In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• ...production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII.
Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control.
Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.
•Nitric oxide is necessary and sufficient for sclerostin protein loss in vitro.•eNOS and iNOS are most relevant to mechanical response in osteocyte-like cells.•Nitric oxide production occurs downstream of calcium influx and CaMKII activation.
•We examined SOST mRNA and sclerostin protein levels in differentiated SaOS2 cultures.•SOST expression was induced by 1,25D after 3h at the mRNA level.•Sclerostin protein levels increased by 12h in ...response to 1,25D.
The osteocyte expressed gene SOST encodes sclerostin, a potent negative regulator of bone formation and inducer of bone resorption. We have recently demonstrated that the human SOST gene is positively regulated in response to 1α,25-dihydroxyvitamin D3 (1,25D). Responsiveness may be mediated at least in part by a single classical DR3-type vitamin D response element (VDRE). In this study we examined the early responsiveness of the SOST gene to both 1,25D and to parathyroid hormone (PTH), a known repressor of SOST expression, in SaOS2 cells differentiated to an osteocyte-like stage of cell maturation. Both SOST mRNA levels and sclerostin protein levels increased in these cultures as early as 3h post-treatment with 1,25D and declined in response to PTH in the same timeframe. For 1,25D, the level of induced SOST appeared dependent on the extent, to which the degradative enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) was induced. Together with the observed rapid decrease in SOST/sclerostin levels in response to PTH, endocrine regulation of sclerostin production appears to be an important determinant of sclerostin levels. These findings confirm that the human SOST gene and sclerostin expression can be considered to be directly 1,25D-responsive in osteocytes.
The knowledge of bone biology has largely changed in the last few decades. Osteocytes are multifunctional bone cells that are surrounded by mineralized bone matrix and for decades it was considered ...that they might be relatively inactive cells. However, nowadays it is known that osteocytes are highly active cells which are indispensable for the normal function of the skeleton, playing main roles in several physiological processes, both within and beyond the bone microenvironment. This review highlights and updates the current state of knowledge of the osteocyte and focuses on its roles in bone remodeling and mineral homeostasis, and also reviews its recently discovered endocrine function. Osteocytes secrete sclerostin (a protein that works as a negative regulator of bone mass), and FGF-23, the most important osteocyte-secreted endocrine factor, since it is able to regulate the phosphate metabolism. Moreover, osteocytes can act as mechanosensory cells, transforming the mechanical strain into chemical signaling towards the effector cells (osteoblasts and osteoclasts). Therefore, the osteocyte plays an important role in bone biology, specifically in the remodeling process, since it regulates both the osteoblast and osteoclast activity.
Finally, the paper discusses the clinical application of the bone biology, updating the new therapies against bone-loss disorders.
BACKGROUND: Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility. The development of biomolecular world found Wnt/β-catenin ...signaling pathway may plays an important role in bone mass regulation. Osteoporosis in geriatric population remains one of global health problems and typically thought of as a disease impacting women, but recently increasing attention is being paid to osteoporosis in males. Osteoporosis in male accounts for higher morbidity and mortality compare to woman population. The association between sclerostin serum and risk for osteoporosis in male geriatric will be described as follows.
METHODS: This study is a case–control study with a total 54 samples of male geriatrics, divided into 27 non- osteoporosis subjects and 27 osteoporosis subjects (age ≥60 years old). Diagnosis of osteoporosis was defined according to the WHO criteria based on bone mineral density. All participants were scanned on a GE lunar prodigy bone densitometer. Sclerostin serum level was measured using enzyme-linked immunosorbent assay (ELISA).
RESULTS: The average age from total 54 samples in case group was 69.81 ± 6.5 years old and control 69.41 ± 5.97 years old. Cutoff value based on receiver operating characteristic curve for sclerostin serum level was 302.5 pg/mL where the sensitivity and specificity for developing osteoporosis in male geriatrics were 59.3% and 81.5%, respectively. Male geriatrics with sclerostin serum ≥302.5 pg/mL is 6.4 times more likely to developed osteoporosis than those with sclerostin serum <302.5 pg/mL (OR = 6.4; p = 0.0020; CI 95% = 1.856–22.068). Multivariate logistic regression analysis after controlling other variables such as bone mass index, age, smoking status, alcohol consumption, physical activity, sun exposure, and type II diabetes mellitus showed that high sclerostin level was an independent susceptibility factors for osteoporosis in male geriatrics population (p = 0.001).
CONCLUSIONS: This study showed that high circulating sclerostin serum (≥302.5 pg/mL) was risk factor for developing osteoporosis in male geriatrics.
•Combination therapy has additive effects on bone mass in dominant Osteogenesis Imperfecta.•TGFβ neutralizing antibody and increased Lrp5 signaling don’t increase bone strength in OI.•There is ...decreased bone formation after six weeks of treatment with TGFβ neutralizing antibody.
Enhancing LRP5 signaling and inhibiting TGFβ signaling have each been reported to increase bone mass and improve bone strength in wild-type mice. Monotherapy targeting LRP5 signaling, or TGFβ signaling, also improved bone properties in mouse models of Osteogenesis Imperfecta (OI). We investigated whether additive or synergistic increases in bone properties would be attained if enhanced LRP5 signaling was combined with TGFβ inhibition. We crossed an Lrp5 high bone mass (HBM) allele (Lrp5A214V) into the Col1a2G610C/+ mouse model of OI. At 6-weeks-of-age we began treating mice with an antibody that inhibits TGFβ1, β2, and β3 (mAb 1D11), or with an isotype-matched control antibody (mAb 13C4). At 12-weeks-old, we observed that combining enhanced LRP5 signaling with inhibited TGFβ signaling produced an additive effect on femoral and vertebral trabecular bone volumes, but not on cortical bone volumes. Although enhanced LRP5 signaling increased femur strength in a 3-point bending assay in Col1a2G610C/+ mice, femur strength did not improve further with TGFβ inhibition. Neither enhanced LRP5 signaling nor TGFβ inhibition, alone or in combination, improved femur 3-point-bending post-yield displacement in Col1a2G610C/+ mice. These pre-clinical studies indicate combination therapies that target LRP5 and TGFβ signaling should increase trabecular bone mass in patients with OI more than targeting either signaling pathway alone. Whether additive increases in trabecular bone mass will occur in, and clinically benefit, patients with OI needs to be determined.
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.
Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, ...brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin’s function in exercise and human health.
Display omitted
•The αV class of integrins are irisin receptors in osteocytes and adipose tissues•Irisin increases sclerostin expression in osteocytes to induce bone resorption•Genetic deletion of FNDC5 (or Irisin) completely blocks OVX-induced trabecular bone loss
Irisin, through its integrin receptor, promotes skeletal remodeling with implications for stemming bone loss.
PDF
