Antonio Maurizi

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation via Tet1 and Tet2 and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2, Col1a1, Bglap2, Sp7, Atf4, and Alpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of the Tet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorption in vivo. Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis.

Generation of the first autosomal dominant osteopetrosis type II (ADO2) disease models

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl(-)/1H(+) antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.

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. CLCN7G215R-, CLCN7R767W-, and CLCN7R286W-specific siRNAs silenced transfected mutant mRNA/EGFP in HEK293 cells, in RAW264.7 cells and in human osteoclasts, with no change of CLCN7WT mRNA and no effect of scrambled siRNA on the mutant transcripts. Osteoclasts from Clcn7G213R ADO2 mice showed reduced bone resorption, a condition rescued by Clcn7G213R-specific siRNA. Treatment of ADO2 mice with Clcn7G213R-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.

Osteoblast‐Derived Extracellular Vesicles Are Biological Tools for the Delivery of Active Molecules to Bone

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine‐tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV‐mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence‐activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro‐osteoclastic cytokine, receptor activator of nuclear factor κ‐B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl–/– osteoblasts lost this pro‐osteoclastic function, indicating its Rankl‐dependence. They integrated ex vivo into murine calvariae, and EV‐shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl–/– mice lack the osteoclast lineage and are negative for its specific marker tartrate‐resistant acid phosphatase (TRAcP). Treatment of rankl–/– mice with wild‐type osteoblast EVs induced the appearance of TRAcP‐positive cells in an EV density‐dependent manner. Finally, osteoblast EVs internalized and shuttled anti‐osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro‐osteoclastic effect, and shuttle anti‐osteoclast drugs, representing a potential means of targeted therapeutic delivery.

The Osteoclast in Bone Metastasis: Player and Target

Bone metastases are frequently the final fate of breast and prostate cancer patients. According to the definition of metastasis as an incurable disease, to date there are no effective treatments for tumor-associated bone metastases and this represents a real challenge for the researchers in the field. The bone is a heterogeneous environment that represents a fertile soil for tumor cells, supporting their growth. Among the different cell types present in the bone, in this review we will focus our attention on the osteoclasts, which are crucial players in the so called “vicious cycle”, a phenomenon triggered by tumor cells eventually leading to both tumor proliferation as well as bone deregulation, thus fueling the development of bone metastasis. The complex network, linking tumor cells to the bone by activating osteoclasts, represents a fruitful target for the treatment of bone metastases. In this review we will describe how tumor cells perturb the bone microenvironment by actively influencing osteoclast formation and activity. Moreover, we will describe the current antiresorptive drugs employed in the treatment of bone metastases as well as new, targeted therapies able to affect both cancer cells and osteoclasts.

A Complex Role for Lipocalin 2 in Bone Metabolism: Global Ablation in Mice Induces Osteopenia Caused by an Altered Energy Metabolism: COMPLEX ROLE FOR LIPOCALIN 2 IN BONE 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.

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.