Abstract Objective To identify Dickkopf-1 (DKK1) as a prognostically relevant candidate in head and neck squamous cell carcinoma and to evaluate whether DKK1 and cytoskeleton-associated protein 4 (CKAP4) expression is associated with cervical lymph node metastasis in tongue squamous cell carcinoma (TSCC). Methods DKK1 was screened using the Human Protein Atlas Pathology Atlas. Immunohistochemical expression of DKK1 and CKAP4 was examined in 54 patients with primary TSCC (cT1-4N0) treated surgically between 2015 and 2020. Nine cases were excluded because of insufficient tissue blocks or inadequate staining quality, leaving 45 evaluable cases. Associations with delayed cervical lymph node metastasis were assessed together with conventional clinicopathological factors, including infiltrative growth pattern (INF) and pathological depth of invasion (pDOI). Results In public database analysis, high DKK1 expression was associated with poorer overall survival in head and neck squamous cell carcinoma. In the TSCC cohort, pDOI ≥5 mm and INF pattern c were significantly associated with cervical lymph node metastasis. Positive DKK1 and CKAP4 expression were also significantly associated with cervical lymph node metastasis. Furthermore, combined DKK1/CKAP4 positivity, when incorporated with INF and pDOI, provided additional risk stratification, and cases with all 3 factors showed a markedly increased likelihood of cervical lymph node metastasis. Conclusions Expression of DKK1 and CKAP4 was associated with cervical lymph node metastasis in TSCC. Combined assessment of DKK1/CKAP4 expression with INF and pDOI may improve pathological risk stratification and may help identify patients who require closer neck evaluation and postoperative management.

Abstract Sex differences in hair growth are clinically evident, but sex-dependent regulation of physiological hair cycling and injury-induced hair regeneration remains incompletely understood. We compared physiological dorsal hair-cycle progression and adhesive material-induced localized hair regeneration in male and female C3H/He mice. Males entered the second and third anagen phases earlier than females, indicating longer telogen phases in females. In contrast, localized hair regrowth after application and removal of a cyanoacrylate adhesive material appeared earlier in females. Ovariectomy induced widespread telogen-to-anagen transition and therefore did not permit isolation of ovarian-hormone effects on the localized response. RNA sequencing of intact dorsal skin identified sex-dependent baseline expression profiles involving inflammation, wound response, and tissue repair. Independent time-course quantitative PCR further demonstrated sex-dependent expression of inflammatory and reparative genes after adhesive material application. Local clodronate liposome administration alone induced delayed perifocal hair growth. When combined with adhesive material application, clodronate treatment markedly delayed wound healing and localized hair regrowth in males, whereas these responses were comparatively preserved in females. These findings show that physiological hair cycling and adhesive material-induced hair regeneration exhibit distinct sex differences and suggest that the localized regenerative response is more macrophage-dependent in males than in females.

ABSTRACT N6‐methyladenosine (m6A) is the most abundant internal chemical modification of eukaryotic RNA and a central mechanism of epitranscriptomic gene regulation. Although m6A was first characterized in messenger RNA (mRNA), it is also deposited on ribosomal RNA (rRNA), circular RNA, and other RNA species, where it regulates RNA stability, decay, splicing, nuclear export, translational efficiency, and ribosome function. m6A‐mediated gene regulation has attracted increasing attention as a mechanism that contributes to development, differentiation, metabolism, and disease. Its relevance is also becoming evident in skeletal biology and bone metabolism. METTL3‐mediated m6A modification of mRNA regulates the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells and contributes to the maintenance of bone mass, while METTL5‐mediated m6A modification of 18S rRNA supports bone formation through OSER1‐dependent antioxidant regulation. m6A modification has also been implicated in osteoclast differentiation and bone‐resorbing activity, acting through mRNA decay, nuclear export, and transcription factor regulation. In this review, we summarize the basic concepts of m6A modification and discuss its emerging roles in the regulation of bone metabolism, with particular focus on osteoblasts, osteoclasts, osteocytes, and bone marrow mesenchymal stem cells. We also consider the implications of m6A‐mediated epitranscriptomic regulation for oral and skeletal biology.

Receptor activator of NF-κB ligand (RANKL) produced by osteoblastic lineage cells is essential for osteoclastogenesis, yet RANKL can be sequestered in intracellular, lysosome-like compartments under basal conditions. How mechanical cues mobilize RANKL toward the cell surface remains poorly defined. Here, we tested whether fluid shear stress alters RANKL subcellular distribution in osteoblast-like MC3T3-E1 cells and examined the involvement of the non-receptor tyrosine kinase c-Src. MC3T3-E1 cells expressing fluorescently tagged RANKL were subjected to fluid shear stress, and RANKL localization was analyzed by microscopy and subcellular fractionation. Fluid shear stress increased c-Src activation (Tyr416 phosphorylation) and promoted redistribution of RANKL toward the cell periphery, accompanied by an increase of RANKL in the membrane fraction. Co-expression experiments showed spatial association of RANKL with c-Src at the cell periphery after shear stimulation. Moreover, constitutively active c-Src (Y527F) enhanced peripheral localization of RANKL even in the absence of shear stress. Together, these data support a model in which shear stress activates c-Src to facilitate RANKL localization from intracellular stores toward membrane-proximal regions, thereby providing a mechanistic link between mechanical cues and osteoblast-derived osteoclastogenic signaling.

Introduction:Periodontitis is a chronic inflammatory disease characterized by progressive alveolar bone loss. Although spheroid culture enhances the osteogenic and regenerative potential of human periodontal ligament-derived multipotent mesenchymal stromal cells (hPDLMSCs), the underlying molecular mechanisms remain unclear. This study aimed to investigate the transcriptional features of spheroid-cultured hPDLMSCs, with a particular focus on cell cycle regulation and Forkhead Box O (FOXO) transcription factors. Methods:Monolayer- and spheroid-cultured hPDLMSCs were subjected to transcriptome analysis using RNA sequencing. Differentially expressed genes and enriched signaling pathways were identified, followed by validation through qRT-PCR and Western blotting. Flow cytometric analysis was then performed to compare cell cycle characteristics between monolayer- and spheroid-cultured hPDLMSCs. The functional roles of FOXO1 and FOXO4 were examined using siRNA-mediated knockdown combined with cell cycle and osteogenic differentiation analyses. Results:Transcriptome analysis revealed significant alterations in cell cycle-related genes and FOXO signaling in spheroid-cultured hPDLMSCs, including downregulation of cyclin family genes associated with cell cycle progression. Spheroid culture induced cell cycle arrest, characterized by an increased G0/G1 phase population and elevated expression of FOXO1, FOXO4, and cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B, and CDKN1C). FOXO1 knockdown promoted cell cycle progression and markedly reduced stemness and osteogenic marker expression as well as alkaline phosphatase activity. Similarly, FOXO4 knockdown decreased stemness-related gene expression but had a limited effect on osteogenic differentiation. Conclusions:FOXO1-mediated cell cycle regulation supports the maintenance of stemness and osteogenic potential in spheroid-cultured hPDLMSCs, suggesting that FOXO1 may serve as a promising target for periodontal tissue regeneration.

Abstract Objective Bone morphogenetic protein-3b (BMP-3b), also known as growth differentiation factor 10, has been implicated in tumor suppression; however, its role in breast cancer and its interaction with transforming growth factor-β1 (TGF-β1) signaling remain incompletely understood. Methods Publicly available datasets were used to examine BMP-3b expression in breast lesions and its association with overall survival in patients with stage III or IV breast cancer. Human MCF-7 and murine 4T1 breast cancer cells were treated with recombinant BMP-3b. Cell proliferation, migration, invasion, epithelial–mesenchymal transition-related proteins, and TGF-β1-induced Smad3 phosphorylation were assessed using Cell Counting Kit-8, scratch wound-healing, Transwell invasion, and Western blot assays. Results BMP-3b expression was lower in ductal carcinoma in situ than in normal mammary tissue. Low BMP-3b expression was associated with poorer overall survival in patients with stage III or IV breast cancer. BMP-3b reduced proliferation of MCF-7 and 4T1 cells and inhibited migration and invasion of 4T1 cells. BMP-3b increased E-cadherin and decreased vimentin expression in both cell lines. It also attenuated TGF-β1-induced migration, invasion, and Smad3 phosphorylation in 4T1 cells. Conclusions BMP-3b suppresses malignant phenotypes of breast cancer cells and modulates TGF- β1/Smad3 signaling. These findings identify BMP-3b as a potential endogenous regulator of breast cancer progression.

Despite strong interest in scalp and body hair and a large hair-care market, effective and practical approaches for inducing hair growth remain limited. Here, we show that pyroxylin treatment shortens the resting stage of the hair cycle and induces localized hair growth in mice. Application of pyroxylin to various skin regions induced injury followed by hair growth, which was also triggered by other adhesive materials. The hair growth was accompanied by hair cycle-related gene expression and gradually synchronized with the cycle of surrounding hair growth. Notably, this approach was effective even in middle-aged and aged mice. Although direct comparisons with established wound-healing models and physiological hair-cycle systems will be needed to further define the scope and limitations of this approach, our findings suggest that pyroxylin treatment provides a useful experimental platform for studying localized hair growth, and they warrant further investigation of its potential for topical hair-growth induction.

Methyltransferase-like 5 (METTL5) is a methyltransferase responsible for rRNA N6-methyladenosine (m6A) modification, mutations in which are associated with skeletal abnormalities and cognitive deficits. Despite METTL5's clinical relevance, the molecular mechanisms underlying METTL5-related genetic disorders remain poorly understood. In this study, we demonstrated that Mettl5 KO led to reduced bone mass and smaller body size in mice and impaired the osteogenic differentiation of mesenchymal stem cells. Mechanistically, Mettl5 deficiency decreased the translation efficiency of oxidative stress-responsive serine-rich protein 1 mRNA, downregulated the expression of key antioxidant genes, and diminished antioxidant capacity. Importantly, administration of the antioxidant N-acetylcysteine (NAC) partially rescued skeletal defects in Mettl5-KO mice. These findings reveal a critical role for METTL5 in antioxidant defense and suggest that NAC supplementation may represent a promising therapeutic strategy for METTL5-related disorders.

The taste system extends beyond the oral cavity, with various taste receptors found in extraoral organs. Mice deficient in the taste receptor type 1 (TAS1R) family member, TAS1R3, and fed a high-fat, high-sugar diet showed high bone mass without altering food consumption. However, the underlying mechanisms, including the cell types responsible for TAS1R3 expression, remain unclear. Here, we demonstrate the expression and function of TAS1R3 in osteoclasts, which are responsible for bone resorption. The expression of Tas1r3 but not Tas1r1 or Tas1r2, is evoked during osteoclast differentiation. Osteoclastogenesis-related genes were downregulated in TAS1R3-deficient mice, whereas the opposite phenotypes were elicited by TAS1R3 overexpression. Contrary to the common heterodimerization with TAS1R1 or TAS1R2, TAS1R3 formed a homodimer that functioned to detect glucose, enhance p38 phosphorylation, and induce osteoclastogenesis. These results provide novel insights into the role of TAS1R3 in bone metabolism and suggest that TAS1R3 may be a viable target for therapeutic agents in bone metabolic diseases.

Mutations in SLIT and NTRK-like family member 1 (SLITRK1) result in Tourette syndrome (TS). Patients with TS exhibit delayed bone maturation and increased fracture risk. To understand the role of SLITRK1 in bone homeostasis, we examined the skeletal phenotype of Slitrk1 null mice and investigated the mechanisms responsible for altered bone cell function. Slitrk1 null mice had thinner cortical bones due to decreased periosteal bone formation, while trabecular bone density remained unchanged. Slitrk1 was expressed within osteoblast-lineage cells. Consequently, deletion of Slitrk1 in osteoblasts impaired ex vivo differentiation capacity. Loss of SLITRK1 in osteoblast cells reduced levels of TAZ, a RUNX2 co-activator necessary for osteoblast differentiation. These findings provide evidence for a cell-autonomous role of SLITRK1 in periosteal osteoblasts regulating cortical bone homeostasis. Our data further demonstrate that periosteal-, endosteal-, and trabecular-bone homeostasis are controlled by different mechanisms and provide new insight into the skeletal manifestations of TS.

Inactivating mutations in the PHEX gene lead to X-linked hypophosphatemia (XLH), which is characterized by impaired skeletal mineralization and low serum phosphate. Subsequent rickets and osteomalacia result in bone deformities and pseudofractures. A hallmark of XLH is an intrinsic defect in osteoblast function resulting in altered bone matrix composition typified by the local accumulation of extracellular matrix proteins and peptide fragments. PHEX is a membrane-bound endopeptidase expressed in osteoblasts and osteocytes. Little is known about PHEX proteolytic substrates or the protein-protein interactions governing PHEX function. Classical affinity purification approaches are challenging in studies of the extracellular environment. Here, we developed an approach for unbiased identification of the extracellular proximal interactome of PHEX in osteoblasts using proximity-dependent biotin identification combined with affinity purification and mass spectrometry. By tagging the PHEX extracellular domain with BioID2 biotin ligase, we labeled and unveiled a PHEX proximity network consisting of 39 high-confidence proteins. Notably, several candidates with documented roles in bone morphogenesis and matrix organization were identified. We validated interaction of PHEX with periostin, a bone-matrix protein associated with collagen-fibril organization, cell adhesion and cell migration. Co-transfection experiments and cell-free enzyme cleavage assays revealed proteolytic cleavage of secreted periostin by PHEX. In conclusion, BioID2 is a powerful strategy to explore cell-matrix relationships in osteoblasts. These results present a novel map of the PHEX interactome and serve as a valuable resource for unraveling the mechanisms underlying PHEX function and XLH.

Royal jelly (RJ), secreted by honeybees, contains major fatty acids such as 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid, which are considered to contribute to bone metabolism. However, these fatty acids are rapidly metabolized in the liver following ingestion, resulting in 2-decenoic acid (2DA) and sebacic acid (SA), respectively. Therefore, elucidating the roles of these metabolites in bone metabolism is of considerable importance. In this study, we focused and investigated the effects of 2DA and SA on osteoblast differentiation using osteoblast-lineage cells. SA treatment significantly upregulated the expression of osteoblast marker genes whereas 2DA had no apparent effect. In addition, SA enhanced alkaline phosphatase activity, a typical marker of osteoblast differentiation and mineralization.

The world’s population is aging. Pneumonia is the leading cause of death among the older adults, with aspiration pneumonia being particularly common. Aspiration pneumonia is caused by a decline in swallowing function. Causes can include age-related sarcopenia of swallowing muscles, cognitive decline, cerebrovascular and other diseases or even changes in individual taste preference. Currently, the main treatment approach for dysphagia is resistance training of swallowing-related muscles. This approach has not been effective and establishment of novel methods are required. In this review, we introduce and discuss the relationship between taste, taste preference, carbonation and swallowing function. Taste and preference improve swallowing function. Recently, it has been shown that a carbonated beverage that combines the functionality of a thickening agent, the appeal of taste, and the stimulation of carbonation improves swallowing function. This may be very useful in the recovery of swallowing function. It is important to note that deliciousness is based not only on taste and preference, but also on visual information such as food form. Umami taste receptors are expressed not only in taste buds but also in skeletal muscle and small intestine. These receptors may be involved in homeostasis of the amino acid metabolic network, i.e., the process of amino acid ingestion, intestine absorption, and storage in skeletal muscle. Proper stimulation of umami receptors in organs other than taste buds may help maintain nutritional status and muscle mass. Umami receptors are therefore a potential therapeutic target for dysphagia.