted on the trabecular bone distal from the growth plate of femur and BV/TV, Tb.N, Tb.Th, and Tb.Sp were calculated (n = 4 mice each group).
Diabetes Associated Periodontal Disease
In our study, we established experimental periodontitis in male adiponectin knockout and diet-induced obesity mice, a model of obesity and type 2 diabetes, and aimed at evaluating the therapeutic potential of adiponectin. We found that systemic adiponectin infusion reduced alveolar bone loss, osteoclast activity and infiltration of inflammatory cells in both periodontitis mouse models. Furthermore, adiponectin treatment decreased the levels of pro-inflammatory cytokines in white adipose tissue of diet-induced obesity mice with experimental periodontitis.
Figure 6. Schematic Diagram of APN Therapeutic Potential in Treating T2DM-Associated Periodontitis. We established experimental periodontitis in APN−/− and DIO mice and treated them with systemic APN infusion. Our results support the notion that systemic administration with APN may constitute a therapeutic strategy to ameliorate T2DM-associated periodontitis due to dual roles of APN at inhibiting osteoclastic activity and hence bone resorption, and at attenuating inflammation.
Figure 7. APN inhibits bone resorption and inflammation in APN−/− mice induced with experimental periodontitis. (A) Alveolar bone loss was determined in palatal bone samples. The distance between the cementoenamel junction and the alveolar crest was measured at 6 sites in APN−/−, APN−/−+PD, APN−/−+PD+APN, as well as WT and WT+PD mice. (B) TRAP staining determined the number of osteoclasts. (C) H&E staining determined the number of inflammatory cells of palatal bone samples in APN−/−, APN−/−+PD, APN−/−+PD+APN, as well as WT and WT+PD mice (black arrows = inflammatory cells).
Figure 8. APN inhibits bone resorption and inflammation in DIO mice induced with experimental periodontitis. (A) Alveolar bone loss was determined in palatal bone samples. The distance between the cementoenamel junction and the alveolar crest was measured at 6 sites in DIO+PD and DIO+PD+APN mice. (B) TRAP staining determined the number of osteoclasts. (C) H&E staining determined the number of inflammatory cells of palatal bone samples in DIO+PD and DIO+PD+APN mice (black arrows = inflammatory cells). (D) qRT-PCR of TNF-α, IL-1, and IL-6 mRNA levels in WAT from DIO+PD and DIO+PD+APN mice, normalized to GAPDH.
AdipoRon (APR), a recently developed orally active small molecule, binds and activates both adiponectin receptors (AdipoR1, R2), with potent effects similar to those of adiponectin. We recently investigated its effect on alveolar bone regeneration after experimental periodontitis in mice.
Figure 9. MicroCT analysis demonstrated that 3 weeks of oral gavage after the establishment of experimental periodontitis, more bone regeneration were detected in APR-treated Diet-induced Obesity (DIO) mice than in vehicle-treated DIO mice.
Epigenetic Modulation in Inflammation and Bone Repair
Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. Our study exhibits that APN can ameliorate the periodontal bone loss and macrophage infiltration in DIO mice with periodontitis.
Figure 10. Adiponectin ameliorated the periodontal bone loss in vivo. A~D: H&E staining of thepalatal bone; E~H: Palatal bone samples stained with 1% methylene blue. The palatal bone samples exhibited no difference between control group and APN−/− group, APN−/− mice with periodontitis exhibited much severe bone loss and system APN treatment significantly decreased alveolar bone loss associated with experimental periodontitis in APN−/− mice. A, E: control group; B,F: APN−/−; C,G: APN−/− with periodontitis; G,H: APN−/− with periodontitis and APN treatment.
We found that JQ1, a novel synthetic bromodomain and extraterminal domain (BET) inhibitor, significantly suppressed lipopolysaccharide (LPS)-stimulated inflammatory cytokine transcription, as well as receptor activator of RANKL-induced osteoclast markers in vitro. JQ1 also inhibited TLR2/4 expression and NF-κB phosphorylation and nuclear translocation. ChIP-qPCR revealed that JQ1 neutralized BRD4 enrichment at several gene promoter regions, including NF-κB, TNF-α, c-Fos, and NFATc1. In a murine periodontitis model, systemic administration of JQ1 significantly inhibited inflammatory cytokine expression in diseased gingival tissues. Alveolar bone loss was alleviated in JQ1-treated mice because of reduced osteoclasts in periodontal tissues. These unprecedented results suggest the BET inhibitor JQ1 as a prospective new approach for treating periodontitis.
Figure 11. JQ1 inhibited the TLR2/4-NF-κB pathway in LPS-treated macrophages. (A) Relative expression of TLR2/4 mRNA induced by LPS (black) was compared with LPS+JQ1 (white) at 0, 0.5, 1, 2, 4, 12, 24, and 48 hr. + p < .05 between baseline and the indicated time; *p < .05 between LPS only and LPS+JQ1. (B) TLR2/4 protein levels were determined at 1/4, 1/2, 1, 2, 4, and 12 hr and balanced by β-actin. (C) Relative NF-κB phosphorylation was analyzed by Western blot at 1/4, 1/2, 1, 2, 4, and 12 hr. (D) Nuclear NF-κB level was compared between the LPS and LPS+JQ1 groups at 5, 15, 30, and 60 min. (E) Immunofluorescence analysis of NF-κB nuclear translocation. NF-κB protein was labeled with FITC (green), and nuclei were labeled with DAPI (blue).
We characterized the epigenetic regulation of PHF8 on SATB2 in BMSCs and the role of PHF8 in osteoblast differentiation and calvarial bone regeneration in mouse calvarial defects filled with BMSCs packed in SSs.
Figure 12. Micro CT and HE staining analysis. (A) Five weeks after surgery, micro CT results indicated the PHF8-modified BMSCs group showed the largest amounts of new bone formation. (B) HE staining demonstrated that PHF8-modified BMSCs group showed more new bone formation than other groups. Original magnification: top panel, 40×; middle panel, 200×; bottom panel, 400×. (C) Volume of newly formed bone detected by micro CT. (D) Percentage of new bone area in different treatment groups measured in HE staining sections. HE, hematoxylin and eosin; S, scaffold; *p<0.05.
Irisin Regulated Bone Metabolism
Irisin, a recently identified novel hormone-like myokine, is the cleaved and secreted portion of fibronectin-type III domain-containing 5 (FNDC5). Our study demonstrated that Irisin induces osteoblast differentiation, mineralization, as well as, inhibits RANKL-induced osteoclast differentiation. Intraperitoneal injections of recombinant irisin induced the appearance of irisin-positive osteoblasts in vivo.
Figure 13. Proposed model of irisin direct and indirect effects on bone metabolism. Exercise, cold exposure, administration with recombinant irisin, or overexpressing FNDC5 can potentially lead to increased levels of irisin in circulation according to the bibliography. In our study, 2 weeks of voluntary exercise increased expression of FNDC5/Irisin and osteogenic markers in bone (Figure 1), increased serum irisin levels in mice lacking adiponectin expression (Figure 2), and upregulated UCP1 expression by subcutaneous WAT while reducing body weight (Figure 3). Recombinant irisin induced osteoblast differentiation (Figure 4) and inhibited osteoclast differentiation (Figure 5) in bone cells lines. Systemic administration of irisin (Figure 6) or FNDC5 overexpression (Figure 7) could potentially regulate bone metabolism in vivo by direct mechanisms on bone cells or indirectly because browning of WAT (mediated by irisin or FNDC5) is anabolic for the skeleton.9–10,22 Recombinant irisin has also been shown to suppress sclerostin,35 which mediates bone response to mechanical unloading through inhibition of the Wnt/β-catenin signaling.
Bone development and mineralization were significantly delayed in Osx-Cre/ Irisinfl/fl mice.
Figure 14. A. Western blot analysis to evaluate beta-catenin protein expression in nuclear extracts of MC3T3-E1 cultures. Lamin B1 was used as a loading control. B. Irisin effects in NFATc1 mRNA expression (left) and NFATc1 protein expression (right) in RAW264.7 cells untreated (blank), treated with irisin or treated with RANKL.C. (a) Representative immunohistochemistry with anti-irisin antibody shows irisin positive osteoblasts (arrow) were found at the edge of growth plate in irisin-treated mice. (b) Circulatory levels of irisin in control and irisin treated mice. (c) µCT images of the distal metaphyseal regions of femurs of control and irisin-treated mice (n=5). (d) Trabecular BV/TV, Tb.Th and Co.Th were measured by µCT in femurs of control and irisin-treated mice. D. The mineralization of skull, hyoid, ribs, xiphoid and coccyx of the irisin cKO mice (arrow) are slower than wild-type on 6 and 10 weeks. E. Cortical BMD, BS/BV and Trabecular BV/TV were measured by µCT in femurs of WT group and cKO group on 6 and 10 weeks.