RANK-dependent autosomal recessive osteopetrosis: characterization of five new cases with novel mutations.

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis.

Transfection of osteoclasts and osteoclast precursors.

Osteoclasts and their precursors have traditionally been considered difficult cells to transfect using standard approaches. Here, we describe several methods for transfection of mature osteoclasts and their precursors using the Amaxa™ Nucleofector system, lentiviruses, and adenoviruses.

The skeleton: a multi-functional complex organ: the role of key signalling pathways in osteoclast differentiation and in bone resorption.

Osteoclasts are the specialised cells that resorb bone matrix and are important both for the growth and shaping of bones throughout development as well as during the process of bone remodelling that occurs throughout life to maintain a healthy skeleton. Osteoclast formation, function and survival are tightly regulated by a network of signalling pathways, many of which have been identified through the study of rare monogenic diseases, knockout mouse models and animal strains carrying naturally occurring mutations in key molecules. In this review, we describe the processes of osteoclast formation, activation and function and discuss the major transcription factors and signalling pathways (including those that control the cytoskeletal rearrangements) that are important at each stage.

Signal peptide mutations in RANK prevent downstream activation of NF-κB.

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins.

Functional interaction between sequestosome-1/p62 and autophagy-linked FYVE-containing protein WDFY3 in human osteoclasts.

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype. Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates. Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.