Search

Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

Bone homeostasis is maintained primarily by the coordinated activities of two main cellular processes: osteoblast- mediated bone formation; and osteoclast-mediated bone resorption. Perturbation of these coordinated activities can lead to profound alterations in bone mass that have clinical relevance (1, 2). For example, osteoporosis is a common cause of vertebral and compression fractures among the elderly. In addition, rare disorders of increased bone mass caused by the absence or dysfunction of osteoclasts, namely osteopetrosis, can cause death during childhood as a result of ablation of the bone marrow space and skeletal abnormalities caused by the absence of normal bone-remodeling activity (3). The elevated osteoclast function is also associated with many other disorders of the skeleton, including periodontal disease, rheumatoid arthritis, and the metastatic spread of cancer to bone (4). Studies in animal models of osteopetrosis, and particularly in mutant mice, have provided significant new insights regarding the fundamental biology of osteoclasts (1, 5, 6). For example, some mutants completely lack osteoclasts, implying the involvement of the mutated genes in the early stages of osteoclast differentiation (7–10); other mutants produce osteoclasts with an immature appearance, highlighting the important roles of these mutated genes in terminal differentiation (11, 12); other mutants produce osteoclasts that appear normal but are defective in their ability to resorb bone, indicating that these genes are required for the maturation of the osteoclast and their normal bone-resorption capacity (13–17). These three different types of osteopetrosis mutants are phenotypically quite similar, except that the first appears to have reduced bone formation, suggesting that the absence of osteoclasts may also affect the activity of osteoblasts in these mice (18). This group of mutants is phenotypically reminiscent of human autosomal-recessive osteopetrosis. The mouse genes Atp6i, Gl, Traf-6, c-Src, and Clcn-7, respectively, when mutated, give rise to osteopetrosis with abnormal osteoclasts (13–17, 19). The human homologues for three of these genes (GL, ATP6i, and CLCN-7, respectively) are in fact disease genes for human autosomal-recessive osteopetrosis (13, 14, 19). However, mutations in these three genes alone do not appear to account for all cases of this disease in humans (13, 14, 19), indicating the existence of additional disease-causing genes for autosomal-recessive osteopetrosis. Osteoclasts are also vital for tooth eruption, a process that requires two highly coordinated processes: the formation of an eruption pathway; and the vertical movement of the developing tooth bud into the oral cavity (20). To form the eruption pathway, active alveolar bone resorption and the recruitment of numerous osteoclasts are required (21). Furthermore, recent studies have shown that a number of genes contribute to this process (21). Interestingly, we know very little about tooth root formation, an important physiological process. There are numerous osteopetrosis models but it is, in fact, not known whether or not these animals have defects in tooth root formation, and whether osteoclasts play any role in this developmental event. In this work, we have characterized a new mouse model of osteopetrosis that is caused by a spontaneous mutation: it is referred to as ntl (‘new toothless’) mouse. First, we investigated whether, how, and why odontomas were developed in this mutant. Second, we attempted to identify, by genetic mapping, the gene that is responsible for this osteopetrosis animal model. Third, we considered whether this model and other similar osteopetrosis models interfere with tooth root formation. Our results support the hypotheses that defects in osteoclasts disrupt the development and growth of the tooth root, and that homeostatic perturbation of the odontogenic precursors that are central to the root formation of mouse incisors is the primary cause of odontoma-like proliferations in this new mutant.

Granulin epithelin precursor (GEP) has been implicated in development, tissue regeneration, tumorigenesis, and inflammation. Herein we report that GEP stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo, and GEP-knockdown mice display skeleton defects. Similar to bone morphogenic protein (BMP) 2, application of the recombinant GEP accelerates rabbit cartilage repair in vivo. GEP is a key downstream molecule of BMP2, and it is required for BMP2-mediated chondrocyte differentiation. We also show that GEP activates chondrocyte differentiation through Erk1/2 signaling and that JunB transcription factor is one of key downstream molecules of GEP in chondrocyte differentiation. Collectively, these findings reveal a novel critical role of GEP growth factor in chondrocyte differentiation and the molecular events both in vivo and in vitro.—Feng, J. Q., Guo, F.-J., Jiang, B.-C., Zhang, Y., Frenkel, S., Wang, D.-W., Tang, W., Xie, Y., Liu, C.-J. Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis.

Background LRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear. Results In order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs. Conclusions Both KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.