Enhanced Bone Resorption Research Articles

Screening for endogenous hypercortisolism in patients with osteoporosis and fractures: why, when and how.

Skeletal comorbidities are frequent and clinically relevant findings in Cushing's syndrome (CS) since an uncoupled suppressed bone formation and enhanced bone resorption leads to a marked skeletal damage with a rapid increase of fracture risk. Reduced Bone Mineral Density (BMD) has been consistently reported and osteopenia or osteoporosis are typical findings in patients with CS. Vertebral Fractures (VFs) are frequently reported and may occur even in patients with an only mild reduction of BMD. Since CS is diagnosed late due to often difficult biochemical and radiological confirmation as well as to signs and symptoms common in other much more frequent diseases an approach suggested for overcoming underdiagnosis is to screen patients with manifestations which may overlap with those of CS such as arterial hypertension, diabetes mellitus and osteoporosis. Our review will focus on the rationale and best practice for screening osteoporotic patients for CS.

Risedronate prevents exercise-induced hypercalcemia but not nausea or vomit in humans: a double blind randomized controlled trial

Reportedly, nausea or vomiting after heavy exercise was associated with post-exercise increased blood calcium (Ca) levels, which was correlated with enhanced bone resorption. We conducted a randomized, double-blind, placebo-controlled trial, enrolling 104 healthy trained male members of the Japan Ground Self-Defense Forces. Risedronate (17.5 mg) or placebo was prescribed 3 and 10 days before heavy exercise lasting approximately 5 h. The primary outcome was the severity of nausea or vomiting assessed by a visual analog scale during or post-exercise. The secondary outcomes included clinical symptoms associated with heat illness, post-exercise serum total Ca (tCa), whole blood ionized Ca (iCa), and serum tartrate-resistant acid phosphatase 5b (TRACP-5b) levels. The mean age was 26 years. The exercise resulted in a 4.5% weight loss. The two groups were comparable in terms of the symptoms, including primary outcome. However, post-exercise tCa and TRACP-5b were significantly lower with risedronate. A similar result was observed for iCa. The post-exercise urinary Ca/Magnesium ratio and the incidence of hypercalcemia (defined as tCa or iCa levels ≥ each median value of all subjects) were significantly lower with risedronate (78.0% vs. 58.5%). A stronger treatment effect of risedronate on blood Ca levels was observed in participants who lost substantial body weight. Post-exercise hypercalcemia is attributed to enhanced bone resorption but not the cause of nausea.

Activation of NF-κB signaling regulates ovariectomy-induced bone loss and weight gain

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss.

Downregulation of the metalloproteinases ADAM10 or ADAM17 promotes osteoclast differentiation

Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal.

Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

Effect of Swimming Free 50 Meters for Males on Calcium Levels and Some Enzymes of Thyroid and Parathyroid Gland

Study purpose. This study aimed to investigate the effect of swimming free 50 meters on calcium levels and activities of some key enzymes involved in calcium metabolism in males. Materials and Methods. Eleven healthy male adults were selected to participate in the study. They freely swam 50 meters daily for four weeks. A one-group pretest-posttest design was used to evaluate the effect of the swimming intervention on markers of calcium metabolism. Measurements were taken for participants before and after swimming. Fasting blood samples were collected before and after swimming to measure serum calcium levels as well as the activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 5'-nucleotidase, thyroid stimulating hormone (TSH) and parathormone (PTH) by standard enzymatic methods. Results. After four weeks of swimming free 50 meters daily, serum calcium levels showed a statistically significant increase compared to baseline (p < 0.05). The activities of ALP, AST, and ALT were significantly higher post-intervention (p < 0.05 for all enzymes). There were no significant changes in 5'-nucleotidase, TSH, and PTH activities. Conclusions. The results indicate that swimming free 50 meters daily for four weeks increases serum calcium levels, likely through enhanced bone resorption as suggested by the elevated activities of ALP, AST, and ALT. However, there were no significant effects on thyroid and parathyroid functions as assessed by TSH and PTH.

Amyloid-β neuropathology induces bone loss in male mice by suppressing bone formation and enhancing bone resorption

Alzheimer's disease (AD) and osteoporosis often coexist in the elderly. Although observational studies suggest an association between these two diseases, the pathophysiologic link between AD and skeletal health has been poorly defined. We examined the skeletal phenotype of 5xFAD mice, an AD model with accelerated neuron-specific amyloid-β accumulation causing full-blown AD phenotype by the age of 8 months. Micro-computed tomography indicated significantly lower trabecular and cortical bone parameters in 8-month-old male, but not female, 5xFAD mice than sex-matched wild-type littermates. Dynamic histomorphometry revealed reduced bone formation and increased bone resorption, and quantitative RT-PCR showed elevated skeletal RANKL gene expression in 5xFAD males. These mice also had diminished body fat percentage with unaltered lean mass, as determined by dual-energy X-ray absorptiometry (DXA), and elevated Ucp1 mRNA levels in brown adipose tissue, consistent with increased sympathetic tone, which may contribute to the osteopenia observed in 5xFAD males. Nevertheless, no significant changes could be detected between male 5xFAD and wild-type littermates regarding the serum and skeletal concentrations of norepinephrine. Thus, brain-specific amyloid-β pathology is associated with osteopenia and appears to affect both bone formation and bone resorption. Our findings shed new light on the pathophysiologic link between Alzheimer's disease and osteoporosis.

Nerve growth factor receptor limits inflammation to promote remodeling and repair of osteoarthritic joints

Osteoarthritis (OA) is a painful, incurable disease affecting over 500 million people. Recent clinical trials of the nerve growth factor (NGF) inhibitors in OA patients have suggested adverse effects of NGF inhibition on joint structure. Here we report that nerve growth factor receptor (NGFR) is upregulated in skeletal cells during OA and plays an essential role in the remodeling and repair of osteoarthritic joints. Specifically, NGFR is expressed in osteochondral cells but not in skeletal progenitor cells and induced by TNFα to attenuate NF-κB activation, maintaining proper BMP-SMAD1 signaling and suppressing RANKL expression in mice. NGFR deficiency hyper-activates NF-κB in murine osteoarthritic joints, which impairs bone formation and enhances bone resorption as exemplified by a reduction in subchondral bone and osteophytes. In human OA cartilage, NGFR is also negatively associated with NF-κB activation. Together, this study suggests a role of NGFR in limiting inflammation for repair of diseased skeletal tissues.

Nlrp3 inflammasome drives regulatory T cell depletion to accelerate periapical bone erosion.

Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated. RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through invitro experiments. Invivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis. Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis. Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion.

Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.

Rosavin regulates bone homeostasis through HDAC1-induced epigenetic regulation of EEF2

Bioactive constituents from Rhodiola rosea L. show a myriad of pharmacological effects on diverse diseases. Rosavin has been linked to reduced osteoclastogenesis, while its role in regulating osteogenesis remains unclear. The present study investigated whether and how Rosavin alleviates ovariectomy (OVX)-induced osteoporosis (OP) in mice. Rosavin had a therapeutic effect on OP in ovariectomized mice and inhibited osteoclast viability and promoted osteoblast viability. Integrated transcriptome sequencing, GO enrichment analysis, and PPI network construction revealed that the HDAC1/EEF2 axis was an important axis of gene action for Rosavin treatment. Mechanistically, HDAC1 suppressed EEF2 expression through histone deacetylation. Rescue experiments exhibited that HDAC1 promoted osteoclast viability, while EEF2 reversed the action of HDAC1 to restore bone homeostasis. In mice with OP, HDAC1 mitigated the effects of Rosavin, resulting in enhanced bone resorption and diminished bone formation, while EEF2 contributed to reduced bone resorption and elevated bone formation in mice. NF-κB and MAPK pathways were inhibited by Rosavin, enhanced by HDAC1, and blocked again by EEF2. To summarize, our results proved that Rosavin maintained bone homeostasis in OP via regulation of histone acetylation of EEF2, thus playing a key role as a therapeutic candidate for OP treatment.

Staphylococci planktonic and biofilm environments differentially affect osteoclast formation

IntroductionThe pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction.Objective and methodInvestigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM).ResultsPriming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM.ConclusionOur data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

Bevacizumab, a vascular endothelial growth factor inhibitor, promotes orthodontic tooth movement in an experimental rat model

ObjectiveThis study aimed to evaluate the impact of bevacizumab on orthodontic tooth movement (OTM) in Wistar rats. Materials and methodsThe OTM model was constructed by placing an orthodontic coil spring between the maxillary first molar and anterior tooth. Bevacizumab (Avastin®; 10 mg/kg twice per week) was started one week before the OTM and continued for 3 weeks. After 1 and 2 weeks, OTM distance and anterior tooth mobility were measured. Thereafter, the maxilla was dissected for micro-CT microarchitectural analysis, followed by histological analysis, and tartrate-resistant acid phosphatase (TRAP) staining. Moreover, the distributions of collagen fibers type-I and –III (Col-I and Col-III) were evaluated using Picro-Sirius red staining. ResultsOrthodontic force prompted bone resorption and formation on the pressure and tension sides, respectively. Bevacizumab therapy resulted in a 42% increase of OTM, particularly after 2 weeks. Furthermore, bevacizumab disturbed the morphometric structure at both pressure and tension sites. The histological evaluation indicated about 35–44% fewer osteoblasts in the bevacizumab group, especially at the tension side, whereas the proportion of TRAP-positive osteoclasts at the pressure side was 34–37% higher than the control. The mature Col-I was reduced at the tension site by 33%, whereas the Col-III/Col-I ratio was enhanced by 20–44% at pressure and tension sites, after 2 weeks, in the bevacizumab group. ConclusionAnti-vascular bevacizumab therapy accentuates OTM in rat model, possibly through the enhancement of bone resorption, at the pressure side, and the reduction of bone formation, at the tension side as well as dysregulation of collagen fibers distribution.

SHP-1 Protein Tyrosine Phosphatase Affects Early Postnatal Bone Development in Mice.

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.

AKT/GSK3β/NFATc1 and ROS signal axes are involved in AZD1390-mediated inhibitory effects on osteoclast and OVX-induced osteoporosis.

As a common disease in modern society, osteoporosis is caused by osteoclast hyperactivation, leading to enhanced bone resorption. Reactive oxygen species (ROS) metobolism and nuclear factor-activated T cells 1 (NFATc1) activities are two crucial processes during osteoclastogenesis. AZD1390 (AZD), an inhibitor of ataxia telangiectasia mutated (ATM), has been reported for antitumor effects, but little is known about how it plays a function in metabolic bone disease. Here, we found that AZD inhibitsthe generation, function and ROS-scavenging enzyme activity of mature osteoclast induced by RANKL stimulation, in a dose-dependent manner.Mechanistic analysis shows thatAZD affects osteoclast function and differentiation by inhibiting RANKL-induced NFATc1 signaling pathway and by increasing ROS-scavenging enzymes production in oxidative stress pathways. Preclinical studies have shown that AZD protects against bone loss in an ovariectomy (OVX) mouse model. Finally, our data confirm that AZD may prevent OVX-induced bone loss by abrogating RANKL-induced AKT/GSK3β/NFATc1 signaling pathways, and by promoting the expression of ROS scavenging enzymes in oxidative stress pathways.Collectively, our research shows that AZD has the potential as a new therapeutic agent for osteoporosis.

Osteoblast/osteocyte-derived interleukin-11 regulates osteogenesis and systemic adipogenesis

Exercise results in mechanical loading of the bone and stimulates energy expenditure in the adipose tissue. It is therefore likely that the bone secretes factors to communicate with adipose tissue in response to mechanical loading. Interleukin (IL)−11 is known to be expressed in the bone, it is upregulated by mechanical loading, enhances osteogenesis and suppresses adipogenesis. Here, we show that systemic IL-11 deletion (IL-11−/−) results in reduced bone mass, suppressed bone formation response to mechanical loading, enhanced expression of Wnt inhibitors, and suppressed Wnt signaling. At the same time, the enhancement of bone resorption by mechanical unloading was unaffected. Unexpectedly, IL-11−/− mice have increased systemic adiposity and glucose intolerance. Osteoblast/osteocyte-specific IL-11 deletion in osteocalcin-Cre;IL-11fl/fl mice have reduced serum IL-11 levels, blunted bone formation under mechanical loading, and increased systemic adiposity similar to IL-11−/− mice. Adipocyte-specific IL-11 deletion in adiponectin-Cre;IL-11fl/fl did not exhibit any abnormalities. We demonstrate that osteoblast/osteocyte-derived IL-11 controls both osteogenesis and systemic adiposity in response to mechanical loading, an important insight for our understanding of osteoporosis and metabolic syndromes.

Bone-sparing effects of tocilizumab in rheumatoid arthritis: a monocentric observational study.

Bone loss is a common feature in several autoimmune and chronic inflammatory diseases, such as rheumatoid arthritis (RA). Indeed, the high levels of pro-inflammatory cytokines seem to enhance bone resorption and to diminish bone formation, thus producing an uncoupling between osteoclast and osteoblast function and favoring the onset of juxtarticular as well as systemic osteoporosis. Many papers underline the high prevalence of osteoporosis in RA, as well as the negative correlation between interleukin 6 (IL-6) serum levels and bone mineral density (BMD). The aim of this study was to assess the effectiveness of one-year treatment with tocilizumab (TCZ), the first approved IL-6 receptor inhibitor, in reducing bone loss in RA. We enrolled 18 patients fulfilling 2010 ACR and EULAR criteria for RA from our arthritis outpatient clinic, assessing clinical and biochemical parameters during a 12-month period. The patients received TCZ 8 mg/kg i.v. every 4 weeks and underwent dual energy X-ray absorptiometry (DXA) for the measurement of bone mineral density (BMD) at baseline and at the end of study. Serum levels of C-reactive protein (CRP), erythrocytes sedimentation rate (ESR), IL-6, serum CrossLaps, osteoprotegerin (OPG), receptor activator of nuclear factor κβ ligand (RANK-L) and dickkopf-1 (DKK-1) were measured at baseline, at 6 months and 1 year. No significant difference in IL-6, RANK-L, DKK-1, OPG and serum CrossLaps levels between baseline, 6 months and 1 year were found. A significant increase of lumbar spine BMD was evidenced after 1 year of TCZ treatment. No difference in total body and femoral neck BMD was documented the end of the study. This study suggest the bone-sparing effect of TCZ in RA affected individuals.

Iron overload induced osteocytes apoptosis and led to bone loss in Hepcidin−/− mice through increasing sclerostin and RANKL/OPG

Numerous studies have demonstrated that iron overload is a risk factor of osteoporosis. However, there has been no systematic and in-depth studies on the effect of iron overload on osteocytes and its role in iron overload-induced bone loss. Therefore, to address this problem, we carried out in vitro and in vivo studies using MLO-Y4 osteocyte-like cells and Hepcidin-/- mice as iron overload models. (1) MLO-Y4 cells were treated with ferric ammonium citrate (FAC). Intracellular reactive oxygen species (ROS) levels and apoptosis of MLO-Y4 cells were determined by flow cytometry. Western blotting was performed to evaluate the effect of FAC on the expression of sclerostin and RANKL/OPG. (2) The conditioned medium of MLO-Y4 cells after treatment with FAC was collected and used to treat pre-osteoblasts and monocytes. Alkaline phosphatase (ALP) staining and alizarin red (AR) staining were used to evaluate osteogenic differentiation capacity, and tartrate-resistant acid phosphatase (TRAP) staining was performed to demonstrate osteoclast differentiation capacity. (3) In vivo studies included a wild type mouse, Hepcidin-/- mice, Hepcidin-/- mice+deferoxamine (DFO), and Hepcidin-/- mice+N-actyl-l-cysteine (NAC) group. Micro-CT was performed to evaluate the bone mineral density (BMD), bone volume, and bone micro-architecture of the mice, and three bending tests were used to assess bone strength. Histological analysis was used to detect alterations in bone turnover. TUNEL staining and scanning electron microscopy (SEM) were performed to evaluate the apoptosis and morphology of osteocytes. Immunohistochemical staining and Western blotting were used to determine alterations in sclerostin and RANKL/OPG expression levels in mice. (1) FAC increased intracellular ROS and apoptosis in MLO-Y4 cells, while FAC enhanced the expression of sclerostin and RANKL/OPG in MLO-Y4 cells. (2) Conditioned medium of MLO-Y4 cells inhibited the osteogenic capacity of osteoblasts while stimulating osteoclast differentiation. (3) By increasing oxidative stress, iron overload promotes the apoptosis of osteocytes and undermines the morphology of osteocytes in Hepcidin-/- mice, further increasing the expression levels of sclerostin and RANKL/OPG in osteocytes, which is considered to be the causative factor for reduced bone formation and enhanced bone resorption. DFO administration reduced iron levels, and NAC treatment decreased oxidative stress in Hepcidin-/- mice. Therefore, DFO or NAC treatment rescued the decrease in BMD, bone volume, and bone strength and attenuated the deterioration of bone architecture in Hepcidin-/- mice by attenuating the effect of iron overload on osteocytes. Osteocyte apoptosis due to increased ROS and resultant sclerostin and RANKL/OPG expression alteration was the main reason for bone loss in Hepcidin-/- mice. Osteocytes are the main targets for the prevention and treatment of iron overload-induced osteoporosis.

Gasdermin D maintains bone mass by rewiring the endo-lysosomal pathway of osteoclastic bone resorption.

Gasdermin D (GSDMD)-mediated pyroptosis induces immunogenic cell death and promotes inflammation. However, the functions of GSDMD in tissue homeostasis remain unclear. Here, we identify a physiological function of GSDMD in osteoclasts via a non-lytic p20-generated protein, which prevents bone loss to maintain bone homeostasis. In the late stage of RANKL-induced osteoclastogenesis, GSDMD underwent cleavage, which is dependent on RIPK1 and caspase-8/-3, to yield this p20 product. Gsdmd-deficient osteoclasts showed normal differentiation but enhanced bone resorption with excessive lysosomal activity. Mice with complete or myeloid-specific Gsdmd deletion exhibited increased trabecular bone loss and more severe aging/ovariectomy-induced osteoporosis. GSDMD p20 was preferentially localized to early endosomes and limited endo-lysosomal trafficking and maturation, relying on its oligomerization and control of phosphoinositide conversion by binding to phosphatidylinositol 3-phosphate (PI(3)P). We have thus identified an anti-osteoclastic function of GSDMD as a checkpoint for lysosomal maturation and secretion and linked this to bone homeostasis and endosome-lysosome biology.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, μCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.