WNT11, a new gene associated with early-onset osteoporosis, is required for osteoblastogenesis

Monogenic early-onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in WNT11 (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G > A p.Ala73Thr) and (NM_004626.2:c.865G > A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor LGR5, was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but WNT3A recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2–LGR5 complex via the non-canonical Wnt pathway.

The Role of Galectin-3 in 1α,25(OH)2D3-Regulated Osteoclast Formation from White Leghorn Chickens In Vitro

Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a β-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)2D3. The results showed that 1α,25(OH)2D3 upregulated the expression of Gal-3 mRNA and receptor activator of nuclear-factor κB ligand (RANKL) expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (CtsK, CAII, MMP-9, and TRAP) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of Gal-3 by small-interfering RNA (siRNA) in BMSCs downregulated the expression of RANKL mRNA and attenuated the effects of 1α,25(OH)2D3 on osteoclastogenesis and bone resorption. Conversely, overexpression of Gal-3 in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of RANKL mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)2D3.

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules, and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.

Hem1 promotes osteoclast fusion and bone resorption in mice

Hem1 (Hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity and for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass due to defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially due to cytoskeletal disorganization and decreased mitochondrial respiration. Transplantation of bone marrow hematopoietic stem and progenitor cells from wild-type into Hem1 KO mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

Regulation of Bone Cell Differentiation and Activation by Microbe-Associated Molecular Patterns

Gut microbiota has emerged as an important regulator of bone homeostasis. In particular, the modulation of innate immunity and bone homeostasis is mediated through the interaction between microbe-associated molecular patterns (MAMPs) and the host pattern recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domains. Pathogenic bacteria such as Porphyromonas gingivalis and Staphylococcus aureus tend to induce bone destruction and cause various inflammatory bone diseases including periodontal diseases, osteomyelitis, and septic arthritis. On the other hand, probiotic bacteria such as Lactobacillus and Bifidobacterium species can prevent bone loss. In addition, bacterial metabolites and various secretory molecules such as short chain fatty acids and cyclic nucleotides can also affect bone homeostasis. This review focuses on the regulation of osteoclast and osteoblast by MAMPs including cell wall components and secretory microbial molecules under in vitro and in vivo conditions. MAMPs could be used as potential molecular targets for treating bone-related diseases such as osteoporosis and periodontal diseases.

Bone remodeling stages under physiological conditions and glucocorticoid in excess: Focus on cellular and molecular mechanisms

This review provides a rationale for the cellular and molecular mechanisms of bone remodeling stages under physiological conditions and glucocorticoids (GCs) in excess. Remodeling is a synchronous process involving bone resorption and formation, proceeding through stages of: (1) resting bone, (2) activation, (3) bone resorption, (4) reversal, (5) formation, (6) termination. Bone remodeling is strictly controlled by local and systemic regulatory signaling molecules. This review presents current data on the interaction of osteoclasts, osteoblasts and osteocytes in bone remodeling and defines the role of osteoprogenitor cells located above the resorption area in the form of canopies and populating resorption cavities. The signaling pathways of proliferation, differentiation, viability, and cell death during remodeling are presented. The study of signaling pathways is critical to understanding bone remodeling under normal and pathological conditions. The main signaling pathways that control bone resorption and formation are RANK / RANKL / OPG; M-CSF – c-FMS; canonical and non-canonical signaling pathways Wnt; Notch; MARK; TGFβ / SMAD; ephrinB1/ephrinB2 – EphB4, TNFα – TNFβ, and Bim – Bax/Bak. Cytokines, growth factors, prostaglandins, parathyroid hormone, vitamin D, calcitonin, and estrogens also act as regulators of bone remodeling. The role of non-encoding microRNAs and long RNAs in the process of bone cell differentiation has been established. MicroRNAs affect many target genes, have both a repressive effect on bone formation and activate osteoblast differentiation in different ways. Excess of glucocorticoids negatively affects all stages of bone remodeling, disrupts molecular signaling, induces apoptosis of osteocytes and osteoblasts in different ways, and increases the life cycle of osteoclasts. Glucocorticoids disrupt the reversal stage, which is critical for the subsequent stages of remodeling. Negative effects of GCs on signaling molecules of the canonical Wingless (WNT)/β-catenin pathway and other signaling pathways impair osteoblastogenesis. Under the influence of excess glucocorticoids biosynthesis of biologically active growth factors is reduced, which leads to a decrease in the expression by osteoblasts of molecules that form the osteoid. Glucocorticoids stimulate the expression of mineralization inhibitor proteins, osteoid mineralization is delayed, which is accompanied by increased local matrix demineralization. Although many signaling pathways involved in bone resorption and formation have been discovered and described, the temporal and spatial mechanisms of their sequential turn-on and turn-off in cell proliferation and differentiation require additional research.

Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimulation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk

Bone and muscle have been recognized as endocrine organs since they produce and secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

Long noncoding RNA HCG18 inhibits the differentiation of human bone marrow-derived mesenchymal stem cells in osteoporosis by targeting miR-30a-5p/NOTCH1 axis

Background Recent studies have demonstrated that long non-coding RNAs (LncRNAs) can influence bone cell differentiation and formation. However, it is unclear whether lncRNA HCG18 is involved in osteoporosis (OP). This study was conducted to investigate the regulation of HCG18 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated and cultured from mouse pathological models and osteoporosis patients. RT-qPCR was performed to detect the expression of HCG18 and miR-30a-5p in BMSCs. The interaction between HCG18 and miR-30a-5p was analyzed by dual luciferase assay and RNA pulldown assay. The interaction between miR-30a-5p and NOTCH1 3′-UTR was analyzed by dual luciferase assay. RT-qPCR and Western blotting were used to detect the expression of osteogenic genes Runx2, OCN and OPN. Hindlimb-unloaded (HU) mice model was established, and HCG18 was knocked down on bone-formation surfaces by using lentivirus mediated shRNA transfection. Results The expression of HCG18 was increased in BMSCs of OP patients, while the expression of miR-30a-5p was decreased. The expression of HCG18 and miR-30a-5p was negatively correlated in BMSCs. During the differentiation from BMSCs to osteoblasts, the expression of HCG18 was significantly downregulated, and the expression of miR-30a-5p was significantly upregulated. Overexpression of HCG18 was able to reverse the osteogenic-induced upregulation of miR-30a-5p expression, and knockdown of HCG18 further promoted the expression of miR-30a-5p. In addition, miR-30a-5p partially abolished the effect of HCG18 on osteogenic differentiation of BMSCs. NOTCH1 was a target protein of miR-30a-5p, and upregulation of NOTCH1 reversed the effect of miR-30a-5p on osteogenic differentiation of BMSCs. Furthermore, this study found that lentivirus mediated HCG18 knockdown on the bone-formation surfaces of hindlimb-unloaded (HU) mice partially alleviated unloading-induced bone loss Conclusions HCG18 inhibited osteogenic differentiation of BMSCs induced by OP via the miR-30a-5p/NOTCH1 axis. HCG18 can be identified as a regulator of osteogenic differentiation of BMSCs.