Wnt/␤-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation. Introduction:The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism. Materials and Methods:In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1 +/− animals. Results: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1 +/− mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold. Conclusions: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.

The dickkopf (dkk) gene family encodes secreted antagonists of Wnt signalling proteins, which have important functions in the control of cell fate, proliferation, and cell polarity during development. Here, we report the isolation, from a regeneration-specific signal peptide screen, of a novel dickkopf gene from the fresh water cnidarian Hydra. Comparative sequence analysis demonstrates that the Wnt antagonistic subfamily Dkk1/Dkk2/Dkk4 and the non-modulating subfamily Dkk3 separated prior to the divergence of cnidarians and bilaterians. In steady-state Hydra, hydkk1/2/4-expression is inversely related to that of hywnt3a. hydkk1/2/4 is an early injury and regeneration responsive gene, and hydkk1/2/4-expressing gland cells are essential for head regeneration in Hydra, although once the head has regenerated they are excluded from it. Activation of Wnt/␤-Catenin signalling leads to the complete downregulation of hydkk1/2/4 transcripts. When overexpressed in Xenopus, HyDkk1/2/4 has similar Wnt-antagonizing activity to the Xenopus gene Dkk1. Based on the corresponding expression patterns of hydkk1/2/4 and neuronal genes, we suggest that the body column of Hydra is a neurogenic environment suppressing Wnt signalling and facilitating neurogenesis.

Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.