Syntaxin 4a Regulates Matrix Vesicle-Mediated Bone Matrix Production by Osteoblasts.

Osteoblasts secrete matrix vesicles and proteins to bone surfaces, but the molecular mechanisms of this secretion system remain unclear. The present findings reveal the roles of important genes in osteoblasts involved in regulation of extracellular matrix secretion. We especially focused on "soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor" (SNARE) genes and identified notable Syntaxin 4a (Stx4a) expression on the basolateral side of the plasma membrane of osteoblasts. Furthermore, Stx4a overexpression was found to increase mineralization by osteoblastic cells, whereas Stx4a knockdown reduced levels of mineralization. Also, BMP-4 and IGF-1 induced the localization of Stx4a to the basolateral side of the cells. To examine the function of Stx4a in osteoblasts, we generated osteoblast-specific Stx4a conditional knockout mice, which demonstrated an osteopenic phenotype due to reduced matrix secretion. Bone mineral density, shown by peripheral quantitative computed tomography (pQCT), was reduced in the femur metaphyseal and diaphyseal regions of Stx4a osteoblast-specific deficient mice, whereas bone parameters, shown by micro-computed tomography (µCT) and bone histomorphometric analysis, were also decreased in trabecular bone. In addition, primary calvarial cells from those mice showed decreased mineralization and lower secretion of matrix vesicles. Our findings indicate that Stx4a plays a critical role in bone matrix production by osteoblasts. © 2016 American Society for Bone and Mineral Research.

A Putative Association of a Single Nucleotide Polymorphism in GPR126 with Aggressive Periodontitis in a Japanese Population.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

Expression pattern of zinc-finger transcription factor Odd-skipped related 2 in murine development and neonatal stage.

The Odd-skipped gene, first identified as a Drosophila pair-rule zinc-finger transcription factor, plays an important role in Drosophila development. The mammalian homolog, Odd-skipped related 2 (Osr2), regulates limb, tooth, and kidney development in mouse embryos. However, the detailed expression pattern of Osr2 during neonatal development remains unclear. In this study, we investigated Osr2 expression patterns in mouse neonatal and embryo tissues using qPCR and in situ hybridization methods. First, we examined the tissue distribution of Osr2 by qPCR, and found it to be highly expressed in the uterus and moderately in the testes, small intestine, and prostate. That expression was also found in eye, kidney, placenta, lung, thymus, lymph node, stomach, and skeletal muscle tissues, and in all embryonic stages. On the other hand, Osr2 was not expressed in brain, heart, liver, or spleen samples. Next, we examined the tissue localization of Osr2 using in situ hybridization. Osr2 was found in the craniofacial region on E13.5, with notable expression in dental germ mesenchyme as well as the renal corpuscle on E17.5. As for neonatal tissues, Osr2 was expressed in the dental papilla, dental follicle, Harderian gland, nasal bone, eyelid dermis, synovial joint, and tibial subcutis. Our findings suggest that Osr2 functions in reproductive system organs, such as the uterus, testes, prostate, placenta, and ovaries. Furthermore, based on its expression in kidney, Harderian gland, eyelid dermis, and tibial subcutis tissues, this transcription factor may be involved in hormone synthesis and function. Together, our results demonstrate the role of Osr2 in postnatal development and embryogenesis.

Activation of the Wnt/ß-catenin pathway and tissue inhibitor of metalloprotease 1 during tertiary dentinogenesis.

Tertiary dentin is deposited inside teeth after various stimuli and serves as a major defensive wall to preserve pulp cells. However, the molecular mechanisms of the activation of quiescent odontoblasts, immature pulp cells and tertiary dentin formation are still unclear. Therefore, we performed a comprehensive gene expression analysis of pulp cells after cavity preparation of 9-week-old rat molars to clarify the critical molecules in tertiary dentinogenesis. As a result, mRNA expression of various molecules was up- or down-regulated. Notably, several members of the matrix metalloprotease family and their endogenous inhibitors were up-regulated after cavity preparation. In situ hybridization showed that tissue inhibitor of metalloprotease 1 (Timp1) was widely and continuously distributed in the pulp beneath the cavity in vivo. We also observed accumulation of ß-catenin in the pulp cells beneath the cavity by fluorescence immunohistochemistry. Furthermore, Timp1 transcription was repressed by a dominant-negative TCF4 in immature undifferentiated mesenchymal cells, but not altered in mature odontoblast-like cells. These results indicate that cavity preparation may activate the Wnt/ß-catenin pathway and the Wnt/ß-catenin pathway and Timp1 may be correlatively involved in pulp repair. Timp1 might play crucial roles in reactivation of immature pulp cells for tertiary dentinogenesis.

Trps1 is necessary for normal temporomandibular joint development.

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Krüppel-like factor 4 regulates membranous and endochondral ossification.

Krüppel-like factor 4 (KLF4/GKLF/EZF) is a zinc finger type of transcription factor highly expressed in the skin, intestine, testis, lung and bone. The role played by Klf4 has been studied extensively in normal epithelial development and maintenance; however, its role in bone cells is unknown. Previous reports showed that Klf4 is expressed in the developing flat bones but its expression diminishes postnatally. We now show that in the developing long bones, Klf4 is expressed in the perichondrium, trabecular osteoblasts and prehypertrophic chondrocytes. In contrast, osteoblasts lining at the surface of the bone collar showed extremely low levels of Klf4 expression. To investigate the possible roles played by Klf4 during skeletal development, we generated transgenic mice expressing Klf4 under mouse type I collagen regulatory sequence. Transgenic mice exhibited severe skeletal deformities and died soon after birth. Transgenic mice showed delayed formation of the calvarial bones; and over-expressing Klf4 in primary mouse calvarial osteoblasts in culture resulted in strong repression of mineralization indicating that this regulation of Klf4 is through an osteoblast-autonomous effect. Surprisingly, long bones of the transgenic mice exhibited delayed marrow cavity formation. Even at E18.5, the presumptive marrow space was occupied by cartilage anlage and invasion of the vascular endothelial cells and osteoclasts were seldom observed. Instead of entering the cartilage anlage, osteoclasts accumulated at the periosteum in the transgenic mice. Significantly, osteocalcin, which is known to chemotact osteoclasts, was up-regulated at the perichindrium as early as E14.5 in the mutants. In vitro studies showed that this induction of osteocalcin by Klf4 was regulated at its transcriptional level. Our results demonstrate that Klf4 regulates normal skeletal development through coordinating the differentiation and migration of osteoblasts, chondrocytes, vascular endothelial cells and osteoclasts.

Hand2 regulates chondrogenesis in vitro and in vivo.

Hand2 is a transcription factor of the basic helix-loop-helix family that plays essential roles during development. Hand2 determines the anterior-posterior axis during limb development and there is also substantial evidence that Hand2 regulates limb skeletogenesis. However, little is known about how Hand2 might regulate skeletogenesis. Here we show that, in a limb bud micromass culture system, over-expression of Hand2 represses chondrogenesis and the expression of chondrocytic genes, Sox9, type II collagen and aggrecan. Furthermore, we show that Hand2 is induced by the activation of canonical Wnt signaling, which strongly represses chondrogenesis. Surprisingly, Hand2 repressed chondrogenesis in a DNA binding- and dimer formation-independent manner. To examine the in vivo role of Hand2 in mice, we targeted the expression of Hand2 to the cartilage using regulatory elements from the collagen II gene. The resulting transgenic mice displayed a dwarf phenotype, with axial, appendicular and craniofacial skeletal deformities. Hand2 strongly inhibited chondrogenesis in the axial and cranial base skeleton. In the sternum, Hand2 inhibited endochondral ossification by slowing chondrocyte maturation. These data support a model of Hand2 regulating endochondral ossification via at least two steps: (1) determination of the site of chondrogenesis by outlining the region of the future cartilage template and (2) regulation of the rate of chondrocyte maturation.