ABSTRACT
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)(2)D(3)] 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 Ca(2+) 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 (VDR(ΔOb/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 VDR(ΔOb/ΔOb) mice because of decreased expression of receptor activator of nuclear factor kappa-B ligand (an essential molecule in osteoclastogenesis) in VDR(ΔOb/ΔOb) osteoblasts. These findings establish that VDR in osteoblasts is a negative regulator of bone mass control.
Subject(s)
Bone and Bones/anatomy & histology , Bone and Bones/metabolism , Osteoblasts/metabolism , Receptors, Calcitriol/metabolism , Animals , Bone Density , Bone Resorption/genetics , Bone Resorption/metabolism , Bone Resorption/pathology , Calcium/metabolism , Female , Gene Expression , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Osteoclasts/metabolism , Receptors, Calcitriol/deficiency , Receptors, Calcitriol/genetics , Rickets/genetics , Rickets/metabolism , Rickets/pathologyABSTRACT
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.
Subject(s)
Drosophila Proteins , Drosophila/embryology , Gene Expression Regulation, Developmental , RING Finger Domains/genetics , Ubiquitin-Protein Ligases , Animals , Cadherins/metabolism , Cells, Cultured , Drosophila/enzymology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Embryo, Nonmammalian/metabolism , Embryonic Development/genetics , Mutation , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
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.
Subject(s)
Adherens Junctions/metabolism , Epithelial Cells/cytology , Trachea/cytology , src-Family Kinases/metabolism , Animals , Cadherins/genetics , Cadherins/metabolism , Drosophila/embryology , Drosophila Proteins/metabolism , Enzyme Activation , Morphogenesis , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Trachea/metabolismABSTRACT
Vitamin D receptor (VDR) mediates a wide variety of vitamin D actions through transcriptional controls of target genes as a ligand-dependent transcription factor. The transactivation by VDR is known to associate with two co-activator complexes, DRIP/TRAP and p160/CBP, through physical interaction with DRIP205 and p160 members (TIF2) components, respectively. However, functional difference between the two co-activator complexes for VDR co-activation remains unclear. In the present study, to address this issue, a series of point mutants in VDR helix 12 were generated to test the functional association. Alanine replacement of VDR valine 418 resulted in loss of DRIP205 interaction, but it was still transcriptionally potent with ability to interact with TIF2. Surprisingly, the V421A mutant was only partially impaired in transactivation without co-activator interaction, implying presence of a putative co-activator/complex. Thus, these findings suggest that ligand-induced transcriptional controls by VDR require a number of known and unknown co-regulator complexes, that may support the tissue-specific function of VDR.
Subject(s)
Nuclear Receptor Coactivator 2/metabolism , Receptors, Calcitriol/metabolism , Transcription Factors/metabolism , Transcriptional Activation/physiology , Acid Phosphatase/metabolism , Amino Acid Substitution , Animals , COS Cells , Carrier Proteins/metabolism , Chlorocebus aethiops , Corticosterone , Humans , Isoenzymes/metabolism , Mediator Complex Subunit 1 , Organ Specificity , Protein Binding , Protein Structure, Secondary/genetics , Receptors, Calcitriol/genetics , Tartrate-Resistant Acid PhosphataseABSTRACT
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.
