The physiological and beneficial actions of vitamin D in bone health have been experimentally and clinically proven in mammals. The active form of vitamin D [1α,25(OH)2D3] binds and activates its specific nuclear receptor, the vitamin D receptor (VDR). Activated VDR prevents the release of calcium from its storage in bone to serum by stimulating intestinal calcium absorption and renal reabsorption. However, the direct action of VDR in bone tissue is poorly understood because serum Ca2+ homeostasis is maintained through tightly regulated ion transport by the kidney, intestine, and bone. In addition, conventional genetic approaches using VDR knockout (VDR-KO, VDR−/−) mice could not identify VDR action in bone because of the animals' systemic defects in calcium metabolism. In this study, we report that systemic VDR heterozygous KO (VDR+/L−) mice generated with the Cre/loxP system as well as conventional VDR heterozygotes (VDR+/−) showed increased bone mass in radiological assessments. Because mineral metabolism parameters were unaltered in both types of mice, these bone phenotypes imply that skeletal VDR plays a role in bone mass regulation. To confirm this assumption, osteoblast-specific VDR-KO (VDRΔOb/ΔOb) mice were generated with 2.3 kb α1(I)-collagen promoter-Cre transgenic mice. They showed a bone mass increase without any dysregulation of mineral metabolism. Although bone formation parameters were not affected in bone histomorphometry, bone resorption was obviously reduced in VDRΔOb/ΔOb mice because of decreased expression of receptor activator of nuclear factor kappa-B ligand (an essential molecule in osteoclastogenesis) in VDRΔOb/ΔOb osteoblasts. These findings establish that VDR in osteoblasts is a negative regulator of bone mass control.

The downregulation of E-cadherin by Src promotes epithelial to mesenchymal transition and tumorigenesis. However, a simple loss of cell adhesion is not sufficient to explain the diverse developmental roles of Src and metastatic behavior of viral Src-transformed cells. Here, we studied the functions of endogenous and activated forms of Drosophila Src in the context of tracheal epithelial development, during which extensive remodeling of adherens junctions takes place. We show that Src42A is selectively activated in the adherens junctions of epithelia undergoing morphogenesis. Src42A and Src64B are required for tracheal development and to increase the rate of adherens junction turnover. The activation of Src42A caused opposing effects: it reduced the E-cadherin protein level but stimulated transcription of the E-cadherin gene through the activation of Armadillo and TCF. This TCF-dependent pathway was essential for the maintenance of E-cadherin expression and for tissue integrity under conditions of high Src activity. Our data suggest that the two opposing outcomes of Src activation on E-cadherin facilitate the efficient exchange of adherens junctions, demonstrating the key role of Src in the maintenance of epithelial integrity. KEY WORDS:Src, E-cadherin, Armadillo, Drosophila, Trachea, Cancer Development 135, 1355Development 135, -1364Development 135, (2008Development 135, ) doi:10.1242 Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan. DEVELOPMENT 1356 strain carrying the trachea-specific btl-Gal4 driver and the UAS-GFPmoesin marker. An additional 142 lines lacking the rough eye phenotype were also tested. GS11022 (Src42A) and GS9618 (Src64B) were analyzed for further study. Fly stocks and geneticsWe used strong loss-of-function alleles of Src genes: Src42A 26-1 (Takahashi et al., 2005), Src42A myri (Tateno et al., 2000) and Src64B P1 (Dodson et al., 1998). The following strains were used in this study: trachealess enhancer trap line 1-eve-1 (Perrimon et al., 1991), UAS-wg (Lawrence et al., 1995), UAS-arm S10 (Pai et al., 1997), UAS-TCF⌬N (van de Wetering et al., 1997), UAS-E-cadherin-GFP (Oda and Tsukita, 1999b), UAS-D␣-catenin-GFP (Oda and Tsukita, 1999a), shg-lacZ , UAS-GFP-moesin (Chihara et al., 2003), YF (Tateno et al., 2000), UAS-Src42A-RNAi (NIG Stock Center) and btl-Gal4 (Shiga et al., 1996). Src42A DN was constructed by introducing the K295M mutation at the catalytic center of the kinase domain and was cloned into the pUAST vector (Brand and Perrimon, 1993). Immunostaining and imagingThe following primary antibodies were used: rat anti-Esg (Fuse et al., 1994); mouse anti-tracheal luminal antigen 2A12, mouse anti-Armadillo N27A1 and mouse anti-septin 4C9H4 (Developmental Studies Hybridoma Bank); rabbit anti-␤-galactosidase (Cappel); mouse anti-GFP B-2 and rabbit anti-GFP (MBL); rabbit anti-Src PY418 (Biosource International); rat anti-E-cadherin (DCAD2) (Oda et al., 1994) and rabbit anti-Src42A (Takahashi et al., 2005). Chicken anti-Src42A antibody ...

Chondromodulin I (ChM-I) was supposed from its limited expression in cartilage and its functions in cultured chondrocytes as a major regulator in cartilage development. Here, we generated mice deficient in ChM-I by targeted disruption of the ChM-I gene. No overt abnormality was detected in endochondral bone formation during embryogenesis and cartilage development during growth stages of ChM-I−/− mice. However, a significant increase in bone mineral density with lowered bone resorption with respect to formation was unexpectedly found in adult ChM-I−/− mice. Thus, the present study established that ChM-I is a bone remodeling factor

Hakai is a RING finger type E3 ubiquitin ligase that is highly conserved in metazoans. Mammalian Hakai was shown to bind and ubiquitinate the intracellular domain of E-cadherin, and this activity is implicated in down-regulation of E-cadherin during v-Src-induced cellular transformation. To evaluate this model in vivo, we studied the function of the Drosophila homologue of Hakai. In cultured S2 cells, Drosophila Hakai and E-cadherin (Shotgun) formed a complex in a way distinct from the interaction described for mammalian counterparts. Hakai null mutants died during larval stages but this lethality could be offset by a HA-tagged Hakai construct. While zygotic Hakai function was dispensable for cell proliferation and differentiation in the wing disc epithelium, maternal Hakai mutants showed a variety of defects in epithelial integrity, including stochastic loss of E-cadherin expression and reduction of aPKC; defects in cell specification and cell migration were also observed. No increase of E-cadherin, however, was observed. Regulation of multiple target proteins under control of Hakai is, therefore, essential for early embryonic morphogenesis in Drosophila.

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