Reciprocal suppression between TGFß signaling and TNF stimulation finetunes the macrophage inflammatory response.

Inflammation plays a crucial role in the development of various disease conditions or is closely associated with them. Inflammatory cytokines like TNF often engage in interactions with other cytokines and growth factors, including TGFß, to orchestrate inflammatory process. Basal/endogenous TGFß signaling is a universal presence, yet the precise way TNF communicates with TGFß signaling to regulate inflammation and influence inflammatory levels in macrophages has remained elusive. To address this question, this study utilized genetic approaches and a combination of molecular and cellular methods, including conditional TGFß receptor knockout mice, human cells, RNAseq, ATACseq and Cut & Run-seq. The results reveal that the TGFß signaling functions as a vital homeostatic pathway, curtailing uncontrolled inflammation in macrophages in response to TNF. Conversely, TNF employs two previously unrecognized mechanisms to suppress the TGFß signaling. These mechanisms encompass epigenetic inhibition and RBP-J-mediated inhibition of the TGFß signaling pathway by TNF. These mechanisms empower TNF to diminish the antagonistic influence exerted by the TGFß signaling pathway, ultimately enhancing TNF's capacity to induce heightened levels of inflammation. This reciprocal suppression dynamic between TNF and the TGFß signaling pathway holds unique physiopathological significance, as it serves as a crucial "braking" mechanism. The balance between TNF levels and the activity of the endogenous TGFß signaling pathway plays a pivotal role in determining the overall extent of inflammation. The potential for therapeutically augmenting the TGFß signaling pathway presents an intriguing avenue for countering the impact of TNF and, consequently, developing innovative strategies for inflammation control.

Histone H3K27 demethylase, Utx, regulates osteoblast-to-osteocyte differentiation.

Osteocytes are master regulators of skeletal homeostasis. However, little is known about the molecular mechanism of their differentiation. Epigenetic regulations, especially H3K27me3 modification, play critical roles in cell differentiation. Here, we found that H3K27me3 in the loci of osteocyte-expressing genes decreased during osteocyte differentiation and that H3K27me3 demethylase, Utx, was bound to the loci of those genes. To investigate the physiological functions of Utx in vivo, we generated late osteoblast-to-osteocyte specific Utx knockout mice using Dmp1-cre mice (UtxΔOcy/ΔOcy). Micro CT analyses showed that UtxΔOcy/ΔOcy displayed osteopenic phenotypes with lower bone volume and trabecular number, and greater trabecular separation. Bone histomorphometric analysis showed that bone mineralization and formation were significantly lower in UtxΔOcy/ΔOcy. Furthermore, Dmp1 expression and the number of osteocytes were significantly decreased in UtxΔOcy/ΔOcy. These results suggest that Utx in Dmp1-expressing osteoblast/osteocyte positively regulates osteoblast-to-osteocyte differentiation through H3K27me3 modifications in osteocyte genes. Our results provide new insight into the molecular mechanism of osteocyte differentiation.

Hyper-Raman spectroscopy of benzene and pyridine revisited.

Hyper-Raman (HR) spectra of benzene-h6, benzene-d6, and pyridine in the liquid phase excited at 1064 nm were measured by a picosecond laser with a high repetition rate. Although benzene and pyridine are important aromatic molecules, the qualities of the HR spectra previously reported were not high enough to be compared with those of IR and Raman spectroscopy. Our HR spectroscopic system significantly improves sensitivity that enables the detection of HR bands of benzene and pyridine not observed before. In addition to band assignments, we interpret HR bands of benzene based on the vibronic coupling theory of (pre-) resonance hyper-Raman scattering. Depolarization ratios of HR bands of benzene and pyridine, obtained from polarized-HR measurements, are first examined from a theoretical point of view of HR spectroscopy. Moreover, we evaluate quantum chemical calculations for HR spectra by comparing experimental and computational spectra. We show that the frequency-dependent polarizability and hyperpolarizability calculations using time-dependent density functional theory well reproduce the HR experiments for bulk aromatic compounds.

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

Regulatory network mediated by RBP-J/NFATc1-miR182 controls inflammatory bone resorption.

Bone resorption is a severe consequence of inflammatory diseases associated with osteolysis, such as rheumatoid arthritis (RA), often leading to disability in patients. In physiological conditions, the differentiation of bone-resorbing osteoclasts is delicately regulated by the balance between osteoclastogenic and anti-osteoclastogenic mechanisms. Inflammation has complex impact on osteoclastogenesis and bone destruction, and the underlying mechanisms of which, especially feedback inhibition, are underexplored. Here, we identify a novel regulatory network mediated by RBP-J/NFATc1-miR182 in TNF-induced osteoclastogenesis and inflammatory bone resorption. This network includes negative regulator RBP-J and positive regulators, NFATc1 and miR182, of osteoclast differentiation. In this network, miR182 is a direct target of both RBP-J and NFATc1. RBP-J represses, while NFATc1 activates miR182 expression through binding to specific open chromatin regions in the miR182 promoter. Inhibition of miR182 by RBP-J servers as a critical mechanism that limits TNF-induced osteoclast differentiation and inflammatory bone resorption. Inflammation, such as that which occurs in RA, shifts the expression levels of the components in this network mediated by RBP-J/NFATc1-miR182-FoxO3/PKR (previously identified miR182 targets) towards more osteoclastogenic, rather than healthy conditions. Treatment with TNF inhibitors in RA patients reverses the expression changes of the network components and osteoclastogenic potential. Thus, this network controls the balance between activating and repressive signals that determine the extent of osteoclastogenesis. These findings collectively highlight the biological significance and translational implication of this newly identified intrinsic regulatory network in inflammatory osteoclastogenesis and osteolysis.

Real-World Comparison of Elobixibat and Lubiprostone Treatment in Patients with Chronic Constipation: A Propensity Score-Matched Analysis.

INTRODUCTION: Elobixibat is a new laxative, but its efficacy and adverse events (AEs) are insufficiently examined compared with those of other laxatives. Hence, by propensity score (PS) matching, we compared the effects and AEs between elobixibat and lubiprostone. METHODS: We retrospectively analyzed 1,887 Japanese patients with chronic constipation (CC) treated at our hospital between October 2013 and April 2020. Enrolled patients were divided into three treatment groups, namely, elobixibat (10 mg daily) (E10 group, n = 293), lubiprostone (24 µg daily) (L24 group, n = 772), and lubiprostone (48 µg daily) (L48 group, n = 822), as their first treatment. We then investigated the changes on the weekly average number of spontaneous bowel movements, stool consistency scores (SCSs), and AEs starting from the baseline until the end of the 2-week treatment. To adjust for patients' background, we performed one-to-one nearest neighbor matching without replacement between elobixibat- and lubiprostone-treated patients according to the individual estimated PSs. RESULTS: After treatment, for SCSs, both the L24 and L48 groups significantly improved compared with the E10 group (p < 0.05), but their stools were soft (Bristol Stool Form Scale: 4.8). Notably, the E10 group had less frequent AEs than the L24 group (26 [9.0%] vs. 43 [14.8%], p = 0.03). Particularly, nausea was significantly less in the E10 group than that in the L48 group (2 [0.7%] vs. 7 [2.4%], p = 0.01). CONCLUSION: Elobixibat is a beneficial drug for patients with mildly symptomatic CC and is safe to use, given its few AEs.

Identification of a Novel Role for Foxo3 Isoform2 in Osteoclastic Inhibition.

Foxo3 acts as an important central regulator that integrates signaling pathways and coordinates cellular responses to environmental changes. Recent studies show the involvement of Foxo3 in osteoclastogenesis and rheumatoid arthritis, which prompted us to further investigate the FOXO3 locus. Several databases document FOXO3 isoform2, an N-terminal truncated mutation of the full-length FOXO3 However, the biological function of FOXO3 isoform2 is unclear. In this study, we established a conditional allele of Foxo3 in mice that deletes the full-length Foxo3 except isoform2, a close ortholog of the human FOXO3 isoform2. Expression of Foxo3 isoform2 specifically in macrophage/osteoclast lineage suppresses osteoclastogenesis and leads to the osteopetrotic phenotype in mice. Mechanistically, Foxo3 isoform2 enhances the expression of type I IFN response genes to RANKL stimulation and thus inhibits osteoclastogenesis via endogenous IFN-ß-mediated feedback inhibition. Our findings identify, to our knowledge, the first known biological function of Foxo3 isoform2 that acts as a novel osteoclastic inhibitor in bone remodeling.

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8+ T Cells.

Although the methylation status of histone H3K27 plays a critical role in CD4+ T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8+ T cell-dependent immune response remains unclear. We therefore generated T cell-specific Utx flox/flox Cd4-Cre Tg (Utx KO) mice to determine the role of Utx in CD8+ T cells. Wild-type (WT) and Utx KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. There was no significant difference in the number of Ag-specific CD8+ T cells upon primary infection between WT and Utx KO mice. However, Utx deficiency resulted in more Ag-specific CD8+ T cells upon secondary infection. Adoptive transfer of Utx KO CD8+ T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including Prdm1 encoding Blimp1, in Utx KO CD8+ T cells. We confirmed that the trimethylation level of histone H3K27 in the Prdm1 gene loci in the Utx KO cells was higher than in the WT cells. The treatment of CD8+ T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8+ T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8+ T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8+ T cells.

Nuclear receptors in bone physiology and diseases.

During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.

Def6 Restrains Osteoclastogenesis and Inflammatory Bone Resorption.

Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions. Def6 deficiency in Def6-/- mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer receptor activator for NF-κB ligand, and Def6-/- osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-α-induced osteoclastogenesis in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-α serum levels correlated negatively with Def6 expression levels in osteoclast precursors obtained from RA patients, and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, B lymphocyte-induced maturation protein-1, and c-Fos by regulating an endogenous IFN-ß-mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction.

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs.

Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3΄-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life. t6A and its derivatives have pleiotropic functions in protein synthesis including aminoacylation, decoding and translocation. We previously discovered a cyclic form of t6A (ct6A) as a chemically labile derivative of t6A in tRNAs from bacteria, fungi, plants and protists. Here, we report 2-methylthio cyclic t6A (ms2ct6A), a novel derivative of ct6A found in tRNAs from Bacillus subtilis, plants and Trypanosoma brucei. In B. subtilis and T. brucei, ms2ct6A disappeared and remained to be ms2t6A and ct6A by depletion of tcdA and mtaB homologs, respectively, demonstrating that TcdA and MtaB are responsible for biogenesis of ms2ct6A.

[A Case Report of Pancreatic Neuroendocrine Tumor Resected with the Preoperative Diagnosis of Gastrointestinal Stromal Tumor of the Jejunum].

A 61-year-old man was examined for cervical pain and CT showed a 9 cm tumor to the third part of the duodenum and proximal jejunum. CT /MRI showed that the tumor was separated from the pancreas body. We scheduled a laparoscopic partial resection of the intestine with a suspected diagnosis of GIST of the intestine. The tumor was adhered to both the proximal jejunum and uncinate process of the pancreas. Therefore, we converted to an open surgery and resected part of the pancreas, duodenum, and proximal jejunum including the tumor. Histopathological examination showed the tumor capsule included the tissue of the pancreas and that the border between the intestine and the tumor was clear, suggesting that the origin of the tumor was the pancreas. We diagnosed the patient as having a grade 2 pancreatic neuroendocrine tumor based on the tumor growth pattern and immunohistochemistry findings. We examined the preoperative CT images retrospectively and found that the tumor had adhered to the uncinate process of the pancreas, which extends over the left side of the superior mesenteric artery. When GIST close to the proximal jejunum is suspected, the possibility of pancreatic neuroendocrine tumor should be considered.

[A Case of Epithelioid Sarcoma of the Sigmoid Mesocolon].

A 73-year-old man visited our hospital with the chief complaints of anorexia and weight loss. Computed tomography showed a 5 cm tumor(diameter)in the sigmoid mesocolon. We performed laparoscopic Hartmann operation; however, because of sacral invasion of tumor, curative surgery was difficult. Therefore, the tumor was excised together with the sigmoid colon. Histopathological examination of the tumor confirmed the diagnosis of epithelioid sarcoma. Postoperatively, the patient received heavy-particle radiotherapy at another facility as treatment for the residual tumor. The size of the residual tumor decreased 6 months after the surgery. Up until 1 year after surgery, the patient's condition has remained stable without any disease progression. Epithelioid sarcoma is a rare soft-tissue tumor and often leads to a poor prognosis. We present a case of epithelioid sarcoma occurring in the sigmoid mesocolon.

Fatostatin, an SREBP inhibitor, prevented RANKL-induced bone loss by suppression of osteoclast differentiation.

Osteoclast differentiation is associated with both normal bone homeostasis and pathological bone diseases such as osteoporosis. Several transcription factors can regulate osteoclast differentiation, including c-fos and Nfatc1. Using genome-wide DNase-seq analysis, we found a novel transcription factor, SREBP2, that participates in osteoclast differentiation in vitro. Here, we asked whether SREBP2 actually plays a role in controlling bone metabolism in vivo. To answer this question, RAW264 cells, primary cultured osteoclasts and the mouse RANKL-induced bone loss model were treated with fatostatin, a small molecule inhibitor specific for the activation of SREBP. When cells were treated with fatostatin, osteoclast differentiation was impaired. Similar results were obtained following treatment with siRNA for Srebf2, the gene coding for SREBP2. In vivo, µCT analyses showed that fatostatin treatment preserved bone mass and structure in the proximal tibial trabecular bone in the mouse RANKL-induced bone loss model. In addition, bone histomorphometric analysis revealed that the protection of bone mass by fatostatin might have been achieved by suppression of RANKL-mediated osteoclast differentiation. These results indicated that the novel transcription factor SREBP2 physiologically functions in osteoclast differentiation in vivo and might be a possible therapeutic target for bone diseases.

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer.

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

Enhanced Photoelectrochemical Property of TiO2 Nanotube Array Photoanode Deposited with Al,Cr-Codoped SrTiO3 Nanocubes.

There is a demand for the effective utilization of solar energy with highly functional photoelectrodes for photoelectrochemical (PEC) applications, such as water splitting and CO2 reduction. TiO2 nanotube arrays (TNTA) with a large surface area have been studied as potential photoelectrodes mainly due to their strong oxidation potential. However, it has disadvantages of fast charge recombination and little responsivity to visible light. In this study, we prepared TNTA by anodizing a Ti plate and decorated the TNTA with Al,Cr-codoped SrTiO3 (STO) nanocubes through a hydrothermal treatment to enhance the PEC properties. We also prepared pristine and undoped STO-decorated TNTA for comparison. The hydrothermal treatment duration was optimized for the TNTA-STO:Al,Cr sample to achieve the best PEC performance. Finally, the possible PEC reaction mechanism was proposed based on the obtained experimental results.

Female sex hormones ameliorate arthritis in SKG mice.

The pathology of rheumatoid arthritis (RA) is ameliorated during pregnancy and deteriorated after delivery. Thus, female sex hormones could be involved in the pathogenesis of RA. However, the effects of estrogen and progesterone on the development and progression of RA have been unclear. In this study, we analyzed the effects of female hormones on the pathogenesis of RA by performing ovariectomy (OVX) and hormone implantation in the SKG mouse model of human RA. OVX mice showed severe arthritis and cartilage destruction with increased serum levels of TNF-α and IL-6, when compared with sham-operated mice. In contrast, estrogen-treated mice exhibited remarkable suppression of arthritis, with no bone erosion, little synovial hyperplasia and little infiltration of immune cells. Moreover, serum levels of TNF-α and IL-6 were decreased. In progesterone-treated mice, mild synovial hyperplasia and no immune cell infiltration were observed, with decreased serum levels of IL-6. These results suggest that female hormones, estrogen and progesterone, can play roles in the remission of RA.

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation.

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire(-/-) mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation.

Testis-specific protein on Y chromosome (TSPY) represses the activity of the androgen receptor in androgen-dependent testicular germ-cell tumors.

Testis-specific protein on Y chromosome (TSPY) is an ampliconic gene on the Y chromosome, and genetic interaction with gonadoblastoma has been clinically established. However, the function of the TSPY protein remains to be characterized in physiological and pathological settings. In the present study, we observed coexpression of TSPY and the androgen receptor (AR) in testicular germ-cell tumors (TGCTs) in patients as well as in model cell lines, but such coexpression was not seen in normal testis of humans or mice. TSPY was a repressor for androgen signaling because of its trapping of cytosolic AR even in the presence of androgen. Androgen treatment stimulated cell proliferation of a TGCT model cell line, and TSPY potently attenuated androgen-dependent cell growth. Together with the finding that TSPY expression is reduced in more malignant TGCTs in vivo, the present study suggests that TSPY serves as a repressor in androgen-induced tumor development in TGCTs and raises the possibility that TSPY could be used as a clinical marker to assess the malignancy of TGCTs.

Tube length optimization of titania nanotube array for efficient photoelectrochemical water splitting.

Anodic TiO2 nanotube arrays (TNTAs) have attracted much attention due to their excellent photoelectrochemical (PEC) properties. In this work, the tube length of TNTAs was optimized for efficient PEC water splitting under two different conditions, in which very few or a massive amount of gas bubbles were generated on the electrodes. As a result, relatively longer TNTAs were found to be preferable for higher PEC performance when a larger number of bubbles were generated. This suggests that the mass transport in the electrolyte is assisted by the generated bubbles, so that the electrode surfaces are more easily exposed to the fresh electrolyte, leading to the higher PEC performance.