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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., Highlights ▸ Rab13 and Rab8 both interact with Endospanin-2 and Endospanin-1. ▸ Rab13 and Rab8 binding to endospanins is specific; Rabs 7, 10, 11 and 32 do not bind. ▸ Rab13 binding to Endospanin-2 is nucleotide-dependent. ▸ Rab13 and Endospanin-2 colocalise in perinuclear vesicles and at the cell periphery.

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, 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.

Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTP-form of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts.

A proteinase was isolated from calf thymus that degraded pADPRT, histone H1 and alpha-casein in a Ca(2+)-dependent manner. In a five-step procedure, a homogenous proteinase was obtained with a subunit structure of 80 and 30 kDa. The amino-acid homology of an internal sequence as well as kinetic and inhibitor assays identified the proteinase as calpain I. It is suggested that even though the general substrate alpha-casein is widely used for the assaying of calpains, more appropriately physiological cellular components (pADPRT and histone H1) specify the thymus proteinase.

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.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

The role of the estrogen receptor α (ERα) in bone-forming cells is incompletely understood at present. To examine the in vivo effects of ERα in these cells, we generated a mouse strain in which the ERα gene is inactivated in osteoblasts via osteocalcin promoter-regulated cyclic recombinase (Cre) activity (ERα(ΔOB/ΔOB)). This enabled micro-computed tomography- and histomorphometry-based analysis of ERα-mediated effects in bone-forming cells in mice, in which the systemic ERα-mediated effects are intact. In female ERα(ΔOB/ΔOB) mice, trabecular and cortical bone volumes were significantly reduced (31.5 and 11.4%, respectively) at 3.5 mo of age compared with control ERα(fl/fl) animals, and their response to ovariectomy was small compared with that of controls. In contrast with females, no differences could be detected in the bone phenotype of young males, whereas in 6-mo-old ERα(ΔOB/ΔOB) males, trabecular bone volume (Tb.BV) was decreased (27.5%). The ERα inactivation-related effects were compared with those of controls having a similar genetic background. Parental osteocalcin-Cre mice did not show Cre-related changes. Our results suggest that in female mice, Tb.BV and cortical bone volume are critically dependent on the ERα regulation of osteoblasts, whereas in male mice, osteoblastic ERα is not required for the regulation of bone formation during rapid skeletal growth, but it is involved in the maintenance of Tb.BV.