Enhanced Bone Resorption Research Articles

Activation of dimeric glucocorticoid receptors in osteoclast progenitors potentiates RANKL induced mature osteoclast bone resorbing activity

Glucocorticoid (GC) therapy is the greatest risk factor for secondary osteoporosis. Pathogenic mechanisms involve an initial increase in bone resorption followed by decreased bone formation. To gain a better understanding of the resorptive activity of GCs, we have used mouse bone marrow macrophages (BMM) to determine if GCs can directly modulate RANKL stimulated osteoclast formation and/or activity. In agreement with previous studies, experiments performed in plastic wells showed that GCs (dexamethasone, hydrocortisone, and prednisolone) inhibited osteoclast number and size during the initial phases of RANKL stimulated osteoclastogenesis; however, in prolonged cultures, decreased apoptosis was observed and escape from GC induced inhibition occurred with an enhanced number of osteoclasts formed, many with an increased area. When BMM cells were seeded on bone slices, GCs robustly enhanced RANKL stimulated formation of resorption pits and release of CTX without affecting the number or size of osteoclasts formed and with no effect on apoptosis. Stimulation of pit formation was not associated with increased life span of osteoclasts or an effect on mRNA expression of several osteoclastic or osteoclastogenic genes. The potentiation of RANKL induced CTX release by dexamethasone was significantly less in BMM cells from mice with conditional knockout of the osteoclastic glucocorticoid receptor and completely absent in cells from GRdim mice, which carry a point mutation in one dimerizing interface of the GC receptor. These data suggest that: 1. Plastic is a poor medium to use for studying direct effects of GCs on osteoclasts 2. GCs can enhance bone resorption without decreasing apoptosis, and 3. A direct enhancement of RANKL mediated resorption is stimulated by the dimeric GC-receptor.

Efficiency and safety of SGLT2 inhibitors

Background According to statistical data of the International Diabetes Federation in 2015 the number of diabetes mellitus (DM) patients was 415 millions. By 2040, projected number of patients will be about 642 millions. Despite the large arsenal of antidiabetic drugs, hypoglycemic means search with maximum efficiency and minimal side effects such as weight gain and hypoglycemia level are continued. Purpose The study of the efficacy and safety of new classes of hypoglycemic drugs due to analysis of publications in medical databases. Results SGLT2 inhibitors are a group of oral hypoglycemic drugs which therapeutic target are kidneys. The mechanism of action is glucose and sodium symport blockage, which ensures reduction of glucose reabsorption and normalize glycaemia. Glucose toxicity reduction at the usage of these drugs is manifested in the recovery of the pancreas beta cells sensitivity, thus increasing insulin synthesis, and sensitization of peripheral tissues to insulin action. The usage of SGLT2 inhibitors not only leads to blood glucose lowering but also to shift the energy balance in a negative way that creates additional benefits in DM patients with obesity. Side effects of SGLT2 inhibitors therapy include urinary tract and genital infections. These drugs also have a diuretic effect, which can lead to the dehydration or hypotension. Some researches highlight the risks for bone fractures. SGLT2 inhibitors increase concentrations of phosphate in serum, probably via increased tubular reabsorption, which has the potential to adversely affect bone. Furthermore, SGLT2 inhibitors increase concentrations of parathyroid hormone, which enhance bone resorption. The question of carcinogenesis is still controversial. Studies have implied that the SGLT receptor may play a role in glucose uptake in many cancers. SGLT2 inhibitors increase the risk for urinary tract infections, which could increase the risk for bladder cancer. Human trials showed excess cases of female breast, male bladder cancer; but they did not reach statistical significance. New data for dapagliflozin suggested that these excess cancers may be due to early diagnosis rather than increased incidence. Canagliflozin rat studies suggested increased risk for renal, adrenal, and Leydig cell tumors, but phase 3 clinical trials do not support increased incidence of these cancers in humans. Evidence for the link between SGLT2 inhibitor use and cancer risk is not conclusive and remains a safety issue. Studies with larger sample size, longer exposure duration, and use of SGLT2 inhibitors in different ethnicities are needed. In May 2015, the FDA issued a warning that SGLT2 inhibitors may lead to ketoacidosis. The decline in circulating insulin levels results in a lowering of the antilipolytic activity of insulin and consequent stimulation of the production of free fatty acids, which are converted to ketone bodies by β-oxidation in the liver. Obviously, not all individuals who take SGLT2 inhibitors are at high-risk for euglycemic ketoacidosis. SGLT2 inhibitors have the potential to reduce cardiovascular risk in DM patients not only through effects on glycemic control, but also via beneficial effects on body weight, blood pressure, lipids. Large studies are underway to elucidate the effects of SGLT2 inhibitors on these outcomes, the results of which are eagerly awaited. Conclusions The resume of the data is ambiguous. On one side, this class of drugs offers new opportunities in the treatment of DM type 2: first of all, a new, insulin independent mechanism of action, absence of negative effect on body weight, favorable pharmacokinetic properties and overall good tolerance. On the other side, mechanism of action of SGLT2 inhibitors is directed to the clinical manifestations of diabetes and not to the cause. Listed, identified and investigated nephroprotective and cardioprotective effects, data on the effects on bone, lack of carcinogenic risk are required confirmation in the long-term. The results of new clinical and epidemiological studies should identify the benefit/risk ratio of a new class of antidiabetic agents in the long-term, and its place in the treatment of patients with DM type 2.

Lead as a Risk Factor for Osteoporosis in Post-menopausal Women.

Lead exposure is increasingly becoming an important risk factor for osteoporosis. In adults, approximately 80-90% of absorbed lead is stored in the bones. These bone lead deposits are released into the blood during periods of enhanced bone resorption like menopause, forming a potential endogenous source of lead exposure. Postmenopausal women are at a higher risk for bone lead release because of hormonal and age related changes in bone metabolism. Estrogen deficiency is associated with increase in osteoclasts number and activity leading to both the early and late form of osteoporosis. Hence, high blood lead level coupled with concomitant environmental exposure exposes women in this age group to lead related adverse outcomes like hypertension, reduced kidney and neurocognitive functions as well as increased risk of atherosclerosis and cardiovascular mortality. A few studies have also identified coexisting variates like ethnicity, occupation, residence, education, smoking, alcohol medications, water etc. as significant determinants of bone and blood lead in women, thus increasing the magnitude of postmenopausal bone changes. Hence, interventions focused on reducing the intensity of bone resorption during menopause will help decrease exposure to endogenous lead. This would play a significant role in decreasing the morbidity and mortality associated with menopause. Also, identification of modifiable factors that prevent bone lead release will reduce the risk of chronic lead exposure and improve the health outcomes of post-menopausal women.

Short-Term Hypoxia Accelerates Bone Loss in Ovariectomized Rats by Suppressing Osteoblastogenesis but Enhancing Osteoclastogenesis.

BackgroundAlthough it has been reported that hypoxic exposure can attenuate hypertension, heart disease, diabetes, and some other diseases, effects of hypoxia on osteoporosis are still unknown.Material/MethodsThe current study investigated whether short-term hypoxic exposure (in comparison with normoxic conditions) affects bone metabolism in normal or ovariectomized (OVX) adult female rats in an vivo study. Micro-computed tomography bone volume/structural analyses, histological examination, and serum bone turnover biochemical assays were used. In addition, the expressions of some associated major regulatory molecules were measured in osteoblastic cultures.ResultsWhile the 14-day hypoxic exposure did not change the bone-remodeling process in normal adult female rats, it decreased bone volume, osteoclast density, and serum bone formation marker (alkaline phosphatase) level, but increased osteoclast density and serum bone resorption marker (C-telopeptide of collagen) level in OVX rats. The bone marrow adipocyte number and serum fatty acid binding protein-4 level were increased in OVX-hypoxic rats compared with OVX-normoxic rats. Consistently, in human MG-63 osteoblastic cultures, the hypoxic condition suppressed protein expression of osteogenic transcriptional factors Runx2 and osterix, elevated protein expression of osteoclastogenic cytokine receptor activator of nuclear factor kappa-B ligand, but reduced that of osteoclastogenic inhibitor osteoprotegerin.ConclusionsOur results suggest that, although no change occurred in the bone-remodeling process in normal adult female rats after hypoxic exposure, under the estrogen-deficient osteoporotic condition, the hypoxic condition can alter the bone microenvironment so that it may further impair osteoblastic differentiation and enhance osteoclastic formation, and thus reduce bone formation, enhance bone resorption, and accelerate bone loss.

Osteoporosis: the current status of mesenchymal stem cell-based therapy.

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.

Bone Metastasis from Renal Cell Carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastasis that are often osteolytic and cause substantial morbidity, such as pain, pathologic fracture, spinal cord compression and hypercalcemia. The presence of bone metastasis in RCC is also associated with poor prognosis. Bone-targeted treatment using bisphosphonate and denosumab can reduce skeletal complications in RCC, but does not cure the disease or improve survival. Elucidating the molecular mechanisms of tumor-induced changes in the bone microenvironment is needed to develop effective treatment. The “vicious cycle” hypothesis has been used to describe how tumor cells interact with the bone microenvironment to drive bone destruction and tumor growth. Tumor cells secrete factors like parathyroid hormone-related peptide, transforming growth factor-β and vascular endothelial growth factor, which stimulate osteoblasts and increase the production of the receptor activator of nuclear factor κB ligand (RANKL). In turn, the overexpression of RANKL leads to increased osteoclast formation, activation and survival, thereby enhancing bone resorption. This review presents a general survey on bone metastasis in RCC by natural history, interaction among the immune system, bone and tumor, molecular mechanisms, bone turnover markers, therapies and healthcare burden.

Toll-Like Receptor 2 Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL.

Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. We, therefore, investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption. S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. Bone resorption induced by S. aureus was abolished by OPG. S. aureus increased the expression of osteoclastogenic cytokines and prostaglandins in the parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption. In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. Our study indicates the importance of using different in vitro approaches for studies of how S. aureus regulates osteoclastogenesis to obtain better understanding of the complex mechanisms of S. aureus induced bone destruction in vivo.

Hajdu-Cheney Syndrome, a Disease Associated with NOTCH2 Mutations.

Notch plays an important function in skeletal homeostasis, osteoblastogenesis, and osteoclastogenesis. Hajdu-Cheney syndrome (HCS) is a rare disease associated with mutations in NOTCH2 leading to the translation of a truncated NOTCH2 stable protein. As a consequence, a gain-of-NOTCH2 function is manifested. HCS is inherited as an autosomal dominant disease although sporadic cases exist. HCS is characterized by craniofacial developmental defects, including platybasia and wormian bones, osteoporosis with fractures, and acro-osteolysis. Subjects may suffer severe neurological complications, and HCS presents with cardiovascular defects and polycystic kidneys. An experimental mouse model harboring a HCSNotch2 mutation exhibits osteopenia secondary to enhanced bone resorption suggesting this as a possible mechanism for the skeletal disease. If the same mechanisms were operational in humans, anti-resorptive therapy could correct the bone loss, but not necessarily the acro-osteolysis. In conclusion, HCS is a devastating disease associated with a gain-of-NOTCH2 function resulting in diverse clinical manifestations.

Osteoclast function and bone-resorbing activity: An overview

Bone resorption is an important cellular function in skeletal development and remodeling of the adult skeleton. Most of the pathological bone disease conditions like osteoporosis reflect increased osteoclast activity; hence, increased bone resorption. Researchers have unraveled most of the intracellular mechanisms responsible for osteoclast bone-resorbing activity in last few decades. Therefore, understanding the fundamentals of osteoclast-induced bone resorption and the cytokines and other substances that modulate the osteoclast activity unequivocally provide insights into the development of drugs to ameliorate pathological bone diseases with enhanced bone resorption. The aim of this review is to examine the literature on osteoclast function and bone-resorbing activity.

Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin’s effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

Expression of osteoclastogenic factor transcripts in osteoblast-like UMR-106 cells after exposure to FGF-23 or FGF-23 combined with parathyroid hormone.

As a bone-derived hormone, fibroblast growth factor-23 (FGF-23) negatively regulates phosphate and calcium metabolism, while retaining growth-promoting action for mesenchymal cell differentiation. Elevated FGF-23 levels, together with hyperparathyroidism, are often observed in chronic kidney disease, which is associated with impaired bone mineralization and enhanced bone resorption. Although overexpression of osteoblast-derived osteoclastogenic cytokines might contribute to this metabolic bone disease, whether FGF-23 alone and FGF-23 plus parathyroid hormone (PTH) directly modulated the expression of osteoblast-derived osteoclastogenic genes remained elusive. Herein, we demonstrated the direct effects of FGF-23 on proliferation and mRNA expression of osteoblast-specific differentiation and osteoclastogenic markers in rat osteoblast-like UMR-106 cells in the presence or absence of PTH. FGF-23 was found to suppress UMR-106 cell proliferation, while increasing FGF-23 expression, the latter of which suggested the presence of positive feedback regulation of FGF-23 expression in osteoblasts. FGF-23 also upregulated the mRNA expression of osteoblast differentiation markers (e.g., Runx2, osterix, AJ18, Dlx5, alkaline phosphatase, and osteopontin), osteoclastogenic factors (e.g., MCSF, MCP-1, IL-6, and TNF-α), and bone resorption regulators (RANKL and osteoprotegerin). However, combined PTH and FGF-23 exposure did not alter the levels of FGF-23-induced transcripts, suggesting that both hormones had no additive effect. In conclusion, FGF-23 directly suppressed osteoblast proliferation, while inducing osteoclastogenic gene expression in UMR-106 cells, and the FGF-23-induced transcripts were not altered by long-standing PTH exposure.

Antiretroviral therapy induces a rapid increase in bone resorption that is positively associated with the magnitude of immune reconstitution in HIV infection.

Antiretroviral therapy (ART) paradoxically intensifies bone loss in the setting of HIV infection. Although the extent of bone loss varies, it occurs with virtually all ART types, suggesting a common pathway that may be aligned with HIV disease reversal. Using an animal model of immunodeficiency we recently demonstrated that immune activation associated with CD4 T-cell reconstitution induces increased production of the osteoclastogenic cytokines RANKL and TNFα by immune cells, driving enhanced bone resorption and loss in bone mineral density. To confirm these findings in humans, we investigated the early kinetics of CD4 T-cell recovery in relation to biomarkers of bone turnover and osteoclastogenic regulators in a prospective 24-week cohort study. Clinical data and blood sampling for HIV-RNA PCR, CD4 T-cell counts, bone turnover biomarkers, and osteoclastogenic regulators were obtained from ART-naïve HIV-infected study participants initiating standard doses of lopinavir/ritonavir plus tenofovir disoproxil fumarate/emtricitabine at baseline and at weeks 2, 8, 12, and 24 post ART. C-terminal telopeptide of collagen (CTx) a sensitive biomarker of bone resorption rose by 200% above baseline at week 12, remaining elevated through week 24 (α<0.01), and was associated with significant increases in plasma levels of osteoclastogenic regulators [receptor activator of NF-kB ligand (RANKL), tumor necrosis factor alpha, (TNFα)]. Importantly, the magnitude of CD4 T-cell recovery correlated significantly with CTx (rs = 0.387, α=0.01). Our data suggest that ART-induced bone loss occurs early, is aligned with early events of immune reconstitution, and these immune changes provide a unifying mechanism to explain in part the skeletal decline common to all ART.

Advanced glycation end products biphasically modulate bone resorption in osteoclast-like cells.

Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 μg/ml) or control BSA (100 μg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs.

Changes in Bone Metabolic Parameters Under Imatinib Treatment in Children with Chronic Myeloid Leukemia (CML)

Objectives: Tyrosine kinase inhibitors (TKIs) demonstrate outstanding efficacy for treatment of CML, however, also exert well-known off-target effects. Among these, inhibition of osteoclast's and osteoblast's growth and differentiation [Vandyke K et al Blood 2010] results in longitudinal growth impairment in children and adolescents [Shima H et al J Pediatr 2011; Millot F et al Eur J Cancer 2014; Tauer JT et al Blood 2014, ASH abstract 522] under longtime imatinib treatment. In children enrolled into trial CML-paed II on ongoing imatinib exposure we investigated blood levels of calcium, phosphate, 25-vitamin D3 [25-vitD3], 1.25-vitamin D3 [1.25-vitD3], parathyroid hormone [PTH], and metabolic markers of bone resorption (osteoclasts: C-terminal cross-linking telopeptide of collagen type I [CTX-I]) and bone formation (osteoblasts: bone alkaline phosphatase [BAP], osteocalcin [OC], procollagen type 1 N propeptide [PINP]) in order to get a deeper insight in alterations associated with bone remodeling in a longitudinal timely fashion.Methods: 119 patients [pts] (70 male/49 female, median age 12 years, range 1-18 yrs) enrolled into the trial received 260-340 mg imatinib/sqm daily within one week after diagnosis (Dx) when CML had been confirmed by either cytogenetics or PCR. 20 to 30 pts out of this cohort could successfully be monitored repeatedly over a median period of 3 years for all parameters planned to be analyzed by collecting blood and urine three-monthly under appropriate circumstances (immediate processing, freezing, light protection, dry ice shipment to study center). Assays were performed in a central laboratory as described previously [Jaeger BA et al Med Sci Monit 2012]. Age normalized reference values were used as standard deviation scores (SDS) for statistical analysis by REML fit linear mixed effect-model with software R version 3.1.1. [https://www.r-project.org/].Results: At Dx mean OC levels were found elevated to the upper normal range (+1.5 SDS) but increased further on up to +5 SDS within the next 3 mo. Also mean BAP-SDS and PINP-SDS increased during follow-up compared to measurements at diagnosis but remained within the normal range. At mo 3 increased CTX-I (+3 SDS) indicated enhanced bone resorption followed by elevated measurements in the upper normal range (+1.5 SDS) over the next 33 mo. 25-vitD3 levels were found stable in the very low but still normal -2 SDS range in the summer time (May - Oct.) but below that range (-2.5 SDS) in the winter time (Nov.-April). 1.25-vitD3 measurements showed a course with a sharp and steep increase from -2 SDS at Dx to + 2 SDS at mo 3 of treatment falling to normal levels (0 SDS) at mo 12 and thereafter. Serum calcium and phosphate remained in the normal range throughout the whole treatment period of 33 mo analyzed while all pts showed low urinary calcium (-6 SDS) at diagnosis and at mo 3 followed by levels in the lowest normal range (-2 SDS) from mo 9 onwards. Urinary phosphate levels also gradually increased from -6 SDS at Dx to normal values at mo 12. PTH ranged within +1SDS to +2 SDS during the total period of monitoring.Conclusion: Children with CML under imatinib treatment exhibit moderate secondary hyperparathyroidism in conjunction with pathologically low 25-vitD3 levels. OC as marker of bone formation is highly elevated possibly due to inhibition of osteoclasts' activity by imatinib via blockade of c-FMS. Bone resorption is also increased, however, to a lesser extent (+ 1.5 SDS) as demonstrated by elevated CTX-I in the upper normal range (+2 SDS). This dysbalanced osseous activity affects bone remodelling and in not-outgrown pts causes longitudinal growth impairment. Most importantly, a typical pattern of changes in most osseous metabolic markers is observed showing an initial increase at mo 3 (phase 1) followed by stable, but not normalized values (phase 2) from mo 9 onwards [see Fig. 1]. This is in line with the clinical observation that skeletal pain may be severe under TKI treatment but usually disappears by the end of the first year. Also in rodent models a typical biphasic spatio-temporal shifting of bone formation and resorption at the epiphyseal line could be demonstrated [Tauer JT et al PLoS One 2015]. These osseous changes mandate close monitoring in pediatric patients under long-term TKI exposure. [Display omitted] DisclosuresSuttorp:Novartis, BMS, Pfizer: Consultancy, Honoraria, Research Funding.

Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss.

HIV infection causes bone loss. We previously reported that immunosuppression-mediated B-cell production of receptor activator of NF-κB ligand (RANKL) coupled with decline in osteoprotegerin correlate with decreased bone mineral density (BMD) in untreated HIV-infection. Paradoxically, antiretroviral therapy (ART) worsens bone loss although existing data suggest that such loss is largely independent of specific antiretroviral regimen. This led us to hypothesize that skeletal deterioration following HIV disease reversal with ART may be related to T-cell repopulation and/or immune-reconstitution. Here we transplant T cells into immunocompromised mice to mimic ART-induced T-cell expansion. T-cell reconstitution elicits RANKL and TNFα production by B-cells and/or T-cells, accompanied by enhanced bone resorption and BMD loss. Reconstitution of TNFα- or RANKL-null T-cells and pharmacological TNFα antagonist all protect cortical, but not trabecular bone, revealing complex effects of T-cell-reconstitution on bone turnover. These findings suggest T-cell repopulation and/or immune-reconstitution as putative mechanisms for bone loss following ART initiation.

Fibrillin-1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche.

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1(Prx1-/-) mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1(Prx1-/-) mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1(Prx1-/-) mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1(Prx1-/-) limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches.

An Osteoclastic Transmembrane Protein-Tyrosine Phosphatase Enhances Osteoclast Activity in Part by Dephosphorylating EphA4 in Osteoclasts.

We have previously shown that PTP-oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP-oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP-oc trapping mutant but not by the wild-type (WT) PTP-oc and that transgenic overexpression of PTP-oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173-Vav3 level (Rac-GTP exchange factor [GEF]) and enhanced bone resorption activity of osteoclasts, reintroduction of WT-Epha4 in Epha4 null osteoclasts led to ∼50% reduction in the pY173-Vav3 level and ∼2-fold increase in bone resorption activity. Overexpression of Y779F-Epha4 mutant in WT osteoclasts markedly increased in pY173-Vav3 and reduced bone resorption activity, but overexpression of Y602F-Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4-mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up-regulate Rac-GTPase and down-regulate Rho-GTPase. PTP-oc overexpression in osteoclasts also increased the GTP-Rac level to 300% of controls, but decreased the GTP-Rho level to ∼50% of controls. PTP-oc overexpression or deficient Epha4 each also reduced pY87-Ephexin level, which is a Rho GEF. Thus, PTP-oc may differentially regulate Rac signaling versus Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac-GTPase versus Rho-GTPase through differential regulation of Vav3 versus Ephexin.

Indicators of the osteoclastogenesis system in men with different clinical types of ankylosing spondylitis

Objective: to evaluate the osteoclastogenesis regulatory system – osteoprotegerin/receptor activator of nuclear factor-κβ ligand (OPG/RANKL) system – in men with different clinical types of ankylosing spondylitis (AS). Subjects and methods. The osteoclastogenesis regulatory system was studied in 60 men, including 40 patients diagnosed with AS complying with the 1984 New York criteria and 20 men without AS. RANKL, a major stimulant of osteoclastogenesis, and OPG, a decoy receptor that binds RANKL and, accordingly, blocks osteoclastogenesis, were investigated. Results. It was shown that in the patients with AS, RANKL concentrations were normal and the content of OPG and OPG/RANKL ratio proved to be significantly higher than those in the men without AS. The highest OPG concentrations were recorded in patients with the axial form of this disease, its moderate activity and early X-ray stage. No relationship was found between the level of RANKL and the clinical characteristics of AS; however, there was a tendency to a slight increase in its concentration in patients with extraskeletal manifestations of AS, its high activity, high functional class, and late X-ray stage. Conclusion. The considerable increase in OPG levels and OPG/RANKL ratio was ascertained to be associated to the fact that 94% of the patients with late-stage AS characterized by the presence of numerous syndesmophytes. These changes must be compensatory in response to modestly increased RANKL level and enhanced bone resorption.

Immunohistochemical localization of tenascin-C in rat periodontal ligament with reference to alveolar bone remodeling.

We investigated the immunohistochemical localization of tenascin-C in 8-week-old rat periodontal ligaments. Tenascin-C immunoreactivity was detected in zones along with cementum and alveolar bone, and more intensely on the resorption surface of alveolar bone than on the formation surface. On the resorbing surface, tenascin-C immunoreactivity was detected in Howship's lacunae without osteoclasts, and in the interfibrous space of the periodontal ligaments, indicating that this molecule works as an adhesion molecule between bone and fibers of periodontal ligaments. Upon experimental tooth movement by inserting elastic bands (Waldo method), the physiological resorption surface of alveolar bone under compressive force showed enhanced bone resorption and enhanced tenascin-C immunoreactivity. However, on the physiological bone formation surface under compressive force, bone resorption was seen only occasionally, and no enhanced tenascin-C immunoreactivity was noted. In an experiment involving excessive occlusal loading to rat molars, transient bone resorption occurred within interradicular septa, but no enhanced tenascin-C immunoreactivity was seen in the periodontal ligaments. These results indicate that tenascin-C works effectively on the bone resorbing surface of physiological alveolar bone remodeling sites, rather than on the non-physiological transient bone resorbing surface. Fibronectin immunoreactivity was distributed evenly in the periodontal ligaments under experimental conditions. Co-localization of tenascin-C and fibronectin immunoreactivity was observed in many regions, but mutually exclusive expression patterns were also seen in some regions, indicating that fibronectin might not be directly involved in alveolar bone remodeling, but may play a role via interaction with tenascin-C.

High-throughput RNA sequencing reveals the effects of 2,2',4,4' -tetrabromodiphenyl ether on retina and bone development of zebrafish larvae.

Background2,2′,4,4′-Tetrabromodiphenyl ether (BDE47) is a prevalent environmental pollutant and has been demonstrated to be a serious toxicant in both humans and animals, but little is known about the molecular mechanism underlying its toxic effect on the early development of vertebrates. BDE47-treated zebrafish larvae were found to present the light-related locomotion reduction in our previous study, therefore, we aimed to use high throughput sequencing to investigate the possible reasons from a transcriptomic perspective.ResultsBy exposing zebrafish embryos/larvae to 5 μg/l and 500 μg/l BDE47, we measured the influence of BDE47 on the mRNA expression profiles of zebrafish larvae until 6 days post-fertilization, using Illumina HiSeq 2000 sequencing. Differential expression analysis and gene enrichment analysis respectively revealed that a great number of genes, and gene sets based on two popular terminologies, were affected by the treatment of 500 μg/l BDE47. Among them, BDE47 caused changes in the retinal metabolism and related biological processes involving eye morphogenesis and visual perception, as confirmed by disordered photoreceptor arrangement and thickened bipolar cell layer of larval retina from histological observations. Other altered genes such as pth1a and collaborative cathepsin family exhibited disrupted bone development, which was consistent with the body curvature phenotype. The transcriptome of larvae was not significantly affected by the treatment of 5 μg/l BDE47, as well as the treatment of DMSO vehicle.ConclusionsOur results suggest that high BDE47 concentrations disrupt the eye and bone development of zebrafish larvae based on both transcriptomic and morphological evidences. The abnormal visual perception may result in the alteration of dark adaption, which was probably responsible for the abnormal larval locomotion. Body curvature arose from enhanced bone resorption because of the intensive up-regulation of related genes. We also proposed the larval retina as a novel potential target tissue for BDE47 exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1194-5) contains supplementary material, which is available to authorized users.