Inhibition of C5AR1 impairs osteoclast mobilization and prevents bone loss.

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.

SUCNR1 signaling in adipocytes controls energy metabolism by modulating circadian clock and leptin expression.

Adipose tissue modulates energy homeostasis by secreting leptin, but little is known about the factors governing leptin production. We show that succinate, long perceived as a mediator of immune response and lipolysis, controls leptin expression via its receptor SUCNR1. Adipocyte-specific deletion of Sucnr1 influences metabolic health according to nutritional status. Adipocyte Sucnr1 deficiency impairs leptin response to feeding, whereas oral succinate mimics nutrient-related leptin dynamics via SUCNR1. SUCNR1 activation controls leptin expression via the circadian clock in an AMPK/JNK-C/EBPα-dependent manner. Although the anti-lipolytic role of SUCNR1 prevails in obesity, its function as a regulator of leptin signaling contributes to the metabolically favorable phenotype in adipocyte-specific Sucnr1 knockout mice under standard dietary conditions. Obesity-associated hyperleptinemia in humans is linked to SUCNR1 overexpression in adipocytes, which emerges as the major predictor of adipose tissue leptin expression. Our study establishes the succinate/SUCNR1 axis as a metabolite-sensing pathway mediating nutrient-related leptin dynamics to control whole-body homeostasis.

HERC1 deficiency causes osteopenia through transcriptional program dysregulation during bone remodeling.

Bone remodeling is a continuous process between bone-forming osteoblasts and bone-resorbing osteoclasts, with any imbalance resulting in metabolic bone disease, including osteopenia. The HERC1 gene encodes an E3 ubiquitin ligase that affects cellular processes by regulating the ubiquitination of target proteins, such as C-RAF. Of interest, an association exists between biallelic pathogenic sequence variants in the HERC1 gene and the neurodevelopmental disorder MDFPMR syndrome (macrocephaly, dysmorphic facies, and psychomotor retardation). Most pathogenic variants cause loss of HERC1 function, and the affected individuals present with features related to altered bone homeostasis. Herc1-knockout mice offer an excellent model in which to study the role of HERC1 in bone remodeling and to understand its role in disease. In this study, we show that HERC1 regulates osteoblastogenesis and osteoclastogenesis, proving that its depletion increases gene expression of osteoblastic makers during the osteogenic differentiation of mesenchymal stem cells. During this process, HERC1 deficiency increases the levels of C-RAF and of phosphorylated ERK and p38. The Herc1-knockout adult mice developed imbalanced bone homeostasis that presented as osteopenia in both sexes of the adult mice. By contrast, only young female knockout mice had osteopenia and increased number of osteoclasts, with the changes associated with reductions in testosterone and dihydrotestosterone levels. Finally, osteocytes isolated from knockout mice showed a higher expression of osteocytic genes and an increase in the Rankl/Opg ratio, indicating a relevant cell-autonomous role of HERC1 when regulating the transcriptional program of bone formation. Overall, these findings present HERC1 as a modulator of bone homeostasis and highlight potential therapeutic targets for individuals affected by pathological HERC1 variants.

Nanoclay-reinforced HA/alginate scaffolds as cell carriers and SDF-1 delivery-platforms for bone tissue engineering.

Bone tissue engineering has come on the scene to overcome the difficulties of the current treatment strategies. By combining biomaterials, active agents and growth factors, cells and nanomaterials, tissue engineering makes it possible to create new structures that enhance bone regeneration. Herein, hyaluronic acid and alginate were used to create biologically active hydrogels, and montmorillonite nanoclay was used to reinforce and stabilize them. The developed scaffolds were found to be biocompatible and osteogenic with mMSCs in vitro, especially those reinforced with the nanoclay, and allowed mineralization even in the absence of differentiation media. Moreover, an in vivo investigation was performed to establish the potential of the hydrogels to mend bone and act as cell-carriers and delivery platforms for SDF-1. Scaffolds embedded with SDF-1 exhibited the highest percentages of bone regeneration as well as of angiogenesis, which confirms the suitability of the scaffolds for bone. Although there are a number of obstacles to triumph over, these bioengineered structures showed potential as future bone regeneration treatments.

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression.

Osteocytes, the most abundant bone cell type, are derived from osteoblasts through a process in which they are embedded in an osteoid. We previously showed that nutrient restriction promotes the osteocyte transcriptional program and is associated with increased mitochondrial biogenesis. Here, we show that increased mitochondrial biogenesis increase reactive oxygen species (ROS) levels and consequently, NRF2 activity during osteocytogenesis. NRF2 activity promotes osteocyte-specific expression of Dmp1, Mepe, and Sost in IDG-SW3 cells, primary osteocytes, and osteoblasts, and in murine models with Nfe2l2 deficiency in osteocytes or osteoblasts. Moreover, ablation of Nfe2l2 in osteocytes or osteoblasts generates osteopenia and increases osteoclast numbers with marked sexual dimorphism. Finally, treatment with dimethyl fumarate prevented the deleterious effects of ovariectomy in trabecular bone masses of mice and restored osteocytic gene expression. Altogether, we uncovered the role of NRF2 activity in osteocytes during the regulation of osteocyte gene expression and maintenance of bone homeostasis.

ACVR1 Function in Health and Disease.

Activin A receptor type I (ACVR1) encodes for a bone morphogenetic protein type I receptor of the TGFß receptor superfamily. It is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterised by progressive heterotopic ossification. ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG), a malignant childhood brainstem glioma, and its function is being studied in other cancer types. Here, we review ACVR1 receptor function and signalling in physiological and pathological processes and its regulation according to cell type and mutational status. Learning from different functions and alterations linked to ACVR1 is a key step in the development of interdisciplinary research towards the identification of novel treatments for these pathologies.

Interplay between BMPs and Reactive Oxygen Species in Cell Signaling and Pathology.

The integration of cell extrinsic and intrinsic signals is required to maintain appropriate cell physiology and homeostasis. Bone morphogenetic proteins (BMPs) are cytokines that belong to the transforming growth factor-ß (TGF-ß) superfamily, which play a key role in embryogenesis, organogenesis and regulation of whole-body homeostasis. BMPs interact with membrane receptors that transduce information to the nucleus through SMAD-dependent and independent pathways, including PI3K-AKT and MAPKs. Reactive oxygen species (ROS) are intracellular molecules derived from the partial reduction of oxygen. ROS are highly reactive and govern cellular processes by their capacity to regulate signaling pathways (e.g., NF-κB, MAPKs, KEAP1-NRF2 and PI3K-AKT). Emerging evidence indicates that BMPs and ROS interplay in a number of ways. BMPs stimulate ROS production by inducing NOX expression, while ROS regulate the expression of several BMPs. Moreover, BMPs and ROS influence common signaling pathways, including PI3K/AKT and MAPK. Additionally, dysregulation of BMPs and ROS occurs in several pathologies, including vascular and musculoskeletal diseases, obesity, diabetes and kidney injury. Here, we review the current knowledge on the integration between BMP and ROS signals and its potential applications in the development of new therapeutic strategies.

Glucose Restriction Promotes Osteocyte Specification by Activating a PGC-1α-Dependent Transcriptional Program.

Osteocytes, the most abundant of bone cells, differentiate while they remain buried within the bone matrix. This encasement limits their access to nutrients and likely affects their differentiation, a process that remains poorly defined. Here, we show that restriction in glucose supply promotes the osteocyte transcriptional program while also being associated with increased mitochondrial DNA levels. Glucose deprivation triggered the activation of the AMPK/PGC-1 pathway. AMPK and SIRT1 activators or PGC-1α overexpression are sufficient to enhance osteocyte gene expression in IDG-SW3 cells, murine primary osteoblasts, osteocytes, and organotypic/ex vivo bone cultures. Conversely, osteoblasts and osteocytes deficient in Ppargc1a and b were refractory to the effects of glucose restriction. Finally, conditional ablation of both genes in osteoblasts and osteocytes generate osteopenia and reduce osteocytic gene expression in mice. Altogether, we uncovered a role for PGC-1 in the regulation of osteocyte gene expression.

Enzymatic crosslinked gelatin 3D scaffolds for bone tissue engineering.

Bone tissue engineering is an emerging medical field that has been developed in recent years to address pathologies with limited ability of bones to regenerate. Here we report the fabrication and characterization of microbial transglutaminase crosslinked gelatin-based scaffolds designed for serving as both cell substrate and growth factor release system. In particular, morphological, biomechanical and biological features have been analyzed. The enzyme ratio applied during the fabrication of the scaffolds affects the swelling capacity and the mechanical properties of the final structure. The developed systems are not cytotoxic according to the biocompatibility tests. The biological performance of selected formulations was studied using L-929 fibroblasts, D1 MSC and MG63 osteoblasts. Moreover, scaffolds allowed efficient osteogenic differentiation and signaling of MSCs. MSC cultured on the scaffolds not only presented lower proliferative and stemness profile, but also increased expression of osteoblast-related genes (Col1a1, Runx2, Osx). Furthermore, the in vitro release kinetics of vascular endothelial growth factor (VEGF) and bone morphogenetic protein -2 (BMP-2) from the scaffolds were also investigated. The release of the growth factors produced from the scaffolds followed a first order kinetics. These results highlight that the scaffolds designed and developed in this work may be suitable candidates for bone tissue regeneration purposes.