Large-scale DNA sequencing identifies rare variants associated with Systemic Lupus Erythematosus susceptibility in known risk genes.

The identification of rare genetic variants associated to Systemic Lupus Erythematosus (SLE) could also help to understand the pathogenic mechanisms at the basis of the disease. In this study we have analyzed a cohort of 200 Italian SLE patients in order to explore the rare protein-coding variants in five genes (TNFAIP3, STAT4, IL10, TRAF3IP2, and HCP5) already investigated for commons variants found associated in our previous studies. Genomic DNA of 200 SLE patients was sequenced by whole exome sequencing. The identified variants were filtered by frequency and evaluated by in silico predictions. Allelic association analysis was performed with standard Fisher's exact test. Introducing a cutoff at MAF < 0.01, a total of 19 rare variants were identified. Seven of these variants were ultra-rare (MAF < 0.001) and six were absent in the GnomAD database. For TNFAIP3 gene, the variant c.A1939C was observed in 4 SLE patients and it is located in a region enriched in phosphorylation sites and affects the predict affinity of specific kinases. In TRAF3IP2 gene, we observed 5 different rare variants, including the novel variant c.G410A, located in the region that mediates interaction with TRAF6, and therefore a possible risk factor for SLE development. In STAT4 gene, we identified 6 different rare variants. Among these, three missense variants decrease the stability of this protein. Moreover, 3 novel rare variants were detected in 3 SLE patients. In particular, c.A767T variant was predicted as damaging by six prediction tools. Concluding, we have observed that even in genes whose common variability is associated with SLE susceptibility, it is possible to identify rare variants that could have a strong effect in the disease development and could therefore allow a better understanding of the functional domain involved.

The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes.

Comparative analysis of recent human genome assemblies highlights profound sequence divergence that peaks within polymorphic loci such as centromeres. This raises the question about the adequacy of relying on human reference genomes to accurately analyze sequencing data derived from experimental cell lines. Here, we generated the complete diploid genome assembly for the human retinal epithelial cells (RPE-1), a widely used non-cancer laboratory cell line with a stable karyotype, to use as matched reference for multi-omics sequencing data analysis. Our RPE1v1.0 assembly presents completely phased haplotypes and chromosome-level scaffolds that span centromeres with ultra-high base accuracy (>QV60). We mapped the haplotype-specific genomic variation specific to this cell line including t(Xq;10q), a stable 73.18 Mb duplication of chromosome 10 translocated onto the microdeleted chromosome X telomere t(Xq;10q). Polymorphisms between haplotypes of the same genome reveals genetic and epigenetic variation for all chromosomes, especially at centromeres. The RPE-1 assembly as matched reference genome improves mapping quality of multi-omics reads originating from RPE-1 cells with drastic reduction in alignments mismatches compared to using the most complete human reference to date (CHM13). Leveraging the accuracy achieved using a matched reference, we were able to identify the kinetochore sites at base pair resolution and show unprecedented variation between haplotypes. This work showcases the use of matched reference genomes for multiomics analyses and serves as the foundation for a call to comprehensively assemble experimentally relevant cell lines for widespread application.

A neuronal action of sirtuin 1 suppresses bone mass in young and aging mice.

The various functions of the skeleton are influenced by extracellular cues, hormones, and neurotransmitters. One type of neuronal regulation favors bone mass accrual by inhibiting sympathetic nervous system (SNS) activity. This observation raises questions about the transcriptional mechanisms regulating catecholamine synthesis. Using a combination of genetic and pharmacological studies, we found that the histone deacetylase sirtuin 1 (SIRT1) is a transcriptional modulator of the neuronal control of bone mass. Neuronal SIRT1 reduced bone mass by increasing SNS signaling. SIRT1 did so by increasing expression of monoamine oxidase A (MAO-A), a SIRT1 target that reduces brain serotonin levels by inducing its catabolism and by suppressing tryptophan hydroxylase 2 (Tph2) expression and serotonin synthesis in the brain stem. SIRT1 upregulated brain catecholamine synthesis indirectly through serotonin, but did not directly affect dopamine ß hydroxylase (Dbh) expression in the locus coeruleus. These results help us to understand skeletal changes associated with selective serotonin reuptake inhibitors (SSRIs) and may have implications for treating skeletal and metabolic diseases.

Multidisciplinary investigation of an ancient renal stone in a mummy from Popoli, central Italy.

The renal stone found in the natural mummy of an anonymous nobleman dating to 19th century was investigated using advanced imaging modalities and analytic investigations. By this multidisciplinary approach we were able to identify the chemical components and their distribution throughout the sample. These results allowed to understand the lifestyle habits of the subject, as well as the exact pathogenesis of his disease.

Improvement of the skeletal phenotype in a mouse model of diastrophic dysplasia after postnatal treatment with N-acetylcysteine.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in the SLC26A2 gene encoding for a sulfate/chloride transporter. When SLC26A2 is impaired intracellular level of sulfate is reduced leading to the synthesis of undersulfated proteoglycans. In normal chondrocytes, the main source of intracellular sulfate is the extracellular uptake through SLC26A2, but a small amount comes from the catabolism of sulfur-containing amino acids and other thiols. Here N-acetylcysteine (NAC), an extensively used drug, is proposed as alternative source of intracellular sulfate in an animal model of DTD (dtd mouse). Mutant and wild type mice were treated twice a day with hypodermic injections of 250 mg NAC/kg body weight for one week after birth. At the end of the treatment, an improvement trend in cartilage proteoglycan sulfation and in the skeletal phenotype of treated dtd mice were observed. Thus, a longer treatment lasted three weeks starting from birth was performed. Treated mutant mice showed a significant increase of cartilage proteoglycan sulfation and a relevant improvement of the skeletal phenotype based on measurements of several bony elements and bone quality by DEXA and micro CT. Moreover, the amelioration of the overall growth plate morphology in treated dtd mice suggested a partial rescue of the endochondral ossification process. Overall, the results prove that NAC is an effective source of intracellular sulfate for dtd mice in the postnatal period. This finding paves the way for a potential pharmacological treatment of DTD patients taking advantage from a drug repositioning strategy.

Combined administration of a small-molecule inhibitor of TRAF6 and Docetaxel reduces breast cancer skeletal metastasis and osteolysis.

Tumour necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in breast cancer and osteoclastic bone destruction. Here, we report that 6877002, a verified small-molecule inhibitor of TRAF6, reduced metastasis, osteolysis and osteoclastogenesis in models of osteotropic human and mouse breast cancer. First, we observed that TRAF6 is highly expressed in osteotropic breast cancer cells and its level of expression was higher in patients with bone metastasis. Pre-exposure of osteoclasts and osteoblasts to non-cytotoxic concentrations of 6877002 inhibited cytokine-induced NFκB activation and osteoclastogenesis, and reduced the ability of osteotropic human MDA-MB-231 and mouse 4T1 breast cancer cells to support bone cell activity. 6877002 inhibited human MDA-MB-231-induced osteolysis in the mouse calvaria organ system, and reduced soft tissue and bone metastases in immuno-competent mice following intra-cardiac injection of mouse 4T1-Luc2 cells. Of clinical relevance, combined administration of 6877002 with Docetaxel reduced metastasis and inhibited osteolytic bone damage in mice bearing 4T1-Luc2 cells. Thus, TRAF6 inhibitors such as 6877002 - alone or in combination with conventional chemotherapy - show promise for the treatment of metastatic breast cancer.

CRELD2 Is a Novel LRP1 Chaperone That Regulates Noncanonical WNT Signaling in Skeletal Development.

Cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER)-resident chaperone highly activated under ER stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage-specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whereas deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating noncanonical WNT4 signaling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor low-density lipoprotein receptor-related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF-ß1 signaling. Therefore, our data provide a novel link between an ER-resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT-responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by 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.

Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry.

BACKGROUND: Recurrence after >5-year disease-free survival affects one-fifth of breast cancer patients and is the clinical manifestation of cancer cell reactivation after persistent dormancy. METHODS: We investigated cellular dormancy in vitro and in vivo using breast cancer cell lines and cell and molecular biology techniques. RESULTS: We demonstrated cellular dormancy in breast cancer bone metastasis, associated with haematopoietic stem cell (HSC) mimicry, in vivo competition for HSC engraftment and non-random distribution of dormant cells at the endosteal niche. Notch2 signal implication was demonstrated by immunophenotyping the endosteal niche-associated cancer cells and upon co-culture with sorted endosteal niche cells, which inhibited breast cancer cell proliferation in a Notch2-dependent manner. Blocking this signal by in vivo acute administration of the γ-secretase inhibitor, dibenzazepine, induced dormant cell mobilisation from the endosteal niche and colonisation of visceral organs. Sorted Notch2HIGH breast cancer cells exhibited a unique stem phenotype similar to HSCs and in vitro tumour-initiating ability in mammosphere assay. Human samples confirmed the existence of a small Notch2HIGH cell population in primary and bone metastatic breast cancers, with a survival advantage for Notch2HIGH vs Notch2LOW patients. CONCLUSIONS: Notch2 represents a key determinant of breast cancer cellular dormancy and mobilisation in the bone microenvironment.

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice.

BACKGROUND: Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). METHODS: The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. FINDINGS: JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. INTERPRETATION: MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.

Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice.

The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. In this work, we have tested this strategy in vivo with the aim to generate two conditional knock-in mice for missense mutations in the Impad1 and Clcn7 genes causing two different skeletal dysplasias. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* and the Clcn7 exon 7 and an inverted exon 7* containing the point mutations were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon. The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation. Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Similarly, the phenotype of homozygous Clcn7 floxed mice overlaps with Clcn7 knock-out mice. Expression studies by qPCR and RT-PCR demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. Thus, the skeletal phenotypes in both murine strains were not caused by the missense mutations, but by aberrant splicing. Our data demonstrate that the Cre mediated genetic switch strategy should be considered cautiously for the generation of conditional knock-in mice.

Osteoblasts Regulate Angiogenesis in Response to Mechanical Unloading.

During mechanical unloading, endothelial cells reduce osteogenesis and increase bone resorption. Here we describe the feedback response of endothelial cells to unloaded osteoblasts. Primary endothelial cells, ex vivo mouse aortic rings and chicken egg yolk membranes were incubated with conditioned medium from mouse primary osteoblasts (OB-CM) subjected to unit gravity or simulated microgravity, to assess its effect on angiogenesis. In vivo injection of botulin toxin A (Botox) in the quadriceps and calf muscles of C57BL/6J mice was performed to mimic disuse osteoporosis. Unloaded osteoblasts showed strong upregulation of the pro-angiogenic factor, VEGF, and their conditioned medium increased in vitro endothelial cell viability, Cyclin D1 expression, migration and tube formation, ex vivo endothelial cell sprouting from aortic rings, and in ovo angiogenesis. Treatment with the VEGF blocker, avastin, prevented unloaded OB-CM-mediated in vitro and ex vivo enhancement of angiogenesis. Bone mechanical unloading by Botox treatment, known to reduce bone mass, prompted the overexpression of VEGF in osteoblasts. The cross talk between osteoblasts and endothelial cells plays a pathophysiologic role in the response of the endothelium to unloading during disuse osteoporosis. In this context, VEGF represents a prominent osteoblast factor stimulating angiogenesis.

Regulation of breast cancer induced bone disease by cancer-specific IKKß.

NFκB is implicated in breast cancer bone metastasis and skeletal remodelling. However, the role of IKKß, a key component of the canonical NFκB pathway, in the regulation of breast cancer osteolytic metastasis has not been investigated. Here, we describe the cancer-specific contribution of IKKß to bone metastasis, skeletal tumour growth and osteolysis associated with breast cancer. IKKß is highly expressed in invasive breast tumours and its level of expression was higher in patients with bone metastasis. IKKß overexpression in parental MDA-MD-231 breast cancer cells, promoted mammary tumour growth but failed to convey osteolytic potential to these cells in mice. In contrast, IKKß overexpression in osteotropic sub-clones of MDA-MB-231 cells with differing osteolytic phenotypes increased incidence of bone metastasis, exacerbated osteolysis and enhanced skeletal tumour growth, whereas its knockdown was inhibitory. Functional and mechanistic studies revealed that IKKß enhanced the ability of osteotropic MDA-MB-231 cells to migrate, increase osteoclastogenesis, and to inhibit osteoblast differentiation via a mechanism mediated, at least in part, by cytoplasmic sequestering of FoxO3a and VEGFA production. Thus, tumour-selective manipulation of IKKß and its interaction with FoxO3a may represent a novel strategy to reduce the development of secondary breast cancer in the skeleton.

RNA interference therapy for autosomal dominant osteopetrosis type 2. Towards the preclinical development.

Autosomal Dominant Osteopetrosis type 2 (ADO2) is a rare bone disease characterized by dense and brittle bones due to impairment of osteoclast bone resorption. Dominant negative mutations of the CLCN7 gene affect about 70% of ADO2 patients. ADO2 has no cure and our recent work established that it is suitable for gene silencing by a specific small interfering RNA that does not affect the normal mRNA, thus inducing a condition of pseudo-haplosufficiency and rescuing the bone phenotype. We performed a systematic study to test the likelihood that the therapy could progress towards clinical trials, treating Clcn7G213R/WT ADO2 mice with Clcn7G213R-specific siRNA and investigating the bone phenotype by µCT and histomorphometry, and safety, by histopathology and serology. We demonstrated that our Clcn7G213R siRNA is not only effective in pre-pubertal ADO2 male mice as we showed in our previous study, but also in adult and ageing mice, in males and females, by intraperitoneal and subcutaneous administration. Furthermore, the study also showed safety following prolonged chronic administration and allowed us to identify specific end-points to be potentially used in clinical trials. These results may pave the way towards regulatory toxicity studies, through which the therapy, that is patent-protected, can obtain approval from public health authorities for the transition to the Phase I/II clinical trials. The study also suggests that similar strategies could be applied to other autosomal dominant bone diseases, opening an avenue for a wider use of the RNA interference therapy in rare genetic disorders.

A Complex Role for Lipocalin 2 in Bone Metabolism: Global Ablation in Mice Induces Osteopenia Caused by an Altered Energy Metabolism.

Lipocalin 2 (Lcn2) is an adipokine that carries out a variety of functions in diverse organs. We investigated the bone phenotype and the energy metabolism of Lcn2 globally deleted mice (Lcn2-/- ) at different ages. Lcn2-/- mice were largely osteopenic, exhibiting lower trabecular bone volume, lesser trabecular number, and higher trabecular separation when compared to wild-type (WT) mice. Lcn2-/- mice showed a lower osteoblast number and surface over bone surface, and subsequently a significantly lower bone formation rate, while osteoclast variables were unremarkable. Surprisingly, we found no difference in alkaline phosphatase (ALP) activity or in nodule mineralization in Lcn2-/- calvaria osteoblast cultures, while less ALP-positive colonies were obtained from freshly isolated Lcn2-/- bone marrow stromal cells, suggesting a nonautonomous osteoblast response to Lcn2 ablation. Given that Lcn2-/- mice showed higher body weight and hyperphagia, we investigated whether their osteoblast impairment could be due to altered energy metabolism. Lcn2-/- mice showed lower fasted glycemia and hyperinsulinemia. Consistently, glucose tolerance was significantly higher in Lcn2-/- compared to WT mice, while insulin tolerance was similar. Lcn2-/- mice also exhibited polyuria, glycosuria, proteinuria, and renal cortex vacuolization, suggesting a kidney contribution to their phenotype. Interestingly, the expression of the glucose transporter protein type 1, that conveys glucose into the osteoblasts and is essential for osteogenesis, was significantly lower in the Lcn2-/- bone, possibly explaining the in vivo osteoblast impairment induced by the global Lcn2 ablation. Taken together, these results unveil an important role of Lcn2 in bone metabolism, highlighting a link with glucose metabolism that is more complex than expected from the current knowledge. © 2018 American Society for Bone and Mineral Research.

TRAF2 in osteotropic breast cancer cells enhances skeletal tumour growth and promotes osteolysis.

NFκB plays an important role in inflammation and bone remodelling. Tumour necrosis factor receptor associated factor 2 (TRAF2), a key component of NFκB signalling, has been identified as an oncogene, but its role in the regulation of breast cancer osteolytic metastasis remains unknown. Here, we report that stable overexpression of TRAF2 in parental and osteotropic sub-clones of human MDA-MB-231 (MDA-231) breast cancer cells increased cell growth and motility in vitro, whereas TRAF2 knockdown was inhibitory. In vivo, TRAF2 overexpression in the parental MDA-231-P cells enhanced tumour growth after orthotopic injection into the mammary fat pad of mice but failed to promote the metastasis of these cells to bone. In contrast, overexpression of TRAF2 in osteotropic MDA-231-BT cells increased skeletal tumour growth, enhanced osteoclast formation and worsened osteolytic bone loss after intra-tibial injection in mice. Mechanistic and functional studies in osteotropic MDA-231-BT and osteoclasts revealed that upregulation of TRAF2 increased the ability of osteotropic MDA-231-BT cells to migrate and to enhance osteoclastogenesis by a mechanism dependent, at least in part, on NFκB activation. Thus, the TRAF2/NFκB axis is implicated in the regulation of skeletal tumour burden and osteolysis associated with advanced breast cancer.

Non-conventional role of haemoglobin beta in breast malignancy.

BACKGROUND: Besides its role as oxygen transporter, recent findings suggest that haemoglobin beta (HBB) may have roles in other contexts. METHODS: We evaluated the impact of HBB expression in primary human breast cancers, and in breast cancer cell lines overexpressing HBB by in vitro and in vivo studies. Publicly available microarray databases were used to perform multivariate survival analyses. RESULTS: A significantly higher expression of HBB was observed in invasive carcinoma histotypes vs in situ counterparts, along with a positive correlation between HBB and the Ki67 proliferation marker. HBB-overexpressing breast cancer cells migrate and invade more, show HIF-1α upregulation and their conditioned media enhances angiogenesis. Blocking the oxygen-binding site of HBB reverts the increase of migration and HIF-1α upregulation observed in HBB-overexpressing breast cancer cells. Orthotopically implanted MDA-MB-231 overexpressing HBB (MDA-HBB) generated tumours with faster growth rate and increased neoangiogenesis. Moreover, local recurrence and visceral metastases were observed only in MDA-HBB-implanted mice. Similar results were observed with 4T1 mouse breast cancer cells. Finally, bioinformatics analyses of public data sets correlated high HBB expression with lower overall survival. CONCLUSIONS: HBB expression increases breast cancer cells aggressiveness and associates with poor prognosis, pointing to HBB as a novel biomarker for breast cancer progression.

Interleukin-1ß, lipocalin 2 and nitric oxide synthase 2 are mechano-responsive mediators of mouse and human endothelial cell-osteoblast crosstalk.

Endothelial cells are spatially close to osteoblasts and regulate osteogenesis. Moreover, they are sensitive to mechanical stimuli, therefore we hypothesized that they are implicated in the regulation of bone metabolism during unloading. Conditioned media from endothelial cells (EC-CM) subjected to simulated microgravity (0.08g and 0.008g) increased osteoblast proliferation and decreased their differentiation compared to unit gravity (1g) EC-CM. Microgravity-EC-CM increased the expression of osteoblast Rankl and subsequent osteoclastogenesis, and induced the osteoblast de-differentiating factor, Lipocalin 2 (Lcn2), whose downregulation recovered osteoblast activity, decreased Rankl expression and reduced osteoclastogenesis. Microgravity-EC-CM enhanced osteoblast NO-Synthase2 (NOS2) and CycloOXygenase2 (COX2) expression. Inhibition of NOS2 or NO signaling reduced osteoblast proliferation and rescued their differentiation. Nuclear translocation of the Lcn2/NOS2 transcription factor, NF-κB, occurred in microgravity-EC-CM-treated osteoblasts and in microgravity-treated endothelial cells, alongside high expression of the NF-κB activator, IL-1ß. IL-1ß depletion and NF-κB inhibition reduced osteoblast proliferation and rescued differentiation. Lcn2 and NOS2 were incremented in ex vivo calvarias cultured in microgravity-EC-CM, and in vivo tibias and calvarias injected with microgravity-EC-CM. Furthermore, tibias of botulin A toxin-treated and tail-suspended mice, which featured unloading and decreased bone mass, showed higher expression of IL-1ß, Lcn2 and Nos2, suggesting their pathophysiologic involvement in endothelial cell-osteoblast crosstalk.

Effective Small Interfering RNA Therapy to Treat CLCN7-dependent Autosomal Dominant Osteopetrosis Type 2.

In about 70% of patients affected by autosomal dominant osteopetrosis type 2 (ADO2), osteoclast activity is reduced by heterozygous mutations of the CLCN7 gene, encoding the ClC-7 chloride/hydrogen antiporter. CLCN7(G215R)-, CLCN7(R767W)-, and CLCN7(R286W)-specific siRNAs silenced transfected mutant mRNA/EGFP in HEK293 cells, in RAW264.7 cells and in human osteoclasts, with no change of CLCN7(WT) mRNA and no effect of scrambled siRNA on the mutant transcripts. Osteoclasts from Clcn7(G213R) ADO2 mice showed reduced bone resorption, a condition rescued by Clcn7(G213R)-specific siRNA. Treatment of ADO2 mice with Clcn7(G213R)-specific siRNA induced increase of bone resorption variables and decrease of trabecular bone mass, leading to an overall improvement of the osteopetrotic bone phenotype. Treatment did not induce overt adverse effects and was effective also with siRNAs specific for other mutants. These results demonstrate that a siRNA-based experimental treatment of ADO2 is feasible, and underscore a translational impact for future strategy to cure this therapeutically neglected form of osteopetrosis.

Biotechnological approach for systemic delivery of membrane Receptor Activator of NF-κB Ligand (RANKL) active domain into the circulation.

Deficiency of Receptor Activator of NF-κB Ligand (RANKL) prevents osteoclast formation causing osteopetrosis. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by metalloproteinase 14 (MMP14). We created a bio-device that harbors primary osteoblasts, cultured on 3D hydroxyapatite scaffolds carrying immobilized MMP14 catalytic domain. Scaffolds were sealed in diffusion chambers and implanted in RANKL-deficient mice. Mice received 1 or 2 diffusion chambers, once or twice and were sacrificed after 1 or 2 months from implants. A progressive increase of body weight was observed in the implanted groups. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker Tartrate-Resistant Acid Phosphatase (TRAcP), consistent with the lack of osteoclasts. In contrast, tibias excised from implanted mice showed TRAcP-positive cells in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. In mice implanted with 4 diffusion chambers total, we noted the highest number and size of TRAcP-positive cells, with quantifiable eroded bone surface and significant reduction of trabecular bone volume. These data demonstrate that our bio-device delivers effective sRANKL, inducing osteoclastogenesis in RANKL-deficient mice, supporting the feasibility of an innovative experimental strategy to treat systemic cytokine deficiencies.