Kazuaki Nonaka

Adiponectin inhibits Toll‐like receptor family‐induced signaling

Recent studies have shown that adiponectin, an adipocyte‐derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll‐like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR‐mediated nuclear factor‐κB (NF‐κB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR‐mediated NF‐κB signaling. gAd‐mediated inhibition of TLR‐mediated IκB phosphorylation and NF‐κB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage‐like cell response to TLR ligands via an unknown endogenous product(s).

Positionally‐dependent chondrogenesis induced by BMP4 is co‐regulated by sox9 and msx2

Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4‐soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up‐regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over‐expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2‐adenovirus to the proximal position inhibited BMP4‐induced chondrogenesis. Over‐expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down‐regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis. Dev Den;217:401–414.

Diurnal Variation of Human Sweet Taste Recognition Thresholds Is Correlated With Plasma Leptin Levels

OBJECTIVE—It has recently been proposed that the peripheral taste organ is one of the targets for leptin. In lean mice, leptin selectively suppresses gustatory neural and behavioral responses to sweet compounds without affecting responses to other taste stimuli, whereas obese diabetic db/db mice with defects in leptin receptor lack this leptin suppression on sweet taste. Here, we further examined potential links between leptin and sweet taste in humans. RESEARCH DESIGN AND METHODS—A total of 91 nonobese subjects were used to determine recognition thresholds using a standard stair-case methodology for various taste stimuli. Plasma leptin levels were determined by an enzyme-linked immunosorbent assay at several timepoints during the day under normal and restricted-meal conditions. RESULTS—The recognition thresholds for sweet compounds exhibited a diurnal variation from 0800 to 2200 h that parallels variation for leptin levels, with the lowest thresholds in the morning and the highest thresholds at night. This diurnal variation is sweet-taste selective—it was not observed in thresholds for other taste stimuli (NaCl, citric acid, quinine, and mono-sodium glutamate). The diurnal variation for sweet thresholds in the normal feeding condition (three meals) was independent of meal timing and thereby blood glucose levels. Furthermore, when leptin levels were phase-shifted following imposition of one or two meals per day, the diurnal variation of thresholds for sweet taste shifted in parallel. CONCLUSIONS—This synchronization of diurnal variation in leptin levels and sweet taste recognition thresholds suggests a mechanistic connection between these two variables in humans.

Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

During early mouse embryogenesis, cranial neural crest cells (CNCC) emigrate from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain into the first branchial arch‐derived maxillary and mandibular processes and there provide cell lineages for several phenotypes, including cartilage, bone, and tooth. Here, we report that Sox9 and Msx2 were coexpressed in a subpopulation of CNCC during their migration. Because Sox9 is a transactivator of chondrogenesis, and Msx genes can act as transcriptional repressors, we hypothesized that Sox9 expression indicates the determination of CNCC‐derived chondrogenic cell lineage and that Msx2 represses chondrogenic differentiation until CNCC migration is completed within the mandibular processes. To test whether Msx2 represses chondrogenesis, we designed experiments to inhibit Msx2 function in migratory CNCC in primary cultures through the expression of loss‐of‐function Msx2 mutants. We showed that infection of migratory CNCC with adenovirus Msx2 mutants accelerated the rate and extent of chondrogenesis, as indicated by the expression level of type II collagen and aggrecan, and the amount of alcian blue staining. Adenovirus infections did not apparently interfere with CNCC proliferation or migration. These findings suggest that an important early event in craniofacial morphogenesis is a transient expression of both Sox9 and Msx2 during emigration into the forming mandibular processes followed by restricted expression of Sox9 within CNCC‐ derived chondroprogenitor cells. We conclude that Msx2 serves as a repressor of chondrogenic differentiation during CNCC migration.

Adiponectin inhibits induction of TNF-α/RANKL-stimulated NFATc1 via the AMPK signaling

We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor‐alpha (TNF‐α)‐ and receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF‐α/RANKL‐induced differentiation of osteoclasts by interfering with TNF receptor‐associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP‐activated protein kinase abrogated gAd inhibition for TNF‐α/RANKL‐induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation.

Essential Roles of Ameloblastin in Maintaining Ameloblast Differentiation and Enamel Formation

During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts.

Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem cell resource for regenerative medicine.

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine.

TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2(flox/flox)) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-beta2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2(flox/flox) mice. Thus, TGF-beta induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.

Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.

TGF-β mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development

Transforming growth factor-beta (TGF-beta) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-beta signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2(fl/fl);Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-beta signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2(fl/fl);Wnt1-Cre mice. Moreover, exogenous TGF-beta can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2(fl/fl);Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2(fl/fl);Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-beta signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-beta signaling.