Elevated tissue expression of RANKL and RANK is associated with poorer survival rates in pancreatic cancer patients.

Pancreatic cancer is a highly lethal malignancy with a growing incidence reported worldwide. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, which is often diagnosed at advanced stages, making its prognosis and medical management difficult. The identification of histopathological biomarkers has allowed a more precise stratification of pancreatic cancer patients, providing additional information about their prognosis and offering possible therapeutic targets to be explored. The prognostic value of the receptor activator of nuclear factor-kappa B (RANK) and its ligand (RANKL) has been evaluated in breast and prostate tumors, however, their usefulness has not been assessed in pancreatic cancer. In the present work, we analyzed the relationship between the protein expression of RANK and RANKL with the survival of 41 patients with pancreatic cancer followed for 60 months, by performing immunohistochemistry and Kaplan-Meier curves. Our results demonstrate a direct association of high expression levels of RANK and RANKL with poorer survival of pancreatic cancer patients in comparison to those with low/medium and null expression levels of both markers. Further studies should be conducted to explore the carcinogenic role of both components in this type of tumor, as well as additional promising translational uses.

Effect of abatacept treatment on serum osteoclast-related biomarkers in patients with rheumatoid arthritis (RA): A multicenter RA ultrasound prospective cohort in Japan.

ABSTRACT: We evaluated the effect of abatacept treatment on osteoclast-related biomarkers and explored whether the biomarkers are associated with the therapeutic response in rheumatoid arthritis (RA) patients treated with abatacept.We enrolled 44 RA patients treated with abatacept from a multicenter prospective ultrasound cohort study of patients who received biologic or targeted synthetic disease-modifying antirheumatic drug therapy. We evaluated the disease activity score (DAS) 28-CRP (C-reactive protein), musculoskeletal ultrasound scores including the total grayscale score (GS)/power Doppler (PD) score and the serum concentrations of isoform 5b of tartrate-resistant acid phosphate (TRACP-5b) and soluble receptor activator of nuclear factor-κB ligand (sRANKL) at baseline and at 3 and 6 months of treatment. "PD responder" was defined as a patient whose Δtotal PD score over 6 months was greater than the median change of that.Abatacept significantly improved DAS28-CRP as well as the total GS/PD score over 6 months. Serum TRACP-5b was significantly elevated and serum sRANKL was significantly decreased at 6 months (P < .0001 and P < .01, respectively). At 6 months, serum sRANKL was significantly decreased in the patients who achieved DAS28-CRP remission and the PD responders but not in those who did not. However, serum TRACP-5b rose regardless of the therapeutic response.Among RA patients treated with abatacept, serum sRANKL decreased in the patients with a good therapeutic response, but serum TRACP-5b elevated paradoxically regardless of the therapeutic response.

Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity.

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

Associations of Circulating Osteoglycin With Bone Parameters and Metabolic Markers in Patients With Diabetes.

Objective: Circulating osteoglycin may facilitate the crosstalk between bone and pancreas to empower adaptation of bone mass to whole body energy balance. We aimed to examine whether osteoglycin is associated with bone and metabolic parameters and if osteoglycin levels differ between patients with type 1 and 2 diabetes (T1D and T2D). Design and methods: A cross-sectional study of 190 patients with diabetes mellitus and stable hemoglobin A1c (HbA1c) (97 T1D and 93 T2D) was conducted. S-osteoglycin was analyzed by ELISA. Unpaired t-tests were performed to test differences between patients with T1D and T2D and linear regression analyses were performed to investigate associations between osteoglycin, glycemic markers, bone turnover markers and characteristics. Results: S-osteoglycin did not differ between patients with T1D and T2D (p=0.10). No associations were present between osteoglycin and age, gender, microvascular complications, HbA1c, or plasma glucose in T1D or T2D patients (p>0.05 for all). S-osteoglycin was not associated with levels of bone turnover markers (C-terminal cross-linked telopeptide of type-I collagen (CTX), P-procollagen type 1 amino terminal propeptide (P1NP), P-osteocalcin (OC), P-sclerostin, S-osteoprotegerin (OPG) or S-Receptor Activator of Nuclear factor Kappa beta Ligand (RANKL)) in neither T1D or T2D patients (p>0.05 for all). Conclusion: Osteoglycin levels were similar in T1D and T2D patients. Osteoglycin did not correlate with glucose, HbA1c or any other biochemical marker of bone turnover. Thus, we did not find evidence supporting the existence of an osteoglycin-bone-pancreas axis. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT01870557.

Effect of vitamin K2 and vitamin D3 on bone mineral density in children with acute lymphoblastic leukemia: a prospective cohort study.

OBJECTIVES: Current treatment protocols in acute lymphoblastic leukemia (ALL) are associated with high remission rates and long life expectancy, enhancing the importance of quality of life and prevention of treatment-related complications in patient care. As osteoporosis is a frequent complication in patients under chemotherapy, we investigated the effect of vitamin K2 (100 mcg menaquinone-7) and vitamin D3 (10 mcg calcitriol) on bone metabolism in children with ALL. METHODS: Twenty-nine consecutive patients recently diagnosed with B precursor ALL (B-ALL) and treated according to the Turkish Acute Lymphoblastic Leukemia Berlin Frankfurt Münster 2000 protocol were randomly assigned into study and control groups. The study group (n=15, M/F: 8/7, age 1-14.5 years, mean 6.5 years) received vitamin K2 and vitamin D3 with their chemotherapy, while the control group (n=14, M/F 9/5, age 2-17 years, mean 7.1 years) received chemotherapy only. Serum calcium, phosphorus, magnesium, alkaline phosphatase, bone-specific alkaline phosphatase, uncarboxylated osteocalcin (ucOC), tartrate resistant acid phosphatase 5b, carboxyl terminal procollagen propeptide (PICP), osteoprotegerin (OPG), and receptor activator nuclear kappa B ligand (RANKL) were measured and bone mineral density (BMD) was determined at baseline and first, second, third and sixth months. RESULTS: The study group had higher serum OPG/RANKL ratio and lower ucOC levels compared to the control group at the first month; PICP levels were higher in the study group at second and third months. CONCLUSIONS: These results suggest an early beneficial effect of the combination of vitamin K2 and vitamin D3 on BMD in ALL patients especially during the period of intensive steroid therapy in the first months.

Influence of nano-hydroxyapatite coating implants on gene expression of osteogenic markers and micro-CT parameters. An in vivo study in diabetic rats.

This study evaluated the response of a nano-hydroxyapatite coating implant through gene expression analysis (runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteopontin (Opn), osteocalcin (Oc), receptor activator of nuclear factor-kappa B (Rank), receptor activator of nuclear factor-kappa B ligand (Rank-L), and osteoprotegerin (Opg)). Three-dimensional evaluation (percent bone volume (BV/TV); percent intersection surface (BIC); bone surface/volume ratio (BS/BV); and total porosity (To.Po)) were also analyzed. Mini implants were surgically placed in tibias of both healthy and diabetic rats. The animals were euthanized at 7 and 30 days. Evaluating all factors the relative expression of Rank showed that NANO surface presented the best results at 7 days (diabetic rats). Furthermore the levels of Runx2, Alp, Oc, and Opn suggest an increase in osteoblasts proliferation, especially in early stages of osseointegration. %BIC in healthy and diabetic (7 days) depicted statistically significant differences for NANO group. BV/TV, BS/BV and To.Po demonstrated higher values for NANO group in all evaluated time point and irrespective of systemic condition, but BS/BV 30 days (healthy rat) and 7 and 30 days (diabetic rat). Microtomographic and gene expression analyses have shown the benefits of nano-hydroxyapatite coated implants in promoting new bone formation in diabetic rats.

PKC-δ deficiency in B cells displays osteopenia accompanied with upregulation of RANKL expression and osteoclast-osteoblast uncoupling.

PKC-δ is an important molecule for B-cell proliferation and tolerance. B cells have long been recognized to play a part in osteoimmunology and pathological bone loss. However, the role of B cells with PKC-δ deficiency in bone homeostasis and the underlying mechanisms are unknown. We generated mice with PKC-δ deletion selectively in B cells by crossing PKC-δ-loxP mice with CD19-Cre mice. We studied their bone phenotype using micro-CT and histology. Next, immune organs were obtained and analyzed. Western blotting was used to determine the RANKL/OPG ratio in vitro in B-cell cultures, ELISA assay and immunohistochemistry were used to analyze in vivo RANKL/OPG balance in serum and bone sections respectively. Finally, we utilized osteoclastogenesis to study osteoclast function via hydroxyapatite resorption assay, and isolated primary calvaria osteoblasts to investigate osteoblast proliferation and differentiation. We also investigated osteoclast and osteoblast biology in co-culture with B-cell supernatants. We found that mice with PKC-δ deficiency in B cells displayed an osteopenia phenotype in the trabecular and cortical compartment of long bones. In addition, PKC-δ deletion resulted in changes of trabecular bone structure in association with activation of osteoclast bone resorption and decrease in osteoblast parameters. As expected, inactivation of PKC-δ in B cells resulted in changes in spleen B-cell number, function, and distribution. Consistently, the RANKL/OPG ratio was elevated remarkably in B-cell culture, in the serum and in bone specimens after loss of PKC-δ in B cells. Finally, in vitro analysis revealed that PKC-δ ablation suppressed osteoclast differentiation and function but co-culture with B-cell supernatant reversed the suppression effect, as well as impaired osteoblast proliferation and function, indicative of osteoclast-osteoblast uncoupling. In conclusion, PKC-δ plays an important role in the interplay between B cells in the immune system and bone cells in the pathogenesis of bone lytic diseases.

Chondroitin Sulfate Prevents STZ Induced Diabetic Osteoporosis through Decreasing Blood Glucose, AntiOxidative Stress, Anti-Inflammation and OPG/RANKL Expression Regulation.

Chondroitin sulfate (CS) has antioxidative, anti-inflammatory, anti-osteoarthritic and hypoglycemic effects. However, whether it has antidiabetic osteoporosis effects has not been reported. Therefore, in this study, we established a STZ-induced diabetic rat model; CS (500 mg kg-1 d-1) was orally administrated for eight weeks to study its preventive effects on diabetic osteoporosis. The results showed that eight weeks of CS treatment improved the symptoms of diabetes; the CS-treated group has increased body weight, decreased water or food intake, decreased blood glucose, increased bone-mineral density, repaired bone morphology and decreased femoral osteoclasts and tibia adipocytes numbers. After CS treatment, bone histomorphometric parameters returned to normal, the levels of serum inflammatory cytokines (IL-1ß, IL-6 and TNF-α) decreased significantly, serum SOD, GPX and CAT activities increased and MDA level increased. In the CS-treated group, the levels of serum ALP, CTX-1, TRACP 5b, osteocalcin and RANKL decreased and the serum RUNX 2 and OPG levels increased. Bone immunohistochemistry results showed that CS can effectively increase the expression of OPG and RUNX2 and reduce the expression of RANKL in diabetic rats. All of these indicate that CS could prevent STZ induced diabetic osteoporosis-mainly through decreasing blood glucose, antioxidative stress, anti-inflammation and regulation of OPG/RANKL expression. CS can therefore effectively prevent bone loss caused by diabetes.

Activation of FoxO1/SIRT1/RANKL/OPG pathway may underlie the therapeutic effects of resveratrol on aging-dependent male osteoporosis.

BACKGROUND: Age-dependent male osteoporosis remains a poorly studied medical problem despite its significance. It is estimated that at least 1 of 5 men will suffer from osteoporotic consequences. Given that multiple mechanisms are involved in the process of senescence, much attention has been given to compounds with polymodal actions. To challenge such a health problem, we tested here the therapeutic potential of resveratrol in male osteoporosis. We also studied the possible molecular mechanisms that may underlie resveratrol effects. METHODS: Thirty male Wistar albino rats were used in the present study. Rats were divided (10/group) into: control (3-4 months old weighing 150-200 g receiving vehicle), aged (18-20 months old, weighing 350-400 g and receiving vehicle), and resveratrol treated aged (18-20 months old, weighing 350-400 g and receiving resveratrol 20 mg/kg/day for 6 weeks) groups. Assessment of serum calcium, phosphate, bone specific alkaline phosphatase, inflammatory cytokines, oxidative stress markers, and rat femur gene expression of FoxO1, SIRT1, RANKL and OPG proteins was carried out. Histopathological assessment of different levels of rat femur was also performed. RESULTS: Age-dependent osteoporosis resulted in significant increase in serum levels of phosphate, bone specific alkaline phosphatase, hsCRP, IL-1ß, IL-6, TNF-α, MDA, NO, and RANKL gene expression. However, there was significant decrease in serum level of GSH, and gene expression of FoxO1, SIRT1 and OPG. Osteoporotic changes were seen in femur epiphysis, metaphysis and diaphysis. Resveratrol restored significantly age-dependent osteoporotic changes. CONCLUSION: We concluded that resveratrol can play an important role in the prevention of male osteoporosis. Resveratrol can counter the molecular changes in male osteoporosis via anti-inflammatory, anti-oxidant and gene modifying effects.

Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in osteocytes.

The present study aimed to investigate the effect of a compressive force (CF) on the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), and vascular endothelial growth factor (VEGF) in murine osteocytes (MLO-Y4) as well as animal study. After application of a CF for 1, 3, 6, and 12 h, gene and protein expression of RANKL, OPG, and VEGF in MLO-Y4 cells were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of a stretch-activated (S-A) channel was examined by gadolinium (Gd3+) administration. In an animal experiment, the expression of these factors in osteocytes of alveolar bone was examined after experimental tooth movement in rats. After CF application, significant increases in RANKL, VEGF and RANKL/OPG ratio were shown. The upregulated gene and protein levels of these factors were reduced by Gd3+ administration. After tooth movement, upregulated RANKL and VEGF were imunohistochemically shown in osteocytes of alveolar bone. These findings suggest that CF application on osteocytes elevates expression of osteoclast-inducing factor and angiogenesis factor in vivo and vitro.

MicroRNA-21 affects mechanical force-induced midpalatal suture remodelling.

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.

Extensive burn injury causes bone collagen network alteration and growth delay related to RANK-L immunoexpression change.

PURPOSE: Extensive burn injury mainly affects children, and hypermetabolic state can lead to growth delay. This study aimed to investigate bone histopathological and morphometric aspects, collagen fibers network and the immunoexpression of biological markers related to bone development in a young experimental model for extensive burn. MATERIALS AND METHODS: A total of 28 male Wistar rats were distributed into Control (C) and subjected to scald burn injury (SBI) groups. Sham or injured animals were euthanized 4 or 14 days post-lesion and proximal epiphyses of the femur were submitted to histological, morphometric (thickness epiphyseal plate), and RUNX-2 and receptor activator of nuclear factor kappa- ß ligand (RANK-L) immunoexpression methods. RESULTS: Histopathological femoral findings showed delayed appearance of the secondary ossification center in SBI, 14 days post-injury. Collagen fibers 4 days after injury were observed in articular cartilage as a pantographic network with a transversally oriented lozenge-shaped mesh, but this network was thinner in SBI. Fourteen days after the injury, the pantographic network of collagen presented square-shaped mesh in C, but this aspect was changed to a wider mesh in SBI. Morphometric analysis of epiphyseal plate revealed that the SBI group had less thickness than the respective controls (p<0.05). RUNX-2 showed no difference between groups, but RANK-L score was higher in all SBI groups. CONCLUSIONS: Extensive burn injury causes delayed bone growth and morphological changes. Alterations in collagen network and enhancement in immunoreactivity of RANK-L result in increased osteoclastogenesis.

CD21-/low B cells associate with joint damage in rheumatoid arthritis patients.

Depletion of B cells is beneficial in rheumatoid arthritis (RA) patients with autoantibodies to citrullinated proteins (ACPA) and/or the Fc portion of immunoglobulins (rheumatoid factor [RF]), suggesting a role for B cells in disease pathogenesis. To date, however, the identity of specifically pathogenic B cell subsets has not been discovered. One candidate population is identified by the low expression or absence of complement receptor 2 (CD21-/low B cells). In this study, we sought to determine whether there was any correlation between CD21-/low B cells and clinical outcome in patients with established RA, either ACPA+ /RF+ (n = 27) or ACPA- /RF- (n = 10). Healthy donors (n = 17) were included as controls. The proportion of the CD21-/low CD27- IgD- memory B cell subset in peripheral blood (PB) was significantly increased in ACPA+ /RF+ RA patients compared with healthy donors, and the frequency of this subset correlated with joint destruction (r = 0.57, P < 0.04). The levels of the chemokines CXCL-9 and CXCL-10 were higher in synovial fluid than in plasma, and PB CD21-/low cells expressed the receptor, CXCR3. In synovial fluid, most of the B cells were CD21-/low , approximately 40% of that population was CD27- IgD- , and a third of those expressed the pro-osteoclastogenic factor receptor activator of the nuclear factor κB ligand (RANKL). This subset also secreted RANKL, in addition to other factors such as IL-6, even in the absence of stimulation. We interpret these data as reason to propose the hypothesis that the CD27- IgD- subset of CD21-/low B cells may mediate joint destruction in patients with ACPA+ /RF+ RA.

Vibration enhances osteoclastogenesis by inducing RANKL expression via NF-κB signaling in osteocytes.

To shorten the duration of orthodontic treatment it is important not only to reduce risks such as dental caries, periodontal disease, and root resorption, but also to decrease pain and discomfort caused by a fixed appliance. Several studies have investigated the effect of vibration applied to fixed appliances to accelerate tooth movement. Although it was reported that vibration accelerates orthodontic tooth movement by enhancing alveolar bone resorption, the underlying cellular and molecular mechanisms remain unclear. In this study, we investigated the effects of vibration on osteoclastogenesis in vitro and in vivo. Vibration applied to pre-osteoclast cell line RAW264.7 cells enhanced cell proliferation but did not affect their differentiation into osteoclasts. Osteocytes in bone are known to be mechanosensitive and to act as receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL). Therefore, in the present study, vibration was applied to cells from the osteocyte-like cell line MLO-Y4. In MLO-Y4 cells, vibration induced phosphorylation of the inhibitor of NF-κB (IκB) and caused nuclear localization of NF-κB p65. Additionally, vibration increased RANKL mRNA expression, but did not affect osteoprotegerin (OPG) mRNA expression in MLO-Y4 cells, thus resulting in an increased RANKL/OPG ratio. Consistent with these findings, vibration applied during experimental tooth movement increased NF-κB activation and RANKL expression in osteocytes on the compression side of alveolar bone in vivo, whereas vibration had no such effects on the tension side. Furthermore, in a co-culture of MLO-Y4 cells and RAW264.7 cells, vibration applied to MLO-Y4 cells enhanced osteoclastogenesis. These findings suggest that vibration could accelerate orthodontic tooth movement by enhancing osteoclastogenesis through increasing the number of pre-osteoclasts and up-regulating RANKL expression in osteocytes on the compression side of alveolar bone via NF-κB activation.

TLR2 Promotes Vascular Smooth Muscle Cell Chondrogenic Differentiation and Consequent Calcification via the Concerted Actions of Osteoprotegerin Suppression and IL-6-Mediated RANKL Induction.

Objective- Vascular smooth muscle cell (VSMC) transformation to an osteochondrogenic phenotype is an initial step toward arterial calcification, which is highly correlated with cardiovascular disease-related morbidity and mortality. TLR2 (Toll-like receptor 2) plays a pathogenic role in the development of vascular diseases, but its regulation in calcification of arteries and VSMCs remains unclear. We postulate that TLR2-mediated inflammation participates in mediating atherosclerotic arterial calcification and VSMC calcification. Approach and Results- We found that ApoE-/- Tlr2-/- genotype in mice suppressed high-fat diet-induced atherosclerotic plaques formation during initiation but progressively lost its preventative capacity, compared with ApoE-/- mice. However, TLR2 deficiency prohibited high-fat diet-induced advanced atherosclerotic calcification, chondrogenic metaplasia, and OPG (osteoprotegerin) downregulation in the calcified lesions. Incubation of VSMCs in a calcifying medium revealed that TLR2 agonists significantly increased VSMC calcification and chondrogenic differentiation. Furthermore, TLR2 deficiency suppressed TLR2 agonist-mediated VSMC chondrogenic differentiation and consequent calcification, which were triggered via the concerted actions of IL (interleukin)-6-mediated RANKL (receptor activator of nuclear factor κB ligand) induction and OPG suppression. Inhibition experiments with pharmacological inhibitors demonstrated that IL-6-mediated RANKL induction is signaled by p38 and ERK1/2 (extracellular signal-regulated kinase 1/2) pathways, whereas the OPG is suppressed via NF-κB (nuclear factor κB) dependent signaling mediated by ERK1/2. Conclusions- We concluded that on ligand binding, TLR2 activates p38 and ERK1/2 signaling to selectively modulate the upregulation of IL-6-mediated RANKL and downregulation of OPG. These signaling pathways act in concert to induce chondrogenic transdifferentiation of VSMCs, which in turn leads to vascular calcification during the pathogenesis of atherosclerosis.

CCAAT/enhancer-binding protein beta (C/EBPß) is an important mediator of 1,25 dihydroxyvitamin D3 (1,25D3)-induced receptor activator of nuclear factor kappa-B ligand (RANKL) expression in osteoblasts.

Receptor activator of nuclear factor kappa B ligand (RANKL) expression in osteoblasts is regulated by 1,25-dihydroxyvitamin D3 (1,25D3). CCAAT/enhancer-binding protein beta (C/EBPß) has been proposed to function as a transcription factor and upregulate RANKL expression, but it is still uncertain how C/EBPß is involved in 1,25D3-induced RANKL expression of osteoblasts. 1,25D3 stimulation increased the expression of RANKL and C/EPBß genes in osteoblasts and enhanced phosphorylation and stability of these proteins. Moreover, induction of RANKL expression by 1,25D3 in osteoblasts was downregulated upon knockdown of C/EBPß. In contrast, C/EBPß overexpression directly upregulated RANKL promoter activity and exhibited a synergistic effect on 1,25D3-induced RANKL expression. In particular, 1,25D3 treatment of osteoblasts increased C/EBPß protein binding to the RANKL promoter. In conclusion, C/EBPß is required for induction of RANKL by 1,25D3. [BMB Reports 2019; 52(6): 391-396].

IL-17 Receptor Signaling in Osteoblasts/Osteocytes Mediates PTH-Induced Bone Loss and Enhances Osteocytic RANKL Production.

Primary hyperparathyroidism (PHPT) is a condition where elevated PTH levels lead to bone loss, in part through increased production of the osteoclastogenic factor IL-17A, by bone marrow (BM) T-helper 17 (Th17) cells, a subset of helper CD4+ T cells. In animals, PHPT is modeled by continuous PTH treatment (cPTH). In mice, an additional critical action of cPTH is the capacity to increase the production of RANKL by osteocytes. However, a definitive link between IL-17A and osteocytic expression of RANKL has not been made. Here we show that cPTH fails to induce cortical and trabecular bone loss and causes less intense bone resorption in conditional knock-out (IL-17RAΔOCY ) male and female mice lacking the expression of IL-17A receptor (IL-17RA) in dentin matrix protein 1 (DMP1)-8kb-Cre-expressing cells, which include osteocytes and some osteoblasts. Therefore, direct IL-17RA signaling in osteoblasts/osteocytes is required for cPTH to exert its bone catabolic effects. In addition, in vivo, silencing of IL-17RA signaling in in DMP1-8kb-expressing cells blunts the capacity of cPTH to stimulate osteocytic RANKL production, indicating that cPTH augments osteocytic RANKL expression indirectly, via an IL-17A/IL-17RA-mediated mechanism. Thus, osteocytic production of RANKL and T cell production of IL-17A are both critical for the bone catabolic activity of cPTH. © 2018 American Society for Bone and Mineral Research.

Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

Parathyroid hormone(1-34) and its analogs differentially modulate osteoblastic Rankl expression via PKA/SIK2/SIK3 and PP1/PP2A-CRTC3 signaling.

Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP(1-36)), is the second osteoanabolic therapy approved by the United States Food and Drug Administration after teriparatide (PTH(1-34)). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP(1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH(1-34) promotes a more robust cAMP response than PTHrP(1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (Sost). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of NF-κB ligand (Rankl), and the latter both in vitro and in vivo Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic Rankl expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced Rankl expression and the effects of PTH(1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH(1-34), PTHrP(1-36), and ABL on Rankl are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and -3, PP1/PP2A, and CRTC3.