Lrig1-expression confers suppressive function to CD4+ cells and is essential for averting autoimmunity via the Smad2/3/Foxp3 axis.

Regulatory T cells (Treg) are CD4+ T cells with immune-suppressive function, which is defined by Foxp3 expression. However, the molecular determinants defining the suppressive population of T cells have yet to be discovered. Here we report that the cell surface protein Lrig1 is enriched in suppressive T cells and controls their suppressive behaviors. Within CD4+ T cells, Treg cells express the highest levels of Lrig1, and the expression level is further increasing with activation. The Lrig1+ subpopulation from T helper (Th) 17 cells showed higher suppressive activity than the Lrig1- subpopulation. Lrig1-deficiency impairs the suppressive function of Treg cells, while Lrig1-deficient naïve T cells normally differentiate into other T cell subsets. Adoptive transfer of CD4+Lrig1+ T cells alleviates autoimmune symptoms in colitis and lupus nephritis mouse models. A monoclonal anti-Lrig1 antibody significantly improves the symptoms of experimental autoimmune encephalomyelitis. In conclusion, Lrig1 is an important regulator of suppressive T cell function and an exploitable target for treating autoimmune conditions.

Facilitation of Reparative Dentin Using a Drug Repositioning Approach With 4-Phenylbutric Acid.

For hard tissue formation, cellular mechanisms, involved in protein folding, processing, and secretion play important roles in the endoplasmic reticulum (ER). In pathological and regeneration conditions, ER stress hinders proper formation and secretion of proteins, and tissue regeneration by unfolded protein synthesis. 4-Phenylbutyric acid (4PBA) is a chemical chaperone that alleviates ER stress through modulation in proteins folding and protein trafficking. However, previous studies about 4PBA only focused on the metabolic diseases rather than on hard tissue formation and regeneration. Herein, we evaluated the function of 4PBA in dentin regeneration using an exposed pulp animal model system via a local delivery method as a drug repositioning strategy. Our results showed altered morphological changes and cellular physiology with histology and immunohistochemistry. The 4PBA treatment modulated the inflammation reaction and resolved ER stress in the early stage of pulp exposure. In addition, 4PBA treatment activated blood vessel formation and TGF-ß1 expression in the dentin-pulp complex. Micro-computed tomography and histological examinations confirmed the facilitated formation of the dentin bridge in the 4PBA-treated specimens. These results suggest that proper modulation of ER stress would be an important factor for secretion and patterned formation in dentin regeneration.

The effects of 4-Phenylbutyric acid on ER stress during mouse tooth development.

Introduction: During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4-Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice. Methods: We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The in vitro cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis. Results and Discussion: Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration.

Facilitating Reparative Dentin Formation Using Apigenin Local Delivery in the Exposed Pulp Cavity.

Apigenin, a natural product belonging to the flavone class, affects various cell physiologies, such as cell signaling, inflammation, proliferation, migration, and protease production. In this study, apigenin was applied to mouse molar pulp after mechanically pulpal exposure to examine the detailed function of apigenin in regulating pulpal inflammation and tertiary dentin formation. In vitro cell cultivation using human dental pulp stem cells (hDPSCs) and in vivo mice model experiments were employed to examine the effect of apigenin in the pulp and dentin regeneration. In vitro cultivation of hDPSCs with apigenin treatment upregulated bone morphogenetic protein (BMP)- and osteogenesis-related signaling molecules such as BMP2, BMP4, BMP7, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) after 14 days. After apigenin local delivery in the mice pulpal cavity, histology and cellular physiology, such as the modulation of inflammation and differentiation, were examined using histology and immunostainings. Apigenin-treated specimens showed period-altered immunolocalization patterns of tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), NESTIN, and transforming growth factor (TGF)-ß1 at 3 and 5 days. Moreover, the apigenin-treated group showed a facilitated dentin-bridge formation with few irregular tubules after 42 days from pulpal cavity preparation. Micro-CT images confirmed obvious dentin-bridge structures in the apigenin-treated specimens compared with the control. Apigenin facilitated the reparative dentin formation through the modulation of inflammation and the activation of signaling regulations. Therefore, apigenin would be a potential therapeutic agent for regenerating dentin in exposed pulp caused by dental caries and traumatic injury.

Signaling Modulation by miRNA-221-3p During Tooth Morphogenesis in Mice.

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including ß-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

Effect of Y2O3 Dispersion Method on the Microstructure Characteristic of Ni-Base Superalloy.

An optimum route to fabricate the Ni-base superalloys with homogeneous dispersion of oxide nanoparticles is investigated. Two methods for developing a uniform dispersion of oxide nanopar-ticles are compared on the basis of the resulting microstructure. Microstructural analysis reveals that the calcined powder from polymeric additive solution with yttrium nitrate and polyvinyl alcohol represented more fine and uniform distribution of Ni, Y and O elements. The densified specimen by spark plasma sintering at 1000 °C using calcined powder exhibits fine microstructure with oxide nanoparticles compared with that using mechanically alloyed powder, presumably by the particle growth or agglomeration prevention from chelating reaction during the calcination step. The oxide particles in the sintered specimen is identified as Y-Al-O phase, formed by the reaction of Y2O3 with Al during calcination and sintering.

Implications of the specific localization of YAP signaling on the epithelial patterning of circumvallate papilla.

Circumvallate papilla (CVP) is a distinctively structured with dome-shaped apex, and the surrounding trench which contains over two hundred taste buds on the lateral walls. Although CVP was extensively studied to determine the regulatory mechanisms during organogenesis, it still remains to be elucidated the principle mechanisms of signaling regulations on morphogenesis including taste buds formation. The key role of Yes-associated protein (YAP) in the regulation of organ size and cell proliferation in vertebrates is well understood, but little is known about the role of this signaling pathway in CVP development. We aimed to determine the putative roles of YAP signaling in the epithelial patterning during CVP morphogenesis. To evaluate the precise localization patterns of YAP and other related signaling molecules, including ß-catenin, Ki67, cytokeratins, and PGP9.5, in CVP tissue, histology and immunohistochemistry were employed at E16 and adult mice. Our results suggested that there are specific localization patterns of YAP and Wnt signaling molecules in developing and adult CVP. These concrete localization patterns would provide putative involvements of YAP and Wnt signaling for proper epithelial cell differentiation including the formation and maintenance of taste buds.

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson's trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1ß, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Stage-specific expression patterns of ER stress-related molecules in mice molars: Implications for tooth development.

The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.

Gene profiling involved in fate determination of salivary gland type in mouse embryogenesis.

Salivary gland (SG) development involves dynamic epithelial-mesenchymal interactions resulting in the formation of highly branched epithelial structures that produce and secrete saliva. The SG epithelium differentiates into saliva-producing terminal buds, i.e., acini, and transporting ducts. Most studies on the salivary gland have focused on branching morphogenesis; however, acinar cell differentiation underlying the determination of serous or mucous salivary glands is unclear. The objective of this study was to identify the mesenchymal signaling molecules involved in the epithelial differentiation of the salivary gland type as serous or mucous. Salivary glands undergoing stage-specific development, including the parotid gland (PG) and the sublingual gland (SLG) at embryonic day 14.5 (E14.5) were dissected. The glands were treated with dispase II to separate the epithelium and the mesenchyme. RNA from mesenchyme was processed for microarray analysis. Thereafter, microarray data were analyzed to identify putative candidate molecules involved in salivary gland differentiation and confirmed via quantitative reverse transcription polymerase chain reaction. The microarray analysis revealed the expression of 31,873 genes in the PG and SLG mesenchyme. Of the expressed genes 21,026 genes were found to be equally expressed (Fold change 1.000) in both PG and SLG mesenchyme. The numbers of genes expressed over onefold in the PG and SLG mesenchyme were found to be 5247 and 5600 respectively. On limiting the fold-change cut off value over 1.5 folds, only 214 and 137 genes were expressed over 1.5 folds in the PG and the SLG mesenchyme respectively. Our findings suggest that differential expression patterns of the mesenchymal signaling molecules are involved in fate determination of the salivary acinar cell types during mouse embryogenesis. In the near future, functional evaluation of the candidate genes will be performed using gain- and loss-of-function mutation studies during in vitro organ cultivation.