Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

We studied whether physiological concentration of short-chain fatty acids (SCFAs) affects colonic transit and colonic motility in conscious rats. Intraluminal administration of SCFAs (100-200 mM) into the proximal colon significantly accelerated colonic transit. The stimulatory effect of SCFAs on colonic transit was abolished by perivagal capsaicin treatment, atropine, hexamethonium, and vagotomy, but not by guanethidine. The stimulatory effect of SCFAs on colonic transit was also abolished by intraluminal pretreatment with lidocaine and a 5-hydroxytryptamine (HT)(3) receptor antagonist. Intraluminal administration of SCFAs provoked contractions at the proximal colon, which migrated to the mid- and distal colon. SCFAs caused a significant increase in the luminal concentration of 5-HT of the vascularly isolated and luminally perfused rat colon ex vivo. It is suggested that the release of 5-HT from enterochromaffin cells in response to SCFAs stimulates 5-HT(3) receptors located on the vagal sensory fibers. The sensory information is transferred to the vagal efferent and stimulates the release of acetylcholine from the colonic myenteric plexus, resulting in muscle contraction.

Gangliosides, sialic acid-containing glyco-

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

Nile red, Sudan III, and oil red O have been used to stain lipid droplets (LDs) for fluorescence microscopy. We noticed that LDs labeled by Nile red are different in appearance from those stained by the latter two dyes. To understand the cause of the difference, we used sequential labeling procedures (first LD stain-photography-quenching-second LD stain-photography), and examined the effect of several factors. Immunofluorescence labeling for adipose differentiation-related protein (ADRP), an LD marker, was also observed comparatively with the lipid stains. As a result, we found that ethanol and isopropanol used for Sudan III and oil red O staining, respectively, and glycerol used for mounting, cause fusion of adjacent LDs even in glutaraldehyde-fixed samples. By the same treatment, immunofluorescence labeling for ADRP was dislocated to the rim of large LDs that were formed as a result of the artifactual fusion. The result indicates that the LD structure can be better observed with Nile red than with Sudan III or oil red O.

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn ؊/؊ ). Epfn ؊/؊ mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn ؊/؊ molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

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.