The Role of Galectin-3 in 1α,25(OH)2D3-Regulated Osteoclast Formation from White Leghorn Chickens In Vitro

Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a β-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)2D3. The results showed that 1α,25(OH)2D3 upregulated the expression of Gal-3 mRNA and receptor activator of nuclear-factor κB ligand (RANKL) expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (CtsK, CAII, MMP-9, and TRAP) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of Gal-3 by small-interfering RNA (siRNA) in BMSCs downregulated the expression of RANKL mRNA and attenuated the effects of 1α,25(OH)2D3 on osteoclastogenesis and bone resorption. Conversely, overexpression of Gal-3 in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of RANKL mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)2D3.

Expression of Wnt10b, β-catenin, Rankl and Runx2 in the Periodontal Tissues During Orthodontic Tooth Movement in Rats

Objectiveto investigate the expression of Wnt10b, Rankl, β-catenin and Runx2 mRNA in periodontal tissues during orthodontic tooth movement (OTM) in rats; Materials and methods36 healthy male SD rats were selected and divided into six groups randomly according to the time of the force applied (0 hour, 12 hours, 24 hours, 5 days, 7 days, 14 days), with 6 rats in each group. 0hour group served as the control group. Except for control group, an orthodontic force of 50g was applied to the upper-left first molar with a nickel-titanium tension spring. The expression of Wnt10b, Rankl, β-catenin and Runx2 during OTM were detected by Real-time PCR (RT-PCR). ResultsCompared with the control group, Wnt10b mRNA expression in the pressure side was inhibited at the initial stage, and then increased, reached peak at day 5. A strong expression of Rankl mRNA in the pressure side can be seen from 12 hours to 14 days. The expression level was consistently higher than control group, and it peaked at day 7. The initial expression of β-catenin and Runx2 mRNA in the periodontal tissues of the experimental groups were small in the tension side, but still higher than the control group. The expression increased gradually with the extension of time, and reached the peak at day 7 after force treatment. ConclusionWnt10b, β-catenin, Rankl and Runx2 are related to periodontal tissues remodeling during OTM in rats; Wnt10b/β-catenin signaling pathways participate in periodontal tissue remodeling during OTM.

Glucocorticoid Receptor Regulates TNFSF11 Transcription by Binding to Glucocorticoid Responsive Element in TNFSF11 Proximal Promoter Region

Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between OPG and RANKL level by decreasing the expression level of OPG and increasing level of RANKL. Here, were aimed to decipher whether glucocorticoid receptor directly influences RANKL promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased RANKL promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the RANKL promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on RANKL promoter activity was diminished by mutation at this site. Overexpression of NR3C1 upregulated RANKL mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of NR3C1 caused slight decrease in RANKL mRNA level, suggesting that NR3C1 directly accounts for RANKL transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal RANKL promoter region. Our study provides evidences that NR3C1 directly upregulates RANKL transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.

Local administration of high-dose diabetes medicine exendin-4 inhibits orthodontic tooth movement in mice

ABSTRACT Objectives To investigate the effects of exendin-4 on orthodontic tooth movement distance, root resorption, and expression levels of osteoclast-related cytokines in a mouse model. Materials and Methods A 10-g NiTi coil spring was placed between the anterior alveolar bone and upper left first molar of 8-week-old male C57BL/6 mice. Twenty microliters of exendin-4 solution (containing 0.2 μg, 4 μg, or 20 μg exendin-4) or phosphate-buffered saline (PBS) were injected on the buccal side of the upper left first molar at 2-day intervals (4 mice per group). Mice were sacrificed on day 12; silicone impressions were taken to record tooth movement distance. The left maxillae of the PBS and 20 μg exendin-4 groups were also excised for histological analysis and quantitative reverse transcription polymerase chain reaction analysis. Results Orthodontic tooth movement distance was smaller in the 20 μg exendin-4 group than in the PBS group (P < .01). Compared with the PBS group, the 20 μg exendin-4 group showed lower osteoclast number (P < .05), odontoclast number (P < .05), and root resorption surface percentage (P < .05). Relative to maxillae with PBS injections, maxillae with 20 μg exendin-4 injections had lower receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression (P < .05), TNF-α mRNA expression (P < .05), and RANKL/osteoprotegerin (OPG) ratio (P < .01). There were no differences in the expression of OPG mRNA. Conclusions Exendin-4 inhibits orthodontic tooth movement. Therefore, additional attention is needed for orthodontic patients who receive exendin-4 for diabetes treatment. GLP-1 receptor may be a treatment target for patients with severe root resorption.

Icariin Combined with Adipose-Derived Stem Cells Ameliorated Osteoporosis in Postmenopausal Rats

Purpose: Osteoporosis is a chronic metabolic disease, it has caused the high incidence of related fractures, which seriously affects the life quality of the patients, especially in early postmenopausal women. Methods: Rat osteoporosis model was induced by bilateral ovarian ablation. Sprague Dawley (SD) rats were randomly divided into five groups (n=9): sham group, model group, the icariin group, ADSCs group, icariin combined with ADSCs group. H&E staining was used to observe the pathological changes of femur. The expression of calcitonin receptor (CALCR) and cathepsin K (CTSK) were investigated by immunohistochemistry. The expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were measured by qRT-PCR, immunohistochemistry and western blot.Results: The results showed that icariin combined with ADSCs ameliorated osteoblast morphology and bone density. Icariin combined with ADSCs significantly attenuated the protein expression of CALCR and CTSK after ovariectomy. Moreover, the expressions of OPG mRNA and protein were increased (p < 0.05), while the expressions of RANKL mRNA and protein were decreased (p < 0.05) in osteoblasts.Conclusions: Icariin combined with ADSCs can effectively inhibit PMOP in rats. The mechanism may be achieved by up-regulated the ratio of OPG / RANKL.

Propranolol and parathyroid hormone synergistically improve bone volume fraction by suppressing resorption

Although the non-selective β-blocker, propranolol, improves bone density with PTH treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal and elevated Igf1 expression in osteoblasts. In vivo, intermittent PTH and propranolol had synergistic effects in the trabecular bone of the distal femur. The most striking finding was a complete suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol. Interestingly, propranolol suppressed differentiation of primary osteoclasts, suggesting a novel direct influence of propranolol on osteoclasts in vivo. Taken together, this work suggests combining propranolol with PTH could be beneficial to patients with extremely low bone density.

Bone Marrow Mesenchymal Stem Cells Decrease the Expression of RANKL in Collagen-Induced Arthritis Rats via Reducing the Levels of IL-22

Objective. To investigate the transplantation effect of bone marrow mesenchymal stem cells (MSCs) on the expression of interlukin-22 (IL-22) and RANKL in collagen-induced arthritis (CIA) rats. Methods. 32 CIA models were established. 16 CIA rats were transplanted with MSCs, and others were used as nontreatment CIA controls. The concentrations of IL-22 and RANKL in serum were detected by ELISA and those in synovial tissue of rats’ joints by immunohistochemical staining. In addition, the expression of RANKL mRNA was measured by RT-PCR in the fibroblast-like synoviocytes (FLSs), cultured with IL-22 in vitro, which were delivered from the joints of CIA rats treated with or without MSCs. Results. The transplantation of MSCs into CIA rats relieved the destruction of joints, measured by AI score, X-ray, and histopathology. MSCs also reduced the expression of IL-22 and RANKL in serum by ELISA (P<0.001) and similarly in FLSs by immunohistochemical staining. In vitro, IL-22 induced significantly the expression of RANKL mRNA in cultured FLSs in a dose-dependent manner, whereas this induction was significantly reduced in FLSs derived from CIA rats transplanted with MSCs (normal controls: F=79.33, P<0.001; CIA controls: F=712.72, P<0.001; and CIA-MSC rats: F=139.04, P<0.001). Conclusion. Our results suggest that the transplantation of MSCs can reduce the expression of RANKL in vivo by downregulating the levels of IL-22, thereby ameliorating the degree of RA bone destruction. This study provides a theoretical basis for a potential therapy of RA with MSCs, and IL-22 and RANKL may become two new targets to treat RA.

Moxibustion of Zusanli (ST36) and Shenshu (BL23) Alleviates Cartilage Degradation through RANKL/OPG Signaling in a Rabbit Model of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic and chronic autoimmune inflammatory disease characterized by severe synovial hyperplasia associated with progressive cartilage degradation. Due to the severe pain and disability caused by RA, effective therapeutic strategies that could simultaneously alleviate the inflammatory response and delay the disease progression are urgently needed. As a major alternative therapy in traditional Chinese medicine, moxibustion has been demonstrated that it could reduce the chronic inflammatory responses of a series of musculoskeletal diseases; however, whether moxibustion has protective effects on RA is still unclear. To investigate the effects of moxibustion on RA, moxibustion was applied to Zusanli (ST36) and Shenshu (BL23) acupoints in a RA rabbit model. HE staining of articular cartilage showed that moxibustion alleviated the cartilage degradation and bone destruction. In addition, moxibustion decreased the osteoclast number in RA rabbits. Real-time PCR revealed that moxibustion decreased the expression of RANKL mRNA while increased the expression of OPG mRNA, indicating a restoration of the balance between osteogenesis and osteoclastogenesis. Taken together, our results indicated that moxibustion had promising antiarthritic effects and could be an useful alternative method in RA therapeutics.