Odontogenic Differentiation-Induced Tooth Regeneration by Psoralea corylifolia L.

Psoralea corylifolia L. (P. corylifolia) has been used as an oriental phytomedicine to treat coldness of hands and feet in bone marrow injury. Hydroxyapatite is usually used for tooth regeneration. In this study, the role of P. corylifolia and bakuchiol, a compound originated from P. corylifolia as differentiation-inducing substances for tooth regeneration, was determined by monitoring odontogenic differentiation in human dental pulp stem cells (hDPSCs). We confirmed that P. corylifolia extracts and bakuchiol increased the odontogenic differentiation of hDPSCs. In addition, the expression of the odontogenic differentiation marker genes alkaline phosphatase (APL), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), and dentin matrix acidic phosphoprotein-1 (DMP-1) was proved by real-time polymerase chain reaction, and protein expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) was proved by western blotting. Further, by confirming the increase in small mothers against decapentaplegia (SMAD) 1/5/8 phosphorylation, the SMAD signaling pathway was found to increase the differentiation of odontoblasts. This study confirmed that P. corylifolia L. extracts and bakuchiol alone promote odontogenic differentiation in hDPSCs. These results suggest that bakuchiol from P. corylifolia is responsible for odontogenic differentiation, and they encourage future in vivo studies on dentin regeneration.

Raman Spectroscopy-Based 3D Analysis of Odontogenic Differentiation of Human Dental Pulp Stem Cell Spheroids.

Numerous efforts have been made to establish three-dimensional (3D) cell cultures that mimic the structure, cell composition, and functions of actual tissues and organs in vitro; however, owing to its intrinsic complexity, precise characterization of 3D differentiation remains challenging and often results in high variations in the resulting differentiated spheroids. This study reports a 3D Raman mapping-based analytical method that helps us to identify the crucial factors responsible for inducing variability in differentiated stem cell spheroids. Human dental pulp stem cell spheroids were generated at various cell densities using the hanging drop (HD) and molded parafilm-based (MP) methods and were then further subjected to odontogenic differentiation. Thereafter, the 3D cellular differentiation in these spheroids was analyzed based on three different Raman peaks, namely, 960, 1156/1528, and 2935 cm-1, which correspond to hydroxyapatite (HA, odontogenic differentiation marker), ß-carotene (precursor of HA), and proteins/cellular components (cell reference). By correlating such cell differentiation-related peaks and water/medium peaks (3400 cm-1), we discovered that the diffusion of the medium containing various nutrients and differentiation factors was crucial in determining the variations in 3D differentiation of stem cell spheroids. Odontogenic differentiation was majorly induced at the outer shell of HD spheroids (up to ∼20 µm), whereas odontogenic differentiation was markedly induced in MP spheroids (up to 40-50 µm). Considering the challenges associated with high variations in spheroid and organoid differentiation, we conclude that the proposed Raman-based 3D analysis plays a pivotal role in stem cell-based regenerative therapy and drug screening.

PFK activation is essential for the odontogenic differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair.

Oral Pathogenic Bacteria-Inducing Neurodegenerative Microgliosis in Human Neural Cell Platform.

Porphyromonas gingivalis is a gram-negative bacterium found in the human oral cavity and is responsible for the development of chronic periodontitis as well as neurological diseases, including Alzheimer's disease (AD). Given the significance of the roles of P. gingivalis in AD pathogenesis, it is critical to understand the underlying mechanisms of P. gingivalis-driven neuroinflammation and their contribution to neurodegeneration. Herein, we hypothesize that P. gingivalis produces secondary metabolites that may cause neurodegeneration through direct or indirect pathways mediated by microglia. To test our hypothesis, we treated human neural cells with bacterial conditioned media on our brain platforms and assessed microgliosis, astrogliosis and neurodegeneration. We found that bacteria-mediated microgliosis induced the production of nitric oxide, which causes neurodegeneration assessed with high pTau level. Our study demonstrated the elevation of detrimental protein mediators, CD86 and iNOS and the production of several pro-inflammatory markers from stimulated microglia. Through inhibition of LPS and succinate dehydrogenase in a bacterial conditioned medium, we showed a decrease in neurodegenerative microgliosis. In addition, we demonstrated the bidirectional effect of microgliosis and astrogliosis on each other exacerbating neurodegeneration. Overall, our study suggests that the mouth-brain axis may contribute to the pathogenesis of AD.

3D Visualization of Dynamic Cellular Reaction of Pulpal CD11c+ Dendritic Cells against Pulpitis in Whole Murine Tooth.

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray's clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.

An endoplasmic reticulum stress regulator, Tmbim6, modulates secretory stage of mice molar.

To understand the role of endoplasmic reticulum (ER)-stress in mice molar development, we studied Tmbim6 that antagonizes the unfolded protein response, using Tmbim6 knockout (KO) mice and in vitro organ cultivation with knocking down using small interfering RNA. During molar development, Tmbim6 is expressed in developing tooth at E14-E16, postnatal0 (PN0), and PN6. Mineral content in Tmbim6 KO enamel was reduced while dentin was slightly increased revealing ultrastructural changes in pattern formation of both enamel and dentin. Moreover, odontoblast differentiation was altered with increased Dspp expression at PN0 followed by altered AMELX localizations at PN5. These results were confirmed by in vitro organ cultivation and showed altered Bmp signaling, proliferation, and actin rearrangement in the presumptive ameloblast and odontoblasts that followed the altered expression of differentiation and ER stress-related signaling molecules at E16.5. Overall, ER stress modulated by Tmbim6 would play important roles in patterned dental hard tissue formation in mice molar within a limited period of development.

Transmembrane BAX Inhibitor Motif-6 (TMBIM6) protects against cisplatin-induced testicular toxicity.

STUDY QUESTION: Is the Transmembrane BAX Inhibitor Motif-6 (TMBIM6) involved in the molecular mechanism by which cisplatin causes reproductive toxicity? SUMMARY ANSWER: TMBIM6 protects against cisplatin-induced testicular toxicity through up-regulation of heme oxygenase-1 (HO-1),-which maintains the levels of steroidogenic enzymes by decreaseing oxidative stress in the endoplasmic reticulum (ER). WHAT IS KNOWN ALREADY: Testosterone production is highly suppressed as a main complication of cisplatin (cis-diamminedichloroplatinum) anticancer therapy. STUDY DESIGN, SIZE, DURATION: Groups of seven wild type or Tmbim6 KO C57BL/6J mice were given a single i.p., injection of cisplatin (30 mg/kg body wt) and testis and serum were collected 3 days later. Tmbim6-lentivirus-mediated testicular expression-rescued KO mice were analyzed to confirm function was restored. Tmbim6-over expressing TM3 mouse Leydig cells were exposed to cisplatin in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: After collection of the specimens serum testosterone level and testicular weight and structure were compared between the groups. Quantitative PCR, immunoblot, and assays for ROS, HO-1 activity and protein disulfide isomerase (PDI) carbonylation were performed. MAIN RESULTS AND THE ROLE OF CHANCE: Phospho protein kinase B (p-Akt), nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), and its downstream gene product HO-1 and the levels of testosterone synthesis-associated enzymes, including steroidogenic acute regulatory protein (StAR), a rate limiting enzyme for testosterone production, were significantly expressed in the presence of Tmbim6 and maintained after cisplatin treament. Excessive post-translational oxidation of protein disulfide isomerase (PDI), altered folding capacitance and ROS accumulation, and ER stress were also decreased in the presence of Tmbim6. Higher levels of ER stress and protein hypercarbonylation were consistently observed in KO testis, compared with WT testis. In the Tmbim6 KO mice, lentivirus-mediated testicular expression of Tmbim6 rescued the above phenotypes. Furthermore, the protective role of Tmbim6 against testicular toxicity was consistently shown in Tmbim6-overexpressing TM3 Leydig cells (testosterone producing cells). We conclude that TMBIM6 protects against cisplatin-induced testicular toxicity by inducing HO-1 and enhancing ER folding capacitance. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a short, 3-day cisplatin treatment condition. Therefore, the results need to be cautiously interpreted with regard to cisplatin-associated chronic toxicity. Moreover, to determine the clinical relevance of the role of TMBIM6, further studies in testicular cancer are needed. WIDER IMPLICATIONS OF THE FINDINGS: Cisplatin-associated ER stress and redox imbalance might be implicated as toxicity mechanisms associated with anticancer therapy. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Research Foundation of Korea (2015R1A2A1A13001849). The authors have no competing interests to disclose.

Compliance with Saliva Collection Protocol in Healthy Volunteers: Strategies for Managing Risk and Errors.

Salivary bioscience technologies such as electrophoresis are widely applied for diagnosing systemic health status. Diagnosis using a saliva sample has emerged as a preferred technique since the sample is easy to collect and the method is inexpensive and non-invasive. Salivary diagnostics have even been identified as potential substitutes for serum protein biomarkers. However, the optimal protocol for collecting saliva has not yet been established. In many scientific settings, such as randomized controlled trials, sampling and statistical errors often occur when handling samples from healthy volunteers. These errors can be due to the psychological behavior of the volunteers, subject nonadherence, questionnaire characteristics, collection methods, and/or sample processing. The purpose of the review presented here is to outline the strategies for managing the risk factors and to minimize the sampling errors during saliva collection in healthy volunteers.

Ixeris dentata Extract Increases Salivary Secretion through the Regulation of Endoplasmic Reticulum Stress in a Diabetes-Induced Xerostomia Rat Model.

This study aimed to investigate the molecular mechanism of diabetes mellitus (DM)-induced dry mouth and an application of natural products from Ixeris dentata (IXD), a recently suggested regulator of amylase secretion in salivary cells. Vehicle-treated or diabetic rats were orally treated with either water or an IXD extract for 10 days to observe the effect on salivary flow. We found that the IXD extract increased aquaporin 5 (AQP5) and alpha-amylase protein expression in the submandibular gland along with salivary flow rate. Similarly, the IXD extract and its purified compound increased amylase secretion in high glucose-exposed human salivary gland cells. Furthermore, increased endoplasmic reticulum stress response in the submandibular gland of diabetic rats was inhibited by treatment with the IXD extract, suggesting that IXD extract treatment improves the ER environment by increasing the protein folding capacity. Thus, pharmacological treatment with the IXD extract is suggested to relieve DM-induced dry mouth symptoms.

Endoplasmic reticulum stress in the regulation of liver diseases: Involvement of Regulated IRE1α and ß-dependent decay and miRNA.

Compromised protein folding capacity in the endoplasmic reticulum (ER) leads to a protein traffic jam that produces a toxic environment called ER stress. However, the ER smartly handles such a critical situation by activating a cascade of proteins responsible for sensing and responding to the noxious stimuli of accumulated proteins. The ER protein load is higher in secretory cells, such as liver hepatocytes, which are thus prone to stress-mediated toxicity and various diseases, including alcohol-induced liver injury, fatty liver disease, and viral hepatitis. Therefore, we discuss the molecular cues that connect ER stress to hepatic diseases. Moreover, we review the literature on ER stress-regulated miRNA in the pathogenesis of liver diseases to give a comprehensive overview of mechanistic insights connecting ER stress and miRNA in the context of liver diseases. We also discuss currently discovered regulated IRE1 dependent decay in regulation of hepatic diseases.

TNFα Increases RANKL Expression via PGE2-Induced Activation of NFATc1.

Tumor necrosis factor α (TNFα) is known to upregulate the expression of receptor activator of NF-κB ligand (RANKL). We investigated the role of the calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway in TNFα-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. TNFα-induced RANKL expression was blocked by the calcineurin/NFAT pathway inhibitors. TNFα increased NFAT transcriptional activity and subsequent RANKL promoter binding. Mutations in the NFAT-binding element (MT(N)) suppressed TNFα-induced RANKL promoter activity. TNFα increased prostaglandin E2 (PGE2) production, which in turn enhanced NFAT transcriptional activity and binding to the RANKL promoter. MT(N) suppressed PGE2-induced RANKL promoter activity. TNFα and PGE2 increased the expression of RANKL, NFAT cytoplasmic-1 (NFATc1), cAMP response element-binding protein (CREB), and cyclooxygenase 2 (COX2); which increment was suppressed by indomethacin, a COX inhibitor. Mutations in the CRE-like element blocked PGE2-induced RANKL promoter activity. PGE2 induced the binding of CREB to the RANKL promoter, whereas TNFα increased the binding of both CREB and NFATc1 to this promoter through a process blocked by indomethacin. The PGE2 receptor antagonists AH6809 and AH23848 blocked TNFα-induced expression of RANKL, NFATc1, and CREB; transcriptional activity of NFAT; and binding of NFATc1 or CREB to the RANKL promoter. These results suggest that TNFα-induced RANKL expression depends on PGE2 production and subsequent transcriptional activation/enhanced binding of NFATc1 and CREB to the RANKL promoter.

Isoproterenol Increases RANKL Expression in a ATF4/NFATc1-Dependent Manner in Mouse Osteoblastic Cells.

Sympathetic nervous system stimulation-induced ß-adrenergic signal transduction is known to induce bone loss and increase of osteoclast activity. Although isoproterenol, a nonspecific ß-adrenergic receptor agonist, has been shown to increase receptor activator of NF-κB ligand (RANKL), the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of the nuclear factor of activated T-cells (NFAT) in isoproterenol-induced RANKL expression in C2C12 and in primary cultured mouse calvarial cells. Isoproterenol increased nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and RANKL expressions at both mRNA and protein levels and increased NFAT reporter activity. NFATc1 knockdown blocked isoproterenol-mediated RANKL expression. Isoproterenol also promoted cAMP response element-binding protein 1 (CREB1) and activating transcription factor 4 (ATF4) phosphorylation. Isoproterenol-mediated transcriptional activation of NFAT was blocked by protein kinase A (PKA) inhibitor H89. Isoproterenol-induced CREB1, ATF4, NFATc1, and RANKL expressions were suppressed by H89. Mutations in cAMP response element-like or NFAT-binding element suppressed isoproterenol-induced RANKL promoter activity. Chromatin immunoprecipitation analysis demonstrated that isoproterenol increased NFAT-binding and ATF4-binding activities on the mouse RANKL promoter, but did not increase CREB1-binding activity. Association of NFATc1 and ATF4 was not observed in a co-immunoprecipitation study. ATF4 knockdown suppressed isoproterenol-induced NFAT binding to the RANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced ATF4 binding to the RANKL promoter. ATF4 knockdown suppressed isoproterenol-induced expressions of NFATc1 and RANKL. These results suggest that isoproterenol increases RANKL expression in an ATF4/NFATc1-dependent manner.

Porcine placenta hydrolysates regulate calcium disturbance in MC3T3-E1 osteoblastic cells.

BACKGROUND: In bone metabolism, Ca(2+) disturbance and oxidative damage are the main biochemical factors related to pathology. Osteoblasts are bone-forming cells that also control bone endocrinology. Endocrine hormones and proteins are matured, folded, and secreted in the endoplasmic reticulum (ER). ER stress has emerged as a new pathological mechanism to explain bone disturbance. Here we studied the role of porcine placenta hydrolysates (PPHs) in the regulation of ER stress. METHODS: Cell viability was determined in vitro using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a caspase kit. The fluorescent Ca(2+)-binding dye Fura-2/AM was used to measure changes in intracellular Ca(2+) ([Ca(2+)]i). ROS levels, NADPH oxidase activity, and superoxide dismutase (SOD) activity were also measured. RESULTS: PPHs protected MC3T3-E1 osteoblastic cells against thapsigargin (Tg)-induced ER stress. Moreover, PPHs regulated caspase-12 and -3 activities, thereby protecting against cell death, and also regulated Tg-induced Ca(2+) release. The Ca(2+) chelator BAPT/AM also regulated caspase-12 and -3 activities and prevented Ca(2) stress-induced cell death. In the presence of PPHs or BAPTA/AM, Ca(2+)-related ROS were also regulated, as demonstrated by alterations in NADPH oxidase and SOD activity. CONCLUSIONS: PPHs appear to regulate bone metabolism disturbance by controlling Ca(2+) concentrations, and thus ER stress and ROS, in osteoblasts cultured in vitro.

Endoplasmic Reticulum Stress and Associated ROS.

The endoplasmic reticulum (ER) is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS). Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI)-endoplasmic reticulum oxidoreductin (ERO)-1, glutathione (GSH)/glutathione disuphide (GSSG), NADPH oxidase 4 (Nox4), NADPH-P450 reductase (NPR), and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.

The cooperation of CREB and NFAT is required for PTHrP-induced RANKL expression in mouse osteoblastic cells.

Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene.

Aging phenotype in AD brain organoids: Track to success and challenges.

Alzheimer's disease (AD) poses a complex challenge, with abnormal protein accumulation in the brain causing memory loss and cognitive decline. Traditional models fall short in AD research, prompting interest in 3D brain organoids (BOs) from human stem cells. These findings hold promise for unveiling the mechanisms of AD, especially in relation to aging. However, an understanding of the aging impact of AD remains elusive. BOs offer insight but face challenges. This review delves into the role of BOs in deciphering aging-related AD and acknowledges limitations. Strategies to enhance BOs for accurate aging modeling in AD brains are suggested. Strengthened by molecular advancements, BOs have the potential to uncover the aging phenotype, advancing AD research.

Triple threat: neutrophil ER stress, NETosis, airway inflammation escalation.

This report aims to propose the novel term 'neutrophil endoplasmic reticulum (ER) stress' (NERS). NERS explores the influence of neutrophil extracellular trap (NET) formation and exacerbation of respiratory ailments. This inquiry aims to advance comprehension in neutrophil biology and respiratory health.

Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

TMBIM6 deficiency leads to bone loss by accelerating osteoclastogenesis.

TMBIM6 is an endoplasmic reticulum (ER) protein that modulates various physiological and pathological processes, including metabolism and cancer. However, its involvement in bone remodeling has not been investigated. In this study, we demonstrate that TMBIM6 serves as a crucial negative regulator of osteoclast differentiation, a process essential for bone remodeling. Our investigation of Tmbim6-knockout mice revealed an osteoporotic phenotype, and knockdown of Tmbim6 inhibited the formation of multinucleated tartrate-resistant acid phosphatase-positive cells, which are characteristic of osteoclasts. Transcriptome and immunoblot analyses uncovered that TMBIM6 exerts its inhibitory effect on osteoclastogenesis by scavenging reactive oxygen species and preventing p65 nuclear localization. Additionally, TMBIM6 depletion was found to promote p65 localization to osteoclast-related gene promoters. Notably, treatment with N-acetyl cysteine, an antioxidant, impeded the osteoclastogenesis induced by TMBIM6-depleted cells, supporting the role of TMBIM6 in redox regulation. Furthermore, we discovered that TMBIM6 controls redox regulation via NRF2 signaling pathways. Our findings establish TMBIM6 as a critical regulator of osteoclastogenesis and suggest its potential as a therapeutic target for the treatment of osteoporosis.

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase).

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.