OBJECTIVES:Serum is an essential supplement of basal cell culture media, providing growth factors, adhesion molecules, hormones, lipids, and minerals that support cell survival and function. This study was conducted to investigate the effects of ultracentrifugation-separated fractions of fetal bovine serum (FBS) on the proliferation and differentiation of dental epithelial cells with the aim of identifying the key serum-derived factors that regulate cellular activities. METHODS:FBS was fractionated by ultracentrifugation at 100,000×g for 18 h at 4 °C into three layers, designated as the FBS-1st (top), -2nd (middle), and 3rd (bottom) fractions. Each fraction was added at 10 % (v/v) to the culture medium of rat dental epithelial HAT-7 cells. Cell proliferation was assessed by cell counting and 5-bromo-2'-deoxyuridine incorporation, and differentiation was evaluated by ameloblastin immunostaining. The involvement of lactoferrin and its receptors was examined using recombinant protein stimulation, reverse transcription polymerase chain reaction, small interfering RNA knockdown, and western blotting. RESULTS:FBS-3rd significantly promoted cell proliferation, whereas FBS-1st and -2nd inhibited it. Ameloblastin staining revealed no significant differences between FBS-1st and the control; however, it was suppressed in the FBS-2nd and -3rd. Proteomic analysis identified lactoferrin as the most abundant protein in FBS-3rd, and exogenous lactoferrin enhanced HAT-7 cell proliferation. The lactoferrin receptor, low-density lipoprotein receptor-related protein 1 (Lrp1), was expressed in HAT-7 cells, and Lrp1 knockdown stopped lactoferrin-induced proliferation. Lactoferrin induced phosphorylation of extracellular signal-regulated kinase 1/2, but not Akt serine/threonine kinase, and mitogen-activated protein kinase kinase inhibition with U0126 suppressed the proliferative effect of lactoferrin. CONCLUSION:Lactoferrin, which was enriched in the FBS-3rd fraction, may contribute to the proliferation of dental epithelial cells via the Lrp1-ERK signaling pathway.

Abstract Although glycerol is a ubiquitous metabolite in mammalian systems, its cellular metabolic pathways and functions have not been fully elucidated. Here, we find that elevated extracellular glycerol modulates intracellular metabolism and pro-inflammatory responses of macrophages. In pro-inflammatory macrophages stimulated with lipopolysaccharide, glycerol is taken up through glycerol channels including Aquaporin 3 (AQP3) and metabolized to glycerol-3-phosphate (G3P), which is then converted to dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase 2 (GPD2). This glycerol-driven pathway enhances mitochondrial ATP production, potentially by supplying electrons to the electron transport chain (ETC) via GPD2, and by upregulating the transcription of genes encoding ETC complexes. In addition, glycerol supplementation elevates intracellular acetyl-CoA levels, promotes histone acetylation at the promoters of pro-inflammatory cytokine genes, and consequently increases cytokine gene expression, suggesting enhanced pro-inflammatory response. In vivo experiments, macrophage-specific AQP3 conditional knockout mice exhibit reduced weight gain and adipose tissue inflammation in a high-fat diet-induced obesity model. Our findings provide novel insights into the metabolic regulation and macrophage inflammation by extracellular glycerol.

BACKGROUND:Aquaporin 3 (AQP3) is highly expressed in both keratinocytes and T cells within psoriatic skin. Previous studies have demonstrated that AQP3 knockout mice show reduced development of psoriatic symptoms in murine models. This study aims to evaluate the effect of AQP3 inhibition on psoriasis progression. METHODS AND RESULTS:AQP3 conditional knockout mice were generated to assess the role of AQP3 expression in keratinocytes and T cells in psoriasis pathogenesis. In an imiquimod (IMQ)-induced psoriasis model, psoriatic symptoms were significantly reduced in mice with keratinocyte-specific AQP3 deletion. Additionally, AQP3 inhibition by administration of anti-AQP3 monoclonal antibody (mAb) effectively alleviated IMQ-induced psoriasis symptoms in wild-type mice. CONCLUSIONS:AQP3 inhibition presents a promising approach for the treatment of psoriasis.

Desmocollin 3 (Dsc3), a desmosomal cadherin, mediates cell-cell adhesion, but its role in dental epithelial cell differentiation remains largely unknown. Using in situ hybridization and immunostaining of mouse incisors, Dsc3 is expressed in the outer enamel epithelium, inner enamel epithelium (IEE) containing transit-amplifying (TA) cells, stratum intermedium, and stellate reticulum, but absent in differentiated ameloblasts. Knockdown of endogenous Dsc3 by siRNA in the odontogenic dental epithelial cell line M3H1 inhibited the expression of the ameloblast makers ameloblastin (Ambn) and amelogenin, indicating the involvement of Dsc3 in ameloblast differentiation. Immunostaining and immunoprecipitation analyses revealed that DSC3 directly co-localized with β-catenin at the cell membrane; this association was disrupted by calpain activity during ameloblast differentiation. von Willebrand factor D and epidermal growth factor domains (Vwde), which was highly expressed in the IEE and particularly in TA cells, upregulated calpain 2 expression in M3H1 cells. VWDE-overexpressing M3H1 cells exhibited enhanced Ambn expression, whereas this induction was suppressed by the calpain inhibitor calpeptin. Western blotting further revealed that VWDE-overexpressing cells induced a 75-kDa β-catenin fragment in the nucleus. These findings suggest that Vwde-calpain signaling regulates DSC3-β-catenin complex, acting as a molecular switch that links cell adhesion to β-catenin-dependent ameloblast differentiation.

Tumor-associated macrophages (TAMs) originating from monocytes are crucial for cancer progression; however, the mechanism of TAM differentiation is unclear. We investigated factors involved in the differentiation of monocytes into TAMs within the tumor microenvironment of triple-negative breast cancer (TNBC). We screened 172 compounds and found that a heat shock protein 90 (HSP90) inhibitor blocked TNBC-induced monocyte-to-TAM differentiation in human monocytes THP-1. TNBC-derived conditional medium (CM) activated cell signaling pathways, including MAP kinase, AKT and STAT3, and increased the expression of TAM-related genes and proteins. These inductions were suppressed by HSP90 inhibition or by knockdown of HSP90 in TNBC. Additionally, we confirmed that TNBC secreted HSP90 extracellularly and that HSP90 itself promoted TAM differentiation. In a mouse tumor model, treatment with an HSP90 inhibitor suppressed tumor growth and reduced TAMs in the tumor microenvironment. Our findings demonstrate the role of HSP90 in TAM differentiation, suggesting HSP90 as a potential target for TNBC immunotherapy due to its regulatory role in monocyte-to-TAM differentiation.

Objectives This study aimed to elucidate the roles of Prrx1 and Prrx2, homeobox transcription factors, in tooth development and determine whether Prrx2 regulates pannexin 3 (Panx3) expression, which is important in preodontoblasts. Methods Tooth sections were prepared from 13.5-, 15.5-, and 18.5-day-old embryonic ICR mice, and Prrx1- and Prrx2-expressing cells were identified by in situ hybridization. To clarify the direct relationship between Prrx2 and Panx3, dual-luciferase reporter assay and electrophoretic mobility shift assay (EMSA) were performed. The effect of endogenous Prrx2 suppression on Panx3 expression was analyzed using an siRNA assay. Results In situ hybridization revealed that in the molars, Prrx1 and Prrx2 were similarly expressed in the bud and cap stages; however, only Prrx2 was expressed in preodontoblasts at the bell stage. In the incisors, Prrx2-expressing cells were observed from dental papilla cells to preodontoblasts. In serial sections, Prrx2-expressing cells in preodontoblasts corresponded to Panx3-expressing cells. Luciferase reporter assay using luciferase reporter plasmids containing Panx3 promoter revealed that Prrx2 overexpression in HEK293 cells significantly increased luciferase activity. EMSA of nuclear extract proteins from Prrx2-overexpressing HEK293 cells or mouse dental papilla-derived cells to the Panx3 promoter showed the protein-probe complex bands. SiRNA assay revealed that Prrx2 knockdown inhibited Panx3 expression. Conclusions Our results suggest that Prrx2 may regulate Panx3 expression during odontoblastic differentiation.

Macrophages play a crucial role in regulating the innate immune system and maintain tissue homeostasis. In the tumor microenvironment, tumor-associated macrophages (TAMs) are known to promote tumor progression, mostly derived from recruiting monocytes. However, the molecular mechanism underlying the differentiation of monocytes into TAM remains unclear.

Background: Although striking effects of vaccination strategy against pandemic COVID-19, a long-term influence by repeated partial virus mRNA injections are unknown. In addition, the molecular mechanism in accelerated ageing process still remains unclear. Through our clinical research, we observe and reported spontaneous SARS-CoV-2 virion and virion part production after Pfizer-BioNTech mRNA vaccination, observed from 3 weeks after the first injection. A significant destruction in ribosomal RNA structures, enhanced by repeated vaccinations, were also identified. The present study is aimed to define molecular mechanisms for SARSCoV- 2 virion production after injection of mRNA vaccination with a comparison to virion proliferation in non-vaccinated patient with severe COVID-19 pneumonia and fibrosis.

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

Aquaporin 3 (AQP3) is a member of the aquaporin water channel family expressed by numerous cell types, including some cancer cells. Accumulating evidence suggests that AQP3 inhibition may impede cancer progression, but drugs targeting AQP3 are still in the early pre-clinical stage of development. Here, we examined the effect of AQP3 inhibition on multiple myeloma (MM), an incurable plasma cell malignancy. Four MM cell lines were cultured in the presence of an anti-AQP3 monoclonal antibody (mAb), the AQP3 inhibitor DFP00173, or corresponding controls, and the effects on cell viability, proliferation, apoptosis, and mitochondrial respiration capacity were compared. Both anti-AQP3 mAb and DFP00173 reduced cell growth, mitochondrial respiration rate, and electron transport chain complex I activity. Both agents also potentiated the antiproliferative efficacy of the anticancer drug venetoclax. Administration of the anti-AQP3 mAb to immunodeficient mice inoculated with RPMI8226 or KMS-11 MM cells significantly suppressed tumor growth. These data provide evidence that AQP3 blockade can suppress MM cell growth in vitro and tumor growth in mice. Thus, AQP3 inhibition may be an effective therapeutic strategy for MM.

The nutritional and palatability relevance of bread prepared with soy flour was examined. There are a few effective nutritional measures that combine palatability, convenience, and functionality in the suppression of muscle loss (contributing to the improvement and prevention of sarcopenia). Therefore, in the present study, we attempted to produce bread using soybeans, which are rich in amino acids involved in the synthesis and degradation of skeletal muscle proteins. Rice flour was also used to avoid gluten intolerance. The bread was baked in an automatic bread maker, and the rheological properties of its breadcrumbs were determined using a creep meter. We found that a 70 g slice of soy bread satisfied approximately one-fifth of the daily nutritional requirement for leucine. Although soy decreased the specific volume of bread by preventing starch construction, the use of preprocessed rice flour recovered the volume, and corn starch improved the taste. We propose that the addition of soy bread to the daily diet may be an effective protein source.

Aquaporin-3 (AQP3), a member of the water- and small molecule-transporting protein family, is involved in inflammatory disease and cancer progression. Its role in pathogenesis has been attributed to AQP3-mediated H2O2 or glycerol transport.

Abstract In the process of caner recovery, Huaier simultaneously promotes significant hair growth and restoration as a typical evidence of its efficacy on induced tissue regeneration and repair function. Hair growth after conventional chemotherapy was a first and significant observation, and that in dose-dependent manner. The molecular basis of hair growth was identified as the activation of Hedgehog signaling pathway and among various perturbed signal transfer network, which initiates systematic tissue repair not only in pathogenic lesions but also to skin problems and inter/intra neural signal transfer. The activation followed by the inhibition at 3-6 months interval resulted in the prevention of subsequent complications, such as recurrence and metastasis by transcriptional control in cancer stem cell production. This regulation was demonstrated to occur irrespectively to SMO (smoothhead) function. The present study thus provides a simple and clear proof of Huaier effects on tissue regeneration, additional to the reported cancer specific-cell death.

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun. Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs. These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.

Macrophage differentiation and molecular mechanism in tumormicroenvironment

Although striking effects of vaccination strategy against COVID-19 world-wide, a long-term influence by sequential viral mRNA injections are unknown. We analysed biological alterations by total RNA sequencing in Pfizer-BioNTech vaccinated normal healthy volunteers and cancer patients, with or without adjuvant Huaier therapy. A significant destruction in ribosomal RNA structures was identified, enhanced by serial shots. Unlike the destruction caused by chemotherapy with platinum (II) complex, progressive destruction in 18S ribosome was identified even at 6 months after vaccination. The influence resulted in massive inhibition of translation and transcription, significantly in intra/inter neural signaling transfer and in lipid metabolism, related to ageing process. Huaier compensated these dysfunctions by miRNA-mediated transcriptional control, by typical activation in PI3K/AKT signaling pathway. Gene Ontology analysis revealed spontaneous virion production in number even after 3 months of the first vaccination. Present study indicated that the adjuvant therapy like Huaier compensates accelerated ageing process by mRNA vaccination.

Background: Clinical significance of anti-cancer effects of Huaier (Trametes robiniophila murr) has been emphasized recently [1-3]. We have already reported the successful Huaier therapy based on the individual genomic potential and miRNA-mediated transcription control [4-7]. In prostate cancer, Huaier also proved to have a significant efficacy on the prevention of cancer progression [6]. Objective: We have been initiating clinical research for thorough understanding of molecular basis of Huaier effects, which contributes to define the responsible molecules and biosystems not only for recovery, but also for prevention of cancer progression and tumorigenesis. Methods: The peripheral blood samples from the volunteer patients were analyzed by total RNA and non-coding small RNA sequencing on BGISEQ-500 Platform [8, 9]. KEGG pathway classification was hired for the analysis of the obtained information in the present study (https://www.genome.jp/kegg/) [10]. Results: In the present study, we focused on the patient and her family correlated with the mutated EGFR [11] and other receptor tyrosine kinases (c-MET [12-14]/erbB-2 [15, 16]). At first, high ratio of SNP variants as A-G transitions (241,896 in total, 37.0%) and A-C transversions (43,213 in total, 6.5 %), with skipped-exon (51%), which influenced significant changes in transcriptional factors and corresponding gene expression in multiple signal transduction pathways. The ratio of alteration ranged from 10% to 30% of total number of transcripts, detected as a drastic down-regulation by Huaier administration. Although the patient was diagnosed as benign meningioma, there were no genomic alteration in RNA polymerase II and its subunits [17], but only quantitative down-regulation in the attached transcriptional factors were observed. Additional to the massive down-regulation in the multiple signaling pathways, we identified additional modifications in neural systems and protein/hormone signal transfer by genetic changes in neural transmitters SLC6A4 solute carrier family 6 member 4 [18], and PRNP (prion protein); CD230 [19]. These alterations were hereditary, familial events. Overall drastic modifications in signal transduction contributed to compensate the defected function by the mutation of EGFR and other receptor tyrosine kinases, related to all the basic mechanisms controlling cell growth, proliferation, metabolism and many other processes regulating a wide variety of cell communications. Huaier seemed to take part in the epigenetic post-transcriptional control for the prevention in carcinogenesis and tumorigenesis for whole family members. Conclusion: The present study provided valuable example for compensation of defecting function by mutated EGFR and receptor tyrosine kinases. We identified hereditary gene expression alterations in EGFR (erbB1), c-MET, HER2/neu (erbB2), together with SLC6A4 solute carrier family 6 member 4 (Htt), and PRNP (prion protein); CD230, in the benign meningioma patient. These mutations were hereditary among family members. Huaier administration contributed the rescue of defected cell communication systems by massive down-regulation in a wide variety of signal transferring pathways. The mutation in transcripts remained even after 2 years of treatment. There are scarce medicine or candidate compounds for mutated EGFR and receptor tyrosine kinases yet, and Huaier is the one successful candidate to prevent carcinogenesis, tumorigenesis, and the other severe health problems correlated with the multiple mutations in those tyrosine kinase regulation systems. The epigenetic post-transcriptional regulation would decide the pathogenicity with hereditary mutations in these transcripts.

The induced normal tissue regeneration was the results of transcriptional control of iPS/ES cell production. Among the gene families to control iPS/ES production, especially c-my expression level played a major role among the other genes or gene families. These results provide a clue to clarify the Huaier effects not only for recovery from cancer, but also for the prevention of many related diseases and disorders caused by daily accumulation of environmental stresses and ageing by controlling normal tissue regeneration by stem cell control.

Lipopolysaccharide (LPS) is the principal component of the outer membrane of gram-negative bacteria. The prior oral administration of LPS attenuates inflammatory responses, such as intestinal injury and atopic dermatitis, in mouse models; however, the underlying mechanism remains unclear. Here, we examined the effect of topical LPS application on allergic contact dermatitis and its mechanism of action using a murine contact hypersensitivity (CHS) model. Prolonged LPS application to the skin significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced CHS. LPS application to the skin also reduced the phagocytosis of fluorescein isothiocyanate (FITC)-dextran by Langerhans and dendritic cells. Cutaneous cell migration into the skin-draining lymph nodes (LNs) induced by FITC painting was reduced by LPS application. During the CHS response, DNFB application induced T-cell proliferation and inflammatory cytokine production in skin-draining LNs, whereas prolonged LPS application inhibited DNFB-induced T-cell growth and interferon gamma production, indicating suppression of DNFB-induced sensitization. These results suggest that prolonged LPS application suppressed DNFB-induced sensitization and subsequently CHS response. Our findings imply that topical application of LPS may prevent allergic dermatitis such as CHS.