Osteoblast-lineage calcium/calmodulin-dependent kinase 2 delta and gamma regulates bone mass and quality.

Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

Type H vessels: functions in bone development and diseases.

Type H vessels are specialized blood vessels found in the bone marrow that are closely associated with osteogenic activity. They are characterized by high expression of endomucin and CD31. Type H vessels form in the cancellous bone area during long bone development to provide adequate nutritional support for cells near the growth plate. They also influence the proliferation and differentiation of osteoprogenitors and osteoclasts in a paracrine manner, thereby creating a suitable microenvironment to facilitate new bone formation. Because of the close relationship between type H vessels and osteogenic activity, it has been found that type H vessels play a role in the physiological and pathological processes of bone diseases such as fracture healing, osteoporosis, osteoarthritis, osteonecrosis, and tumor bone metastasis. Moreover, experimental treatments targeting type H vessels can improve the outcomes of these diseases. Here, we reviewed the molecular mechanisms related to type H vessels and their associated osteogenic activities, which are helpful in further understanding the role of type H vessels in bone metabolism and will provide a theoretical basis and ideas for comprehending bone diseases from the vascular perspective.

Unloading-Induced Skeletal Interoception Alters Hypothalamic Signaling to Promote Bone Loss and Fat Metabolism.

Microgravity is the primary factor that affects human physiology in spaceflight, particularly bone loss and disturbances of the central nervous system. However, little is known about the cellular and molecular mechanisms of these effects. Here, it is reported that in mice hindlimb unloading stimulates expression of neuropeptide Y (NPY) and tyrosine hydroxylase (TH) in the hypothalamus, resulting in bone loss and altered fat metabolism. Enhanced expression of TH and NPY in the hypothalamus occurs downstream of a reduced prostaglandin E2 (PGE2)-mediated ascending interoceptive signaling of the skeletal interoception. Sympathetic antagonist propranolol or deletion of Adrb2 in osteocytes rescue bone loss in the unloading model. Moreover, depletion of TH+ sympathetic nerves or inhibition of norepinephrine release ameliorated bone resorption. Stereotactic inhibition of NPY expression in the hypothalamic neurons reduces the food intake with altered energy expenditure with a limited effect on bone, indicating hypothalamic neuroendocrine factor NPY in the facilitation of bone formation by sympathetic TH activity. These findings suggest that reduced PGE2-mediated interoceptive signaling in response to microgravity or unloading has impacts on the skeletal and central nervous systems that are reciprocally regulated.

Synthesis and anticancer properties of celastrol derivatives involved in the inhibition of VEGF.

In this study, fourteen celastrol derivatives (1-14) were synthesised by esterification of celastrol at the 29th position. The in vitro anticancer activity of them was determined by the MTT assay. All the synthetic compounds showed significant antiproliferative activity against six cancer cells, with IC50 of the submicron molar level. The most promising compound (2) blocked the cell cycle in the G2 phase and inhibited the expression of VEGF and MMP-9 in gastric cancer cell line MGC-803. In flow cytometry analysis, compound 2 induced cancer cell apoptosis in a dose-dependent manner. In the mouse tumour xenograft model, compound 2 showed significant anti-tumour activity in vivo at the dosage of 2.5 mg/kg and 1 mg/kg, with a higher inhibition rate than 5-FU (10 mg/kg). What's more, the anticancer mechanism involved in the inhibition of VEGF and the toxicity evaluation of compound 2 were also investigated.

Sympathetic Innervation Regulates Osteocyte-Mediated Cortical Bone Resorption during Lactation.

Bone undergoes constant remodeling by osteoclast bone resorption coupled with osteoblast bone formation at the bone surface. A third major cell type in the bone is osteocytes, which are embedded in the matrix, are well-connected to the lacunar network, and are believed to act as mechanical sensors. Here, it is reported that sympathetic innervation directly regulates lacunar osteocyte-mediated bone resorption inside cortical bone. It is found that sympathetic activity is elevated in different mouse models of bone loss, including lactation, ovariectomy, and glucocorticoid treatment. Further, during lactation elevated sympathetic outflow induces netrin-1 expression by osteocytes to further promote sympathetic nerve sprouting in the cortical bone endosteum in a feed-forward loop. Depletion of tyrosine hydroxylase-positive (TH+ ) sympathetic nerves ameliorated osteocyte-mediated perilacunar bone resorption in lactating mice. Moreover, norepinephrine activates ß-adrenergic receptor 2 (Adrb2) signaling to promote secretion of extracellular vesicles (EVs) containing bone-degrading enzymes for perilacunar bone resorption and inhibit osteoblast differentiation. Importantly, osteocyte-specific deletion of Adrb2 or treatment with a ß-blocker results in lower bone resorption in lactating mice. Together, these findings show that the sympathetic nervous system promotes osteocyte-driven bone loss during lactation, likely as an adaptive response to the increased energy and mineral demands of the nursing mother.

Metformin attenuates high-fat diet induced metabolic syndrome related osteoarthritis through inhibition of prostaglandins.

High-fat diet induces bone marrow inflammation and osteoarthritis phenotype in knee joint, but the underlying mechanisms is unknown. Here, we report that high-fat diet induces aberrant bone formation and cartilage degeneration in knee joint. Mechanistically, a high-fat diet increases the number of macrophages and the secretion of prostaglandins in subchondral bone, promoting bone formation. Metformin treatment is able to decrease the number of macrophages and also the level of prostaglandins induced by high-fat diet in subchondral bone. Importantly, metformin rescues aberrant bone formation and cartilage lesions by decreasing the number of osteoprogenitors and type-H vessels, which also results in relief of osteoarthritis pain response. Thus, we demonstrate prostaglandins secreted by macrophages may be a key reason for high-fat diet induced aberrant bone formation and metformin is a promising therapy for high-fat diet induced osteoarthritis.

Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones.

H3R is becoming an attractive and promising target for epilepsy treatment as well as the discovery of antiepileptics. In this work, a series of 6-aminoalkoxy-3,4-dihydroquinolin-2(1H)-ones was prepared to screen their H3R antagonistic activities and antiseizure effects. The majority of the target compounds displayed a potent H3R antagonistic activity. Among them, compounds 2a, 2c, 2h, and 4a showed submicromolar H3R antagonistic activity with an IC50 of 0.52, 0.47, 0.12, and 0.37 µM, respectively. The maximal electroshock seizure (MES) model screened out three compounds (2h, 4a, and 4b) with antiseizure activity. Meanwhile, the pentylenetetrazole (PTZ)-induced seizure test gave a result that no compound can resist the seizures induced by PTZ. Additionally, the anti-MES action of compound 4a fully vanished when it was administrated combined with an H3R agonist (RAMH). These results showed that the antiseizure role of compound 4a might be achieved by antagonizing the H3R receptor. The molecular docking of 2h, 4a, and PIT with the H3R protein predicted their possible binding patterns and gave a presentation that 2h, 4a, and PIT had a similar binding model with H3R.

Oxylipin-PPARγ-initiated adipocyte senescence propagates secondary senescence in the bone marrow.

The chronic use of glucocorticoids decreases bone mass and quality and increases bone-marrow adiposity, but the underlying mechanisms remain unclear. Here, we show that bone-marrow adipocyte (BMAd) lineage cells in adult mice undergo rapid cellular senescence upon glucocorticoid treatment. The senescent BMAds acquire a senescence-associated secretory phenotype, which spreads senescence in bone and bone marrow. Mechanistically, glucocorticoids increase the synthesis of oxylipins, such as 15d-PGJ2, for peroxisome proliferator-activated receptor gamma (PPARγ) activation. PPARγ stimulates the expression of key senescence genes and also promotes oxylipin synthesis in BMAds, forming a positive feedback loop. Transplanting senescent BMAds into the bone marrow of healthy mice is sufficient to induce the secondary spread of senescent cells and bone-loss phenotypes, whereas transplanting BMAds harboring a p16INK4a deletion did not show such effects. Thus, glucocorticoid treatment induces a lipid metabolic circuit that robustly triggers the senescence of BMAd lineage cells that, in turn, act as the mediators of glucocorticoid-induced bone deterioration.

An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion.

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

Endocrine role of bone in the regulation of energy metabolism.

Bone mainly functions as a supportive framework for the whole body and is the major regulator of calcium homeostasis and hematopoietic function. Recently, an increasing number of studies have characterized the significance of bone as an endocrine organ, suggesting that bone-derived factors regulate local bone metabolism and metabolic functions. In addition, these factors can regulate global energy homeostasis by altering insulin sensitivity, feeding behavior, and adipocyte commitment. These findings may provide a new pathological mechanism for related metabolic diseases or be used in the diagnosis, treatment, and prevention of metabolic diseases such as osteoporosis, obesity, and diabetes mellitus. In this review, we summarize the regulatory effect of bone and bone-derived factors on energy metabolism and discuss directions for future research.

Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.

Parathyroid hormone attenuates osteoarthritis pain by remodeling subchondral bone in mice.

Osteoarthritis, a highly prevalent degenerative joint disorder, is characterized by joint pain and disability. Available treatments fail to modify osteoarthritis progression and decrease joint pain effectively. Here, we show that intermittent parathyroid hormone (iPTH) attenuates osteoarthritis pain by inhibiting subchondral sensory innervation, subchondral bone deterioration, and articular cartilage degeneration in a destabilized medial meniscus (DMM) mouse model. We found that subchondral sensory innervation for osteoarthritis pain was significantly decreased in PTH-treated DMM mice compared with vehicle-treated DMM mice. In parallel, deterioration of subchondral bone microarchitecture in DMM mice was attenuated by iPTH treatment. Increased level of prostaglandin E2 in subchondral bone of DMM mice was reduced by iPTH treatment. Furthermore, uncoupled subchondral bone remodeling caused by increased transforming growth factor ß signaling was regulated by PTH-induced endocytosis of the PTH type 1 receptor-transforming growth factor ß type 2 receptor complex. Notably, iPTH improved subchondral bone microarchitecture and decreased level of prostaglandin E2 and sensory innervation of subchondral bone in DMM mice by acting specifically through PTH type 1 receptor in Nestin+ mesenchymal stromal cells. Thus, iPTH could be a potential disease-modifying therapy for osteoarthritis.

Over time the cartilage between our bones gets worn down, and this can lead to a painful joint disorder known as osteoarthritis. Nearly 40 million people with osteoarthritis in the United States experience chronic pain. Although there are a number of drugs available for these patients, none of them provide sustained pain relief, and some have substantial side effects when ingested over a long period of time. Bone tissue is continuously broken down into minerals, such as calcium, that can be reabsorbed into the blood. In 2013, a group of researchers found that the tissue in the layer of bone below the cartilage ­ known as the subchondral bone ­ is reabsorbed and replaced incorrectly in patients with osteoarthritis. This irregular 'remodeling' stimulates nerve cells to grow into the subchondral layer, leading to increased sensitivity in the joint. A protein called parathyroid hormone, or PTH for short, plays an important role in the loss and formation of bone. A drug containing PTH is used to treat patients with another bone condition called osteoporosis, and could potentially work as a treatment for osteoarthritis pain. To investigate this, Sun et al. ­ including some of the researchers involved in the 2013 study ­ tested this drug on a mouse model that mimics the symptoms of osteoarthritis. This revealed that PTH significantly decreases the number of nerves present in the subchondral bone, which caused the mice to experience less pain. PTH also slowed down the progression of osteoarthritis, by preventing the cartilage on the subchondral layer from deteriorating as quickly. Sun et al. found that the subchondral bones of treated mice also had a more stable structure and reduced levels of a protein involved in the reabsorption of bone. The results suggest that PTH is able to correct the errors in bone remodeling caused by osteoarthritis, and that this drug could potentially alleviate patients' chronic pain. This drug has already been approved by the US Food and Drug Administration (FDA), and could be used in clinical trials to see if PTH has the same beneficial effects on patients with osteoarthritis.

Mechanical stress determines the configuration of TGFß activation in articular cartilage.

Our incomplete understanding of osteoarthritis (OA) pathogenesis has significantly hindered the development of disease-modifying therapy. The functional relationship between subchondral bone (SB) and articular cartilage (AC) is unclear. Here, we found that the changes of SB architecture altered the distribution of mechanical stress on AC. Importantly, the latter is well aligned with the pattern of transforming growth factor beta (TGFß) activity in AC, which is essential in the regulation of AC homeostasis. Specifically, TGFß activity is concentrated in the areas of AC with high mechanical stress. A high level of TGFß disrupts the cartilage homeostasis and impairs the metabolic activity of chondrocytes. Mechanical stress stimulates talin-centered cytoskeletal reorganization and the consequent increase of cell contractile forces and cell stiffness of chondrocytes, which triggers αV integrin-mediated TGFß activation. Knockout of αV integrin in chondrocytes reversed the alteration of TGFß activation and subsequent metabolic abnormalities in AC and attenuated cartilage degeneration in an OA mouse model. Thus, SB structure determines the patterns of mechanical stress and the configuration of TGFß activation in AC, which subsequently regulates chondrocyte metabolism and AC homeostasis.

Regulation of bone marrow mesenchymal stem cell fate by long non-coding RNA.

Bone mesenchymal stem cells (BMSCs) are progenitor cells isolated from bone marrow, which keep potential to differentiate into several kinds of cells including osteoblasts and adipocytes. A dynamic mutual regulation exists between osteogenesis and adipogenesis processes. Long non-coding RNA (lncRNA) performs diverse functions in biological activities including regulation of BMSCs commitment. Evidence has shown that lncRNA regulates key signaling pathways including TGFß/BMP, Wnt and Notch pathways, and several transcription factors in BMSCs differention. Dysregulation of lncRNA in BMSCs leads to disruption of osteo-adipogenesis difffrentiation and results in impairment of bone homeostasis. In this review, we focus on the role of lncRNA in several critical signaling pathways that involved in regulation of osteo-adipogenesis of BMSC and prospects the potential clinical application of lncRNA.

Inhibition of cyclooxygenase-2 activity in subchondral bone modifies a subtype of osteoarthritis.

Osteoarthritis (OA) causes the destruction of joints. Its pathogenesis is still under investigation, and there is no effective disease-modifying therapy. Here, we report that elevated cyclooxygenase-2 (COX-2) expression in the osteocytes of subchondral bone causes both spontaneous OA and rheumatoid arthritis (RA). The knockout of COX-2 in osteocytes or treatment with a COX-2 inhibitor effectively rescues the structure of subchondral bone and attenuates cartilage degeneration in spontaneous OA (STR/Ort) mice and tumor necrosis factor-α transgenic RA mice. Thus, elevated COX-2 expression in subchondral bone induces both OA-associated and RA-associated joint cartilage degeneration. The inhibition of COX-2 expression can potentially modify joint destruction in patients with arthritis.