Satoshi Wakisaka

Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification

Background: Molecular mechanisms controlling the late stages of endochondral ossification are unclear. Results: Osterix functions as a downstream and transcriptional partner of Runx2 and induces MMP13 during chondrocyte differentiation. Conclusion: Osterix is essential for late-stage endochondral ossification. Significance: Osterix affects the ossification of cartilage matrices and matrix vesicles and might be involved in the development of osteoarthritis and related disorders. Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

Distribution of peptidergic nerves

The dental pulp is a very unique tissue compared with other tissues. It is enclosed in a rigid dentine chamber and has a special character in that all external mechanical, chemical and thermal stimuli are recognized as pain. Numerous papers have been published in the past on morphological, physiological and pharmacological characteristics of the nerves in the dental pulp. According to the classical classification, the peripheral nerves are divided into sensory nerves and autonomic ones; the latter are also subdivided into sympathetic and parasympathetic nerves. Morphological analysis has revealed the distribution and morphology of nerves in the dental pulp of various mammals (Bradlow, 1939; Arwill, 1968; Corpron and Avery, 1973; Byers and Matthews, 1981; Gunji, 1982). However, these morphological studies could not clarify what kind of neurotransmitters or neuroregulators are involved in the intrapulpal nerves. The details of sympathetic and parasympathetic innervation in the dental pulp have been reported by several investigators, and are discussed elsewhere in this book.

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway.

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.

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.

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.

Activation of the Wnt/β-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 β-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/β-catenin pathway and the Wnt/β-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.

Morphological characteristics of the vallate papillae of the one-humped camel (Camelus dromedarius).

In this study, the morphology of the vallate papillae of camel was investigated using gross, light and scanning electron microscopy as well as immunohistochemistry. Vallate papillae were arranged along an identical line on each side of the lingual torus and revealed remarkable individual differences. However, each papilla – round or flat, small or large, single or paired – was surrounded by a prominent groove and an annular pad. Based on our findings, postnatal development and formation of new papillae occur in camel. Microscopically, taste buds were constantly observed along the medial wall epithelium, and in the papillary wall epithelium on both sides of the secondary groove apparently separating the vallate papillae. In addition, an aggregation of taste buds was occasionally observed at the bottom of the lateral wall epithelium. Using SEM, we observed several pits and microplicae on the surface of papillae as well as distinct taste pores on the peripheral parts of the dorsal surface. We demonstrated immunoreactivity of α‐gustducin only in mature taste buds. We conclude that the morphological features and microstructure of vallate papillae are a characteristic feature in camel compared to other ruminants. These features might have evolved to assist the camel in the manipulation and tasting of thin organic stiff plants that grow in its environment and therefore might have related to the feeding habits of the animal.

Morphological variations of the vallate papillae in some mammalian species

The morpho-structural characteristics of the vallate papillae of the tongue of rat, dog, donkey and buffalo were investigated by macroscopy and their microstructure by light and scanning electron microscopy (SEM). The numbers of vallate papillae varied among the different species. In rat, a single vallate papilla surrounded by incomplete groove and an annular fold was observed. Taste buds were detected along the entire length of the medial and lateral groove epithelium, but not in the papillary dome. In dog, some papillae lacking the annular pad had irregular ridges and grooves toward the center of the papillary surface, while other papillae had small secondary papillary grooves arising from the center of the papilla. Taste buds were located in the medial and lateral epithelium of both primary and secondary grooves as well as in the dome epithelium. In donkey, two papillae were frequently observed around the midline of the tongue root, and an additional papilla was found occasionally in the middle and associated with secondary papilla. In buffalo, several papillae were relatively small and variable in shape. With SEM, small ridges and grooves were found in the papillae of donkey and buffalo. In both species, taste buds were constantly observed along the medial wall epithelium, but no taste buds were found in the lateral wall. We conclude that the vallate papillae exhibited peculiar characteristics, which are species specific and might have a correlation with the variable feeding habits among these animals.

Kruppel-like factor 4 expression in osteoblasts represses osteoblast-dependent osteoclast maturation.

Kruppel-like factor 4 (KLF4) is a zinc-finger-type transcription factor with a restricted expression pattern during skeletal development. We have previously shown that KLF4 represses osteoblast mineralization concomitant with a down-regulation in the expression of a number of osteoblastic genes, both in vivo and in vitro. In addition to the cell-autonomous effects of KLF4 in osteoblasts, transgenic osteoblastic-KLF4 mice show severe defects in osteoclast maturation. Wild-type bone-marrow-derived macrophages co-cultured with KLF4-expressing osteoblasts exhibit reduced formation of multinuclear osteoclasts as compared with control cultures overexpressing green fluorescent protein. Significantly, the transduction of Runx2, a master regulator of osteoblastogenesis, together with KLF4 into osteoblasts restores the reduction in osteoclastogenesis induced by KLF4 alone. Various extracellular matrix molecules are down-regulated by KLF4 overexpression but this down-regulation can be partially restored by the co-transduction of Runx2. These results suggest that osteoblastic-KLF4 affects osteoclast maturation by regulating cell-matrix interactions and reinforce the importance of the regional down-regulation of KLF4 expression in the subset of osteoblasts for normal skeletal modeling and remodeling.

A novel adipokine C1q/TNF-related protein 1 (CTRP1) regulates chondrocyte proliferation and maturation through the ERK1/2 signaling pathway.

Adipose tissue-derived adipokines play important roles as regulators of skeletal growth and development. CTRP1, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the CTRP1 in chondrocyte proliferation and maturation using a mouse chondrocytic cell line, N1511. The CTRP1 protein was strongly expressed and predominantly distributed in the reserve and proliferative chondrocytes in the fetal growth plate and its mRNA decreased during the maturation of N1511 chondrocytes. Recombinant CTRP1 promoted proliferation of immature proliferating N1511 chondrocytes in a dose-dependent manner, whereas it inhibited maturation of maturing N1511 chondrocytes. The stimulatory effect of CTRP1 on chondrocyte proliferation was associated with activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway. On the other hand, the inhibitory effect of CTRP1 on chondrocyte maturation is associated with suppression of the ERK1/2 pathway. These results suggest a novel physiological role for CTRP1 in endochondral ossification.