Tyrosine kinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAM signals.

Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCgamma-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.

Fluconazole-Induced Protein Changes in Osteogenic and Immune Metabolic Pathways of Dental Pulp Mesenchymal Stem Cells of Osteopetrosis Patients.

Osteopetrosis is a rare inherited disease caused by osteoclast failure, resulting in increasing bone density in humans. Patients with osteopetrosis possess several dental and cranial complications. Since carbonic anhydrase II (CA-II) deficiency is a major cause of osteopetrosis, CA-II activators might be an attractive potential treatment option for osteopetrosis patients. We conducted comprehensive label-free quantitative proteomics analysis on Fluconazole-treated Dental Pulp Mesenchymal Stem/Stromal Cells from CA-II-Deficient Osteopetrosis Patients. We identified 251 distinct differentially expressed proteins between healthy subjects, as well as untreated and azole-treated derived cells from osteopetrosis patients. Twenty-six (26) of these proteins were closely associated with osteogenesis and osteopetrosis disease. Among them are ATP1A2, CPOX, Ap2 alpha, RAP1B and some members of the RAB protein family. Others include AnnexinA1, 5, PYGL, OSTF1 and PGAM4, all interacting with OSTM1 in the catalytic reactions of HCO3 and the Cl- channel via CAII regulation. In addition, the pro-inflammatory/osteoclast regulatory proteins RACK1, MTSE, STING1, S100A13, ECE1 and TRIM10 are involved. We have identified proteins involved in osteogenic and immune metabolic pathways, including ERK 1/2, phosphatase and ATPase, which opens the door for some CA activators to be used as an alternative drug therapy for osteopetrosis patients. These findings propose that fluconazole might be a potential treatment agent for CAII- deficient OP patients. Altogether, our findings provide a basis for further work to elucidate the clinical utility of azole, a CA activator, as a therapeutic for OP.

Ultrastructural Analyses of Alveolar Bone in a Patient With Osteomyelitis Secondary to Osteopetrosis: A Review of the Literature.

Osteopetrosis is a generic term for generalized sclerotic conditions caused by rare genetic disorders. Decreased osteoclastic activities disturb bone remodeling, resulting in greater mineral density and greater compressive strength; therefore, bone fracture is a major physical symptom of osteopetrosis. Osteomyelitis of the maxilla or mandible is a common and well-documented complication of osteopetrosis. Local infection, such as odontogenic infection, is more likely to lead to osteomyelitis, and treatment strategies can be challenging. However, detailed ultrastructural analyses of bone from patients with osteopetrosis and odontogenic infection are limited. This report describes a case of osteomyelitis of the maxilla and mandible secondary to osteopetrosis in an adult patient and presents ultrastructural data of alveolar bone tissue analyzed by contact microradiography, electron probe microanalysis, and x-ray diffraction. Cases of osteomyelitis of the jaw secondary to osteopetrosis also are reviewed.

Diagnostic dilemma: osteopetrosis with superimposed rickets causing neonatal hypocalcemia.

Osteopetrosis is a rare genetic condition of reduced osteoclastic bone resorption which causes defective bone remodeling and skeletal sclerosis during growth, having effects on many organs and tissues. Mutation of T-cell immune regulator 1 (TCRG1) gene is the most common genetic defect leading to osteopetrosis, with poor prognosis. The autosomal recessive form presents in the infantile period (also known as malignant infantile osteopetrosis--MIOP), and is characterized by fractures, short stature, hepatosplenomegaly, compressive neuropathies, hypocalcemia and pancytopenia. Being a rare disease with non-specific clinical manifestations, the diagnosis is difficult and usually delayed. Rickets is a characteristic feature of MIOP which results from the defect in osteoclasts to provide a normal Ca/P balance resulting in the poor mineralization of the osteoid. Various treatment options have been suggested for osteopetrosis, but hematopoietic stem cell transplantation still remains the only curative treatment option presently. The authors report the case of a 46-day-old girl with late-onset neonatal hypocalcemia and rickets that was later diagnosed as osteopetrosis. This case report emphasizes that infantile osteopetrosis is an important cause of neonatal hypocalcemia. As irreversible complications develop within the first months of life, immediate diagnosis and early intervention are crucial and may be life-saving.

RANKL cytokine: from pioneer of the osteoimmunology era to cure for a rare disease.

Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.

Chloroquine increases osteoclast activity in vitro but does not improve the osteopetrotic bone phenotype of ADO2 mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We created mouse models of ADO2 by introducing a knock-in (p.G213R) mutation in the Clcn7 gene, which is analogous to one of the common mutations (G215R) found in humans. The mutation leads to severe osteopetrosis and lethality in homozygous mice but produces substantial phenotypic variability in heterozygous mice on different genetic backgrounds that phenocopy the human disease of ADO2. ADO2 is an osteoclast-intrinsic disease, and lysosomal enzymes and proteins are critical for osteoclast activity. Chloroquine (CQ) is known to affect lysosomal trafficking, intracellular signaling and the lysosomal and vesicular pH, suggesting it might improve ADO2 osteoclast function. We tested this hypothesis in cell culture studies using osteoclasts derived from wild-type (WT or ADO2+/+) and ADO2 heterozygous (ADO2+/-) mice and found that CQ and its metabolite desethylchloroquine (DCQ), significantly increased ADO2+/- osteoclasts bone resorption activity in vitro, whereas bone resorption of ADO2+/+ osteoclasts was increased only by DCQ. In addition, we exploited our unique animal model of ADO2 on 129 background to identify the effect of CQ for the treatment of ADO2. Female ADO2 mice at 8 weeks of age were treated with 5 doses of CQ (1, 2.5, 5, 7.5 and 10 mg/kg BW/day) via drinking water for 6 months. Bone mineral density and bone micro-architecture were analyzed by longitudinal in vivo DXA and micro-CT at baseline, 3 and 6 months. Serum bone biomarkers (CTX, TRAP and P1NP) were also analyzed at these time points. CQ treatment at the doses tested failed to produce any significant changes of aBMD, BMC (whole body, femur and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group (water only). Further, levels of bone biomarkers were not significantly changed due to CQ treatment in these mice. Our findings indicate that while CQ increased osteoclast activity in vitro, it did not improve the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

In silico experiments of bone remodeling explore metabolic diseases and their drug treatment.

Bone structure and function are maintained by well-regulated bone metabolism and remodeling. Although the underlying molecular and cellular mechanisms are now being understood, physiological and pathological states of bone are still difficult to predict due to the complexity of intercellular signaling. We have now developed a novel in silico experimental platform, V-Bone, to integratively explore bone remodeling by linking complex microscopic molecular/cellular interactions to macroscopic tissue/organ adaptations. Mechano-biochemical couplings modeled in V-Bone relate bone adaptation to mechanical loading and reproduce metabolic bone diseases such as osteoporosis and osteopetrosis. V-Bone also enables in silico perturbation on a specific signaling molecule to observe bone metabolic dynamics over time. We also demonstrate that this platform provides a powerful way to predict in silico therapeutic effects of drugs against metabolic bone diseases. We anticipate that these in silico experiments will substantially accelerate research into bone metabolism and remodeling.

CLC-7: a potential therapeutic target for the treatment of osteoporosis and neurodegeneration.

CLC-7 is a member of the voltage-gated chloride channels family. It resides mainly in the late endosomes, lysosomes and the ruffled membrane of osteoclasts. Mice deficient in the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. In the present review, some of the known features of CLC-7 such as structure, function and its roles in physiological or pathophysiological processes are highlighted.

High dose calcitriol in osteopetrorickets.

Osteopetrorickets is a rare autosomal recessive disorder of osteoclast function characterized by abnormally dense bone and failure of resorption of calcified cartilage. Rickets is a paradoxical complication of osteopetrosis, resulting from the inability of the osteoclasts to maintain a normal calcium-phosphorus balance in the extracellular fluid. We report a patient with an unusual case of infantile osteopetro-rickets who was admitted with anterior fontanel bulging and was treated with haploidentical bone marrow transplantation.

Biological Effects of Anti-RANKL Antibody and Zoledronic Acid on Growth and Tooth Eruption in Growing Mice.

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

Interferon Gamma-1b Does Not Increase Markers of Bone Resorption in Autosomal Dominant Osteopetrosis.

In autosomal dominant osteopetrosis type 2 (ADO2) CLCN7 mutations cause impaired osteoclast function. Severe consequences include skeletal fragility despite high bone mass, osteomyelitis, osteonecrosis, bone marrow failure, and severe cranial nerve impingement. There is no effective medical treatment for ADO2. We recruited subjects with ADO2 into a 14-week, open-label, pilot clinical trial of interferon gamma-1b. Doses were titrated based on tolerability and if fasting serum C-telopeptide (CTX) was <25% above baseline at week 8, targeting doses of 100 µg/m2 three times a week. The primary outcomes were change from baseline in CTX and N-telopeptide/creatinine ratio (NTX/Cr) at week 14. Secondary outcomes included changes in urine calcium/creatinine ratio, bone formation markers and tolerability. Nine adults and three children were recruited. Severe manifestations of ADO2 included histories of fractures (100%), osteomyelitis (16.7%), vision loss (50%), and anemia (58.3%). Baseline CTX and NTX/Cr were generally low-normal. Procollagen type I N-terminal propeptide was elevated or in the upper-normal range in 11 of 12 (91.6%) subjects. Elevations of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were common. One subject withdrew due to rash. Five subjects achieved doses of 50 µg/m2 3 days a week, while six reached the full dose of 100 µg/m2 3 days a week. Only 3 of 11 (27.3%) completing subjects achieved the primary outcome of increasing CTX ≥25% above baseline at week 14. The mean ± SD change from baseline in CTX at week 14 was +2.2% ± 43.2%, p = 0.86). Likewise, there was no significant change in NTX/Cr (mean change -2.1%, p = 0.81). Interferon gamma-1b was poorly tolerated. Most subjects had adverse events, and the Mental Health and Mental Component Scales of the SF-36v2 health survey declined slightly (p < 0.05). Over 14 weeks, interferon gamma-1b failed to significantly increase bone turnover markers in ADO2 and was poorly tolerated. Consequently, interferon gamma-1b is unlikely to be effective for decreasing bone mass in ADO2. © 2019 American Society for Bone and Mineral Research.

Bone remodelling: a signalling system for osteoclast regulation.

Two physiological regulators of osteoclast maturation have recently been identified: the secreted protein osteoprotegerin and the cell-surface ligand to which it binds. These proteins are likely to play an important part in the control of bone resorption, but are also likely to have important roles in other tissues.

Decompressive Cranioplasty in a Patient with Osteopetrosis.

BACKGROUND: Osteopetrosis is a heterogeneous group of uncommon congenital disorders that causes bony sclerosis and remodeling. Patients who are symptomatic can show significant neurologic consequences with the involvement of cranial nerves and symptoms of increased intracranial pressure (ICP). CASE DESCRIPTION: We report an unusual case of a 26-year-old woman with an autosomal-dominant type of osteopetrosis who presented with headache and severe visual deterioration, both attributed to increased ICP. A hemicranioplasty was preformed, resulting in the resolution of her symptoms of ICP and stabilization of her vision. Postoperative imaging showed expansion of the ventricles and the subarachnoid spaces with an improvement of the associated cerebellar herniation. CONCLUSIONS: In conclusion, in patients with symptomatic osteopetrosis, cranioplasty can be considered as an option to treat high ICP-related symptoms.

Malignant infantile osteopetrosis: a rare cause of neonatal hypocalcemia.

Malignant infantile osteopetrosis is a rare autosomal recessive disorder characterized by presentation in the first few months of life with manifestations relating to an underlying defect in osteoclastic bone resorption. This report describes a 10 day-old boy in whom neonatal hypocalcemia was present and whose brother had died with the diagnosis of osteopetrosis.

Interferon gamma-1b: new indication. Severe malignant osteopetrosis: too many unknowns.

(1) Severe malignant osteopetrosis is a very rare disease. The principal manifestations are anaemia, infections, sensory disorders and fractures, due to generalised bone condensation. The disease is generally fatal in childhood. The only treatment capable of modifying the natural outcome is bone marrow transplantation. The benefits of high-dose steroids and calcitriol are usually modest and transient. (2) Severe malignant osteopetrosis is a new licensed indication for interferon gamma-1b, a drug known to reduce the incidence of severe infections in children with chronic septic granulomatosis. (3) An unblinded trial involving 15 children with a mean age of about one year compared calcitriol plus interferon gamma-1b with calcitriol alone. The time to treatment failure was longer with the combination, based on a combined endpoint chosen to make the statistical analysis more sensitive. (4) A clinical trial involving 15 patients, who were compared with a historical series of 94 untreated patients, provided ambiguous results. (5) In these trials the main adverse effect of interferon gamma-1b was a flu-like syndrome. (6) Given the gravity of severe malignant osteopetrosis, the limited available treatment options, and the rarity of serious adverse events with interferon gamma-1b, evaluation of this therapy should continue.

Thrombocytopenia in the toothless (osteopetrotic) rat and its rescue by treatment with colony-stimulating factor-1.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor.

Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.

Treatment of congenital osteopetrosis in the rabbit with high-dose 1,25-dihydroxyvitamin D.

Osteopetrosis is a congenital metabolic bone disease characterized by skeletal sclerosis resulting from defective osteoclast-mediated bone resorption. Osteopetrosis has been described in several animal species (mouse, rat, and rabbit) and in children. Bone marrow transplantation, originally shown to reverse the skeletal sclerosis in some animal mutations, has been effective in curing osteopetrosis in some children. Unfortunately, not all children with osteopetrosis are candidates for or respond to bone marrow transplantation. Recent studies have shown that several animal mutations and some children inheriting osteopetrosis have significantly elevated serum levels of 1,25-(OH)2D. Based on the possibility that there may be a resistance to 1,25-(OH)2D, high-dose calcitriol therapy has been used to treat some children and stimulated some parameters of resorption. In this study, we have examined the effects of high-dose calcitriol therapy on various serum and skeletal parameters in the osteopetrotic rabbit. Mutant rabbits and normal littermates were given continuous infusions of calcitriol via subcutaneously implanted osmotic minipumps for 2 weeks at a dose of 0.5, 2.5, or 25 micrograms/kg/per day. Untreated mutant rabbits are hypocalcemic and hypophosphatemic in the presence of elevated serum 1,25-(OH)2 levels in comparison with their normal littermates. Calcitriol infusions resulted in dose-dependent increases in circulating 1,25-(OH)2D levels in both normal and mutant rabbits. However, evaluation of other serum parameters and the skeletal response demonstrated significant differences between osteopetrotic and normal rabbits. At the highest dose, normal animals rapidly became hypercalcemic and osteoporotic, accompanied by weight loss and a failure to thrive; mutants remained hypocalcemic and osteopetrotic but did not exhibit the deleterious physical effects seen in treated normal littermates. Although the number of osteoclasts increased in both mutants and normals, osteoclast phenotype in the former remained abnormal. These data indicate that although very high levels of circulating 1,25-(OH)2D were achieved in osteopetrotic mutants, activation of osteoclast-mediated bone resorption with subsequent improvement of skeletal sclerosis was not observed.

Auditory ossicle abnormalities and hearing loss in the toothless (osteopetrotic) mutation in the rat and their improvement after treatment with colony-stimulating factor-1.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.