The fibulins are a family of secreted glycoproteins associated with basement membranes, elastic fibers, and other matrices. They are expressed in a variety of tissues. Association with these matrix structures is mediated by their ability to interact with many extracellular matrix constituents. The seven members of the family are defined by the presence of two structural modules, a tandem repeat of epidermal growth factor-like modules and a unique C-terminal fibulin-type module. They act not only as intermolecular bridges within the extracellular matrix to form supramolecular structures, but also as mediators for cellular processes and tissue remodeling. These important functions of fibulins in a wide range of biological processes have been shown in in vitro systems, gene knockout mice, and human genetic disorders. In this review, we describe the structure and function of these proteins and discuss the implication of fibulins in development and diseases.

Cartilage plays an important role in mechanical load resistance and in skeletal structure support. It also serves as the skeletal template for endochondral ossification by which most bones in the body, such as long bones, are formed. In endochondral ossification, cartilage development is initiated by mesenchymal cell condensation, followed by a series of proliferation and differentiation processes. Cells undergoing condensation differentiate into chondrocytes, which then proliferate, produce type II collagen and form the proliferative zone of the cartilage molds. As development proceeds, chondrocytes in the center of the cartilage molds (prehypertrophic zone) cease proliferating and differentiate into type X collagen-producing hypertrophic chondrocytes to form the hypertrophic zone. Terminally differentiated hypertrophic chondrocytes mineralize the surrounding matrix. Eventually these cells die by apoptosis and are replaced by osteoblasts that form trabecular bone.The regulation of chondrocyte proliferation and differentiation must be tightly coordinated to allow formation of properly sized cartilage and bone (1). Parathyroid hormone-related peptide (PTHrP) 2 and parathyroid hormone (PTH) sustain chondrocyte proliferation and delay differentiation of the growth plate (2). PTHrP is expressed by perichondrial cells and chondrocytes in the upper region of growing cartilage. Mutant mice that are deficient in PTHrP (3), PTH (4), or its receptor (5) have short proliferative zones and accelerated chondrocyte differentiation, which results in abnormal endochondral bone formation. In contrast, mice that overexpress PTHrP have enlarged proliferative zones and delayed chondrocyte terminal differentiation (6). Humans with an activating mutation in the PTH/ PTHrP receptor develop Jansen metaphyseal chondrodysplasia, characterized by disorganization of the growth plates and delayed chondrocyte terminal differentiation (7). These results suggest that PTH/PTHrP signaling regulates skeletal development by promoting cell proliferation and inhibiting hypertrophic differentiation of chondrocytes.The binding of PTH/PTHrP to its receptor activates both G s and G q family heterotrimeric G proteins (8, 9). The activation of G s is necessary for cAMP production and protein kinase A (PKA) activation, which leads to phosphorylation of the cAMPresponse element-binding (CREB) family of transcription factors. CREB then induces genes such as the cyclin D1 and cyclin A genes. The activated cyclin/cyclin-dependent kinases in turn phosphorylate the retinoblastoma protein and its relative factors, which then dissociates the E2F transcription factor and subsequently activates the target genes necessary for DNA replication and cell cycle progression. Thus, CREB is a direct target of PKA and a downstream target of PTH/PTHrP/cAMP signaling and is required for chondrocyte proliferation (10, 11). How proliferation signaling is down-regulated in the prehypertrophic zone to stop proliferation and allow the switch to the postmitotic state is not well unde...

Pannexin 3 functions as an essential protein for Ca2+ and ATP transport and cell–cell communication during osteoblast differentiation

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain ␣-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas.Gangliosides, sialic acid-containing glycosphingolipids, are enriched in the nervous tissues of mammals and birds, and also distributed in various tissues and cells (1), playing regulatory roles with their polymorphic carbohydrate structures. In particular, b-series gangliosides including GD3, 1 GD2, GD1b, GT1b, and GQ1b have been considered to be critical as receptors for bacteria toxins (2) and are adhesion-associated (3, 4), neurite-inducing (5), cell growth-promoting (6, 7) and apoptosis-mediating molecules (8).To clarify the physiological functions of these b-series gangliosides in vivo, we have established a gene knockout mice line for ␣-2,8-sialyltransferase (9), which is responsible for the generation of all b-series gangliosides as well as c-series gangliosides. As expected, all b-series structures were deleted in the mutant mice, and a-series species were slightly increased instead. However, these b-series ganglioside-lacking mice showed an almost intact morphology of the brain and other nervous tissues, and no clear abnormal behaviors were detectable during the early period of life. Despite that GD3 has been considered to mediate Fas-induced apoptosis in lymphoid cells (8,10), no differences in the apoptotic reaction of lymphocytes between wild-type and mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are important in the repair of damaged nerves rather than in the differentiation of the nervous system. EXPERIMENTAL PROCEDURESGeneration of GD3 Synthase Gene Knockout Mice-The chromosomal GD3 synthase gene was isolated from the gt11 phage library using GD3 synthase cDNA (pD3T-31) and mapped as described previously (11). To distinguish the true GD3 synthase gene from pseudo-genes, in situ hybridization was performed, and the identity was confirmed based on the correspondence of the gene assignment between humans and mice. The neo r gene was inserted between the BalI and ...

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin ␤1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7. The extracellular matrix (ECM)4 plays active roles during organ development and in mature tissue functions. Effects on cell behavior and gene expression are often mediated through interactions between ECM molecules and cell surface receptors, leading to signal transduction across the plasma membrane. It is well known that many ECM proteins, including collagens, elastin, as well as other glycoproteins and proteoglycans are crucial for morphogenesis during embryonic development, and dysfunctions of these molecules cause congenital defects in humans. We have been interested in ECM molecules important in both tooth development and diseases. Previously, we identified the enamel matrix-specific protein ameloblastin that is essential for maintaining differentiated dental epithelial cells (ameloblasts) and for enamel formation (1-3). In this report, we characterized a new dentin matrix protein that we named TM14, which we identified in mouse tooth germ cDNA microarrays by differential hybridization (4).Mature teeth consist of two major mineralized tissues, dentin and enamel, the hardest tissue in the body. The development of these tissues is initiated by reciprocal interactions between the dental epithelium and mesenchyme, leading to the terminal differentiation of matrix-producing ameloblasts and odontoblasts, ...

Background:The role of dental epithelium in stem cell differentiation has not been clearly elucidated. Results: SP cells differentiated into odontoblasts by epithelial BMP4, whereas iPS cells differentiated into ameloblasts when cultured with dental epithelium. Conclusion: Stem cells can be induced to odontogenic cell fates when co-cultured with dental epithelium. Significance: This is the first report to show induction of ameloblasts from iPS cells.

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