Kalman G. Büki

The critical role of IL-34 in osteoclastogenesis.

It has been widely believed that the cytokines required for osteoclast formation are M-CSF (also known as CSF-1) and RANKL. Recently, a novel cytokine, designated IL-34, has been identified as another ligand of CSF1R. This study was to explore the biological function, specifically osteoclastogenesis and bone metabolism, of the new cytokine. We produced recombinant mouse IL-34 and found that together with RANKL it induces the formation of osteoclasts both from splenocytes as well as dose-dependently from bone marrow cells in mouse and these cells also revealed bone resorption activity. It also promotes osteoclast differentiation from human peripheral blood mononucleated cells. Finally, we show that systemic administration of IL-34 to mice increases the proportion of CD11b+ cells and reduces trabecular bone mass. Our data indicate that IL-34 is another important player in osteoclastogenesis and thus may have a role in bone diseases. Strategies of targeting CSF1/CSF1R have been developed and some of them are already in preclinical and clinical studies for treatment of inflammatory diseases. Our results strongly suggest the need to revisit these strategies as they may provide a new potential pharmaceutical target for the regulation of bone metabolism in addition to their role in the treatment of inflammatory diseases.

Inactivation of the androgen receptor in bone-forming cells leads to trabecular bone loss in adult female mice

Removal of the androgen receptor (AR) from bone-forming cells has been shown to reduce trabecular bone volume in male mice. In female mice, the role of AR in the regulation of bone homeostasis has been poorly understood. We generated a mouse strain in which the AR is completely inactivated only in mineralizing osteoblasts and osteocytes by breeding mice carrying osteocalcin promoter-regulated Cre-recombinase with mice possessing loxP recombination sites flanking exon 2 of the AR gene (AR(ΔOB/ΔOB) mice). In female AR(ΔOB/ΔOB) mice, the trabecular bone volume was reduced owing to a smaller number of trabeculae at 6 months of age compared with the control AR(fl/fl) animals. In male AR(ΔOB/ΔOB) mice, an increase in trabecular bone separation could already be detected at 3.5 months of age, and at 6 months, the trabecular bone volume was significantly reduced compared with that of male AR(fl/fl) mice. No AR-dependent changes were observed in the cortical bone of either sex. On the basis of micro-computed tomography and histomorphometry, we conclude that in male mice, the AR is involved in the regulation of osteoclast number by osteoblasts, whereas in female mice, the lack of the AR in the bone-forming cells leads to a decreased number of trabeculae upon aging.

Fam3c modulates osteogenic differentiation by down-regulating Runx2

Fam3c, a cytokine-like protein, is a member of the Fam3 family (family with sequence similarity 3) and has been implicated to play a crucial role in Epithelial-to- mesenchymal transition (EMT) and subsequent metastasis during cancer progression. A few independent genome-wide association studies on different population cohorts predicted the gene locus of Fam3c to be associated with bone mineral density and fractures. In this study, we examined the role of Fam3c during osteoblast differentiation. Fam3c was found to be expressed during osteogenic differentiation of both primary bone marrow stromal cells and MC3T3-E1 pre-osteoblasts. In differentiating osteoblasts, knockdown of Fam3c increased alkaline phosphatase expression and activity whereas overexpression of Fam3c reduced it. Furthermore, overexpression of Fam3c caused reduction of Runx2 expression at both mRNA and protein levels. Fam3c was localized in the cytoplasm and it was not secreted outside the cell during osteoblast differentiation and therefore, may function intracellularly. Furthermore, Fam3c and TGF-β1 were found to regulate each other reciprocally. Our findings therefore suggest a functional role of Fam3c in the regulation of osteoblast differentiation.

Fam3c modulates osteogenic cell differentiation and affects bone volume and cortical bone mineral density.

Fam3c, a cytokine-like growth factor, has been suggested to have a role in epithelial-to-mesenchymal transition (EMT), tumor growth and metastasis. A single-nucleotide polymorphism affecting bone mineral density has been found in the first intron of the Fam3c gene in a study analyzing an Asian population cohort. Other independent studies on different population cohorts have found the fam3c locus to be associated with bone mineral density and fractures. In order to investigate the role of Fam3c in bone biology, we have generated a Fam3c knock-out (KO) mouse strain. The Fam3c KO mice were found to have normal appearance, behavior and fertility, but small changes in bone morphology and content were also observed. Micro-CT analysis of tibiae of the female mice revealed decreased number of trabeculae. In male mice the changes in the bone phenotype were smaller, but hematological changes were observed. Furthermore, there was a negative correlation between body weight and tibial trabecular and cortical bone volume in the male KO mice. There was a small increase in cortical bone mineral density, but in the lateral direction of tibiae the breaking strength was reduced. Fam3c KO bone marrow cells showed accelerated osteogenic differentiation and mineralization in vitro. The reduced number of bone trabeculae in Fam3c KO mice and the stimulated osteogenic differentiation indicate a role for Fam3c in osteoblast differentiation and bone homeostasis.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

Novel interaction of Rab13 and Rab8 with endospanins

Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP‐bound and inactive GDP‐bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two‐hybrid system. We found that Rab13 binds to the C‐terminus of Endospanin‐2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin‐2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin‐1, a close homolog of Endospanin‐2. Rab13 and Endospanin‐2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin‐2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13–Endospanin‐2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin‐2 and Endospanin‐1. The role of this interaction in cell physiology, however, remains to be elucidated.