Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Effects of osteoblast-derived extracellular vesicles on aggressiveness, redox status and mitochondrial bioenergetics of MNNG/HOS osteosarcoma cells.

Osteosarcoma is the most common primary bone malignancy. The crosstalk between osteosarcoma and the surrounding tumour microenvironment (TME) drives key events that lead to metastasization, one of the main obstacles for definitive cure of most malignancies. Extracellular vesicles (EVs), lipid bilayer nanoparticles used by cells for intercellular communication, are emerging as critical biological mediators that permit the interplay between neoplasms and the tumour microenvironment, modulating re-wiring of energy metabolism and redox homeostatic processes. We previously showed that EVs derived from the human osteosarcoma cells influence bone cells, including osteoblasts. We here investigated whether the opposite could also be true, studying how osteoblast-derived EVs (OB-EVs) could alter tumour phenotype, mitochondrial energy metabolism, redox status and oxidative damage in MNNG/HOS osteosarcoma cells.These were treated with EVs obtained from mouse primary osteoblasts, and the following endpoints were investigated: i) cell viability and proliferation; ii) apoptosis; iii) migration and invasive capacity; iv) stemness features; v) mitochondrial function and energy metabolism; vi) redox status, antioxidant capacity and oxidative molecular damage. OB-EVs decreased MNNG/HOS metabolic activity and viability, which however was not accompanied by impaired proliferation nor by increased apoptosis, with respect to control. In addition, OB-EV-treated cells exhibited a significant reduction of motility and in vitro invasion as compared to untreated cells. Although the antioxidant N-acetyl-L-cysteine reverted the cytotoxic effect of OB-EVs, no evidence of oxidative stress was observed in treated cells. However, the redox balance of glutathione was significantly shifted towards a pro-oxidant state, even though the major antioxidant enzymatic protection did not respond to the pro-oxidant challenge. We did not find strong evidence of mitochondrial involvement or major energy metabolic switches induced by OB-EVs, but a trend of reduction in seahorse assay basal respiration was observed, suggesting that OB-EVs could represent a mild metabolic challenge for osteosarcoma cells. In summary, our findings suggest that OB-EVs could serve as important means through which TME and osteosarcoma core cross-communicate. For the first time, we proved that OB-EVs reduced osteosarcoma cells' aggressiveness and viability through redox-dependent signalling pathways, even though mitochondrial dynamics and energy metabolism did not appear as processes critically needed to respond to OB-EVs.

Liquid biopsies in primary and secondary bone cancers.

Liquid biopsies are a powerful tool to non-invasively analyze tumor phenotype and progression as well as drug resistance. In the bone oncology field, liquid biopsies would be particularly important to develop, since standard biopsies can be very painful, dangerous (e.g., when found in proximity to the spinal cord), and hard to collect. In this review, we explore the recent advances in liquid biopsies in both primary (osteosarcoma and Ewing sarcoma) and secondary bone cancers (breast, prostate, and lung cancer-induced bone metastases), presenting their current role and highlighting their unexpressed potential, as well as the barriers limiting their possible adoption, including costs, scalability, reproducibility, and isolation methods. We discuss the use of circulating tumor cells, cell-free circulating tumor DNA, and extracellular vesicles for the purpose of improving diagnosis, prognosis, evaluation of therapy resistance, and driving therapy decisions in both primary and secondary bone malignancies.

Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy.

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2-/- mice (MDXxLcn2-/-), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2-/- mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Lipocalin 2 increases after high-intensity exercise in humans and influences muscle gene expression and differentiation in mice.

Lipocalin 2 (LCN2) is an adipokine that accomplishes several functions in diverse organs. However, its importance in muscle and physical exercise is currently unknown. We observed that following acute high-intensity exercise ("Gran Sasso d'Italia" vertical run), LCN2 serum levels were increased. The Wnt pathway antagonist, DKK1, was also increased after the run, positively correlating with LCN2, and the same was found for the cytokine Interleukin 6. We, therefore, investigated the involvement of LCN2 in muscle physiology employing an Lcn2 global knockout (Lcn2-/- ) mouse model. Lcn2-/- mice presented with smaller muscle fibres but normal muscle performance (grip strength metre) and muscle weight. At variance with wild type (WT) mice, the inflammatory cytokine Interleukin 6 was undetectable in Lcn2-/- mice at all ages. Intriguingly, Lcn2-/- mice did not lose gastrocnemius and quadriceps muscle mass and muscle performance following hindlimb suspension, while at variance with WT, they lose soleus muscle mass. In vitro, LCN2 treatment reduced the myogenic differentiation of C2C12 and primary mouse myoblasts and influenced their gene expression. Treating myoblasts with LCN2 reduced myogenesis, suggesting that LCN2 may negatively affect muscle physiology when upregulated following high-intensity exercise.

Anti-osteoblastogenic, pro-inflammatory and pro-angiogenic effect of extracellular vesicles isolated from the human osteosarcoma cell line MNNG/HOS.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

Extracellular Vesicles From Osteotropic Breast Cancer Cells Affect Bone Resident Cells.

Extracellular vesicles (EVs) are emerging as mediators of a range of pathological processes, including cancer. However, their role in bone metastases has been poorly explored. We investigated EV-mediated effects of osteotropic breast cancer cells (MDA-MB-231) on bone resident cells and endothelial cells. Pretreatment of osteoblasts with conditioned medium (CM) of MDA-MB-231 (MDA) cells promoted pro-osteoclastogenic and pro-angiogenic effects by osteoblast EVs (OB-EVs), as well as an increase of RANKL-positive OB-EVs. Moreover, when treating osteoblasts with MDA-EVs, we observed a reduction of their number, metabolic activity, and alkaline phosphatase (Alp) activity. MDA-EVs also reduced transcription of Cyclin D1 and of the osteoblast-differentiating genes, while enhancing the expression of the pro-osteoclastogenic factors Rankl, Lcn2, Il1b, and Il6. Interestingly, a cytokine array on CM from osteoblasts treated with MDA-EVs showed an increase of the cytokines CCL3, CXCL2, Reg3G, and VEGF, while OPG and WISP1 were downregulated. MDA-EVs contained mRNAs of genes involved in bone metabolism, as well as cytokines, including PDGF-BB, CCL3, CCL27, VEGF, and Angiopoietin 2. In line with this profile, MDA-EVs increased osteoclastogenesis and in vivo angiogenesis. Finally, intraperitoneal injection of MDA-EVs in mice revealed their ability to reach the bone microenvironment and be integrated by osteoblasts and osteoclasts. In conclusion, we showed a role for osteoblast-derived EVs and tumor cell-derived EVs in the deregulation of bone and endothelial cell physiology, thus fueling the vicious cycle induced by bone tumors. © 2019 American Society for Bone and Mineral Research.

Extra-skeletal manifestations in mice affected by Clcn7-dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications.

Autosomal dominant osteopetrosis type 2 (ADO2) is a high-density brittle bone disease characterized by bone pain, multiple fractures and skeletal-related events, including nerve compression syndrome and hematological failure. We demonstrated that in mice carrying the heterozygous Clcn7 G213R mutation, whose human mutant homolog CLCN7 G215R affects patients, the clinical impacts of ADO2 extend beyond the skeleton, affecting several other organs. The hallmark of the extra-skeletal alterations is a consistent perivascular fibrosis, associated with high numbers of macrophages and lymphoid infiltrates. Fragmented clinical information in a small cohort of patients confirms extra-skeletal alterations consistent with a systemic disease, in line with the observation that the CLCN7 gene is expressed in many organs. ADO2 mice also show anxiety and depression and their brains exhibit not only perivascular fibrosis but also ß-amyloid accumulation and astrogliosis, suggesting the involvement of the nervous system in the pathogenesis of the ADO2 extra-skeletal alterations. Extra-skeletal organs share a similar cellular pathology, confirmed also in vitro in bone marrow mononuclear cells and osteoclasts, characterized by an impairment of the exit pathway of the Clcn7 protein product, ClC7, through the Golgi, with consequent reduced ClC7 expression in late endosomes and lysosomes, associated with high vesicular pH and accumulation of autophagosome markers. Finally, an experimental siRNA therapy, previously proven to counteract the bone phenotype, also improves the extra-skeletal alterations. These results could have important clinical implications, supporting the notion that a systematic evaluation of ADO2 patients for extra-skeletal symptoms could help improve their diagnosis, clinical management, and therapeutic options.