Objective. To identify the mediator responsible for the impact of chronic inflammation on skeletal development in children (bone loss, defective peak bone mass accrual, stunted growth), we evaluated the effects of chronic interleukin-6 (IL-6) overexpression on the skeletons of growing prepubertal mice.Methods. We studied IL-6-transgenic mice that had high circulating IL-6 levels since birth. Trabecular and cortical bone structure were analyzed by microcomputed tomography. Epiphyseal ossification, growth plates, and calvariae were studied by histology/ histomorphometry. Osteoclastogenesis, osteoblast function/differentiation, and the effects of IL-6 on bone cells were studied in vitro. Osteoblast gene expression was evaluated by reverse transcriptase-polymerase chain reaction. The mineral apposition rate was evaluated dynamically in cortical bone by in vivo double fluorescence labeling.Results. In prepubertal IL-6-transgenic mice, we observed osteopenia, with severe alterations in cortical and trabecular bone microarchitecture, as well as uncoupling of bone formation from resorption, with decreased osteoblast and increased osteoclast number and activity. Increased osteoclastogenesis and reduced osteoblast activity, secondary to decreased precursor proliferation and osteoblast function, were present. IL-6-transgenic mice also showed impaired development of growth plates and epiphyseal ossification centers. Intramembranous and endochondral ossification and the mineral apposition rate were markedly affected, showing the presence of defective ossification.Conclusion. Chronic overexpression of IL-6 alone induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development. We hypothesize that IL-6-modifying drugs may reduce skeletal defects and prevent the growth retardation associated with these diseases.

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Interleukin-6 (IL-6) and c-src impair osteoblast maturation in vitro and in vivo. Given the similar effects of these factors, they are likely to establish a functional loop to maintain osteoblasts in a less mature status. Here we describe a pathway whereby c-src stimulates IL-6 expression through the sTAT3 factor, which, in response to IL-6 induces insulin-like growth factor 5 (IGFBP5), a c-src activating factor that amplifies this loop only in immature osteoblasts. In contrast, in mature osteoblasts, IGFBP5 is enhanced by Runx2, but is no longer able to stimulate c-src activation, as this tyrosine kinase at this stage is downregulated. We find that the IGFBP5 produced by osteoblasts stimulates osteoclastogenesis and bone resorption, acting as an osteoblast-osteoclast coupling factor. Finally, we demonstrate that the integrated actions of c-src, IL-6 and IGFBP5 also have a role in vivo. We conclude that this pathway is relevant for bone metabolism, both in physiological and in pathological conditions.

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk. Introduction:The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis. Materials and Methods:We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out. Results: Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACPcontaining conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling. Conclusions: We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.

Src is the representative member of the Src-family kinases (SFKs), a group of tyrosine kinases involved in several cellular processes. Its main function has been for long confined to the plasma membrane/cytoplasm compartment, being a myristoylated protein anchored to the cell membrane and functioning downstream to receptors, most of them lacking intrinsic kinase activity. In the last decades, new roles for some SFKs have been described in the nuclear compartment, suggesting that these proteins can also be involved in directly regulating gene transcription or nucleoskeleton architecture. In this review, we focused on those nuclear functions specifically attributable to Src, by considering its function as both tyrosine kinase and adapting molecule. In particular, we addressed the Src involvement in physiological as well as in pathological conditions, especially in tumors.

Osteosarcoma is the most common primary bone cancer and the most frequent cause of bone cancer-related deaths in children and adolescents. Osteosarcoma cells are able to establish a crosstalk with resident bone cells leading to the formation of a deleterious vicious cycle. We hypothesized that osteosarcoma cells can release, in the bone microenvironment, transforming Extracellular Vesicles (EVs) involved in regulating bone cell proliferation and differentiation, thereby promoting tumor growth. We assessed EV production by three osteosarcoma cell lines with increasing aggressiveness in order to investigate their roles in the communication between osteosarcoma cells and normal recipient cells. Osteosarcoma-derived EVs were used to treat the murine fibroblast cell line NIH3T3 and to study the induction of tumor-like phenotypes. Our results showed that osteosarcoma cell lines are able to produce EVs that fuse to recipient cells, with a very high uptake efficiency. The treatment of recipient NIH3T3 with osteosarcoma-derived EVs induced substantial biological and functional effects, as an enhanced proliferation and survival capability under starved conditions, high levels of activated survival pathways, an increased migration, adhesion, and 3D sphere formation and the acquired capability to grow in an anchorage-independent manner. Moreover, in murine NIH3T3 we found human mRNAs of TNF-α, IL-6, and TGF-β, as well as a de novo expression of murine MMP-9 and TNF-α following the treatment of human osteosarcoma-derived EVs.

Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low- versus high-risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well-characterized membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so-far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high-stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan-Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival (OS) of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N-myristoyltransferase enzymes expression and activity and the Src nuclear content. In conclusion, these results described a so-far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N-myristoyltransferase enzymes and the Src subcellular localization was described as well.