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Tripartite motif (TRIM) family proteins are involved in various biological processes and the pathophysiology of cancers. However, the roles of TRIM39, a TRIM family member, in breast cancer is not well-understood. Here, we performed immunohistochemical study of TRIM39 protein in clinical estrogen receptor-positive (ER

Background Singing in an indoor space may increase the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We conducted a case-control study of karaoke-related coronavirus disease 2019 (COVID-19) outbreaks to reveal the risk factors for SARS-CoV-2 infection among individuals who participate in karaoke. Methods Cases were defined as people who enjoyed karaoke at a bar and who tested positive for SARS-CoV-2 by reverse-transcription polymerase chain reaction between 16 May and 3 July 2020. Controls were defined as people who enjoyed karaoke at the same bar during the same period as the cases and tested negative. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. ORs of key variables adjusted for each other were also estimated (aOR). Results We identified 81 cases, the majority of whom were active elderly individuals (median age, 75 years). Six cases died (case fatality ratio, 7%). Among the cases, 68 (84%) were guests, 18 of whom had visited ≧2 karaoke bars. A genome analysis conducted in 30 cases showed 6 types of isolates within 4 single-nucleotide variation difference. The case-control study revealed that singing (aOR, 11.0 [95% CI, 1.2–101.0]), not wearing a mask (aOR, 3.7 [95% CI, 1.2–11.2]), and additional hour spent per visit (aOR, 1.7 [95% CI, 1.1–2.7]) were associated with COVID-19 infection. Conclusions A karaoke-related COVID-19 outbreak that occurred in 2 different cities was confirmed by the results of genome analysis. Singing in less-ventilated, indoor and crowded environments increases the risk of acquiring SARS-CoV-2 infection. Wearing a mask and staying for only a short time can reduce the risk of infection during karaoke.

Octamer transcription factor 1 (OCT1) is a transcriptional factor reported to be a poor prognostic factor in various cancers. However, the clinical value of OCT1 in breast cancer is not fully understood. In the present study, an immunohistochemical study of OCT1 protein was performed using estrogen receptor (ER)-positive breast cancer tissues from 108 patients. Positive OCT1 immunoreactivity (IR) was associated with the shorter disease-free survival (DFS) of patients (p = 0.019). Knockdown of OCT1 inhibited cell proliferation in MCF-7 breast cancer cells as well as its derivative long-term estrogen-deprived (LTED) cells. On the other hand, the overexpression of OCT1 promoted cell proliferation in MCF-7 cells. Using microarray analysis, we identified the non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) as a novel OCT1-taget gene in MCF-7 cells. Immunohistochemical analysis showed that NCAPH IR was significantly positively associated with OCT1 IR (p &lt, 0.001) and that positive NCAPH IR was significantly related to the poor DFS rate of patients (p = 0.041). The knockdown of NCAPH inhibited cell proliferation in MCF-7 and LTED cells. These results demonstrate that OCT1 and its target gene NCAPH are poor prognostic factors and potential therapeutic targets for patients with ER-positive breast cancer.

[Background] Tripartite motif-containing (TRIM) proteins typically function as E3 ubiquitin ligases and involved in many biological processes including cancer biology. We previously reported that TRIM25/Efp which stimulated the degradation of a negative cell-cycle regulator, 14-3-3 sigma,is a poor prognostic factor for breast cancer patients (Nature 417, 871, 2002; Clin Cancer Res 11, 6148, 2005) and that TRIM44 overexpressed in breast cancer enhanced NF-κB signaling (Int J Mol Sci 18, pii:E1931, 2017). In the present study, we focused on the prognostic value and the roles in breast cancer of a TRIM family protein, TRIM39. [Methods] Tissue samples of invasive breast cancer were obtained from 124 Japanese female primary breast cancer patients who underwent surgical treatment at Toranomon Hospital, Tokyo, Japan. This study was approved by the ethical committee in Toranomon Hospital. We performed immunohistochemical analysis with a polyclonal antibody against TRIM39. Immuno-stained slides were evaluated for intensity and proportion of nuclear staining. We examined the relationship between immunoreactivity of TRIM39 and clinico-pathological parameters and patients’ prognosis. In order to investigate TRIM39 biological function in breast cancer cells, we used MCF-7 and MDA-MB-231, breast cancer cell lines. We knocked down expression of TRIM39 and evaluated cell proliferation and cell migration. We investigated gene expression profiles affected by knockdown of TRIM39 with microarray analysis (Affymetrix, Clariom S). [Results] Among clinico-pathological parameters, higher pathological stage, higher pathological tumor size and higher nuclear grade were significantly associated with strong immunoreactivity of TRIM39. TRIM39 immunoreactivity was significantly associated with distant disease-free survival (p Citation Format: Takuya Ogura, Kotaro Azuma, Junichiro Sato, Keiichi Kinowaki, Kazuhiro Ikeda, Hidetaka Kawabata, Satoshi Inoue. TRIM39 is a poor prognostic factor for breast cancer patients and promotes proliferation and migration of breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-11.

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

We previously reported that a strong immunoreactivity of tripartite motif-containing 44 (TRIM44) predicts the poor prognosis of patients with invasive breast cancer, and proposed that TRIM44 activates nuclear factor-κB (NF-κB) signaling as a causative mechanism. In the present study, we examined the clinicopathological roles of A20, which is known to be an NF-κB responsive gene, with TRIM44, in an updated cohort. Tissue samples of invasive breast cancer were obtained from 140 Japanese female breast cancer patients who underwent surgical treatment. Immunoreactivities of A20 and TRIM44 were analyzed using specific antibodies for each protein. A positive A20 immunoreactivity was significantly associated with a shorter disease-free survival (P = 0.043) and was positively correlated with TRIM44 immunoreactivity (P = 0.039). Combined use of the immunoreactivities for two proteins revealed that double-positive status for both A20 and TRIM44 immunoreactivities was associated with a shorter disease-free survival (P = 0.012) and was an independent factor for poor prognosis. These results indicate that a combined A20 and TRIM44 immunoreactivity predicted the prognosis of patients with invasive breast cancer. Moreover, the positive correlation between A20 and TRIM44 immunoreactivities suggested that the activation of NF-κB signaling by TRIM44 could occur in clinical breast cancer tissues.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

Background We previously identified estrogen-responsive finger protein (Efp) as an estrogen-induced gene, and showed that the positive immunoreactivity of Efp is a poor prognostic factor for patients with breast cancer. We also demonstrated that Efp has distinctive roles in innate immunity by activating pattern recognition receptor retinoic acid-inducible gene I (RIG-I). The clinical value of RIG-I protein expression in breast cancer had not been evaluated in relationship with patients’ prognosis. Patients and Methods Tissue samples of estrogen receptor-positive invasive breast cancer were obtained from 145 female patients with breast cancer who underwent surgical treatment. Immunoreactivities of RIG-I and Efp were analyzed with the antibodies generated for the present study. Results Positive immunoreactivity of RIG-I was correlated with lower disease-free survival (P = .032) and was an independent poor prognostic factor (P = .043). RIG-I immunoreactivity was positively correlated with that of Efp (P = .0004). Patients with positive immunoreactivities of both RIG-I and Efp proteins were associated with a lower disease-free survival rate (P = .005). Conclusions Positive immunoreactivity of RIG-I has clinical significance as a poor prognostic factor in patients with estrogen receptor-positive breast cancer. A positive correlation of RIG-I and Efp immunoreactivities was observed, and the combination of their immunoreactivities can be used to predict patients’ prognosis.

Impairment of skeletal muscle function causes disabilities in elderly people. Therefore, in an aged society, prevention and treatment of sarcopenia are important for expanding healthy life expectancy. In addition to aging, adipose tissue disfunction and inflammation also contribute to the pathogenesis of sarcopenia by causing the combined state called ‘sarcopenic obesity’. Muscle quality as well as muscle mass contributes to muscle strength and physical performance. Mitochondria in the skeletal muscles affect muscle quality by regulating the production of energy and reactive oxygen species. A certain portion of the mitochondrial respiratory chain complexes form a higher-order structure called a “supercomplex”, which plays important roles in efficient energy production, stabilization of respiratory chain complex I, and prevention of reactive oxygen species (ROS) generation. Several molecules including phospholipids, proteins, and certain chemicals are known to promote or stabilize mitochondrial respiratory chain supercomplex assembly directly or indirectly. In this article, we review the distinct mechanisms underlying the promotion or stabilization of mitochondrial respiratory chain supercomplex assembly by supercomplex assembly factors. Further, we introduce regulatory pathways of mitochondrial respiratory chain supercomplex assembly and discuss the roles of supercomplex assembly factors and regulatory pathways in skeletal muscles and adipose tissues, believing that this will lead to discovery of potential targets for prevention and treatment of muscle disorders such as sarcopenia.

[Background] Many of the tripartite motif (TRIM) proteins, like Efp/TRIM25 which was identified by our group previously (Nature 417, 871-875, 2002), function as E3 ubiquitin ligases, and are thought to be involved in various physiological and pathological processes such as immunity and oncogenesis. In regard to tripartite motif containing 44 (TRIM44), which is an atypical TRIM family protein lacking RING finger domain, some evidences suggest that it is implicated in the progression of several human malignancies. But its pathophysiological significance in breast cancer remains unknown. [Methods] In the present study, immunohistochemical analysis using anti-TRIM44 antibody was performed in clinical breast cancer tissues from 129 patients with the approval of institutional ethical committees (approval number: 845). We then explored the pathophysiological role of TRIM44 in breast cancer by modulating TRIM44 expression in MCF-7 and MDA-MB-231 breast cancer cells. [Results] TRIM44 strong immunoreactivity was significantly associated with nuclear grade, distant disease-free survival and overall survival of the breast cancer patients. With multivariate analysis it was shown that the TRIM44 status was an independent prognostic factor for distant disease-free survival and overall survival. The proliferation of MCF-7 and MDA-MB-231 cells was significantly decreased by siRNA-mediated TRIM44 knockdown. TRIM44 knockdown also suppressed migration of MDA-MB-231 cells. Microarray analysis and qRT-PCR revealed that TRIM44 knockdown upregulated CDK19 (Cyclin Dependent Kinase 19), which is reported to be a tumor suppressor gene, whereas downregulated MMP1 (Matrix Metallopeptidase 1) in MDA-MB-231 cells. Notably, TRIM44 knockdown impaired nuclear factor-kappa B (NF-κB)-mediated transcriptional activity stimulated by tumor necrosis factor α (TNFα). Moreover, TRIM44 knockdown substantially attenuated the TNFα-dependent phosphorylation of p65 subunit of NF-κB and IκBα in both MCF-7 and MDA-MB-231 cells. [Discussion] Our clinical study showed that prognosis of breast cancer patients is correlated with the immunoreactivity detected by anti-TRIM44 antibody. This result suggested that expression of TRIM44 protein could be used as a potential biomarker of breast cancer. We demonstrated that NF-κB signaling pathway is modulated by TRIM44. Since NF-κB augmentation is shown to be related to aggressive character of breast cancer, stimulation of NF-κB signaling with TRIM44 might be underlying mechanism of poor prognosis. Our in vitro study showed TRIM44 knockdown caused attenuated proliferation and migration of breast cancer cells, raising the possibility of TRIM44 as a potential therapeutic target for breast cancer. These findings provide new clues to develop alternative effective strategies for breast cancer management. Citation Format: Kawabata H, Azuma K, Ikeda K, Sugitani I, Kinowaki K, Fujii T, Osaki A, Saeki T, Horie-Inoue K, Inoue S. TRIM44 is a possible poor prognostic factor for breast cancer patients and positively regulates NF-κB signaling pathway [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-06.

TRIM44 has oncogenic roles in various cancers. However, TRIM44 expression and its function in renal cell carcinoma (RCC) are still unknown. Here in this study, we investigated the clinical significance of TRIM44 and its biological function in RCC. TRIM44 overexpression was significantly associated with clinical M stage, histologic type (clear cell) and presence of lymphatic invasion (P = .047, P = .005, and P = .028, respectively). Moreover, TRIM44 overexpression was significantly associated with poor prognosis in terms of cancer‐specific survival (P = .019). Gain‐of‐function and loss‐of‐function studies using TRIM44 and siTRIM44 transfection showed that TRIM44 promotes cell proliferation and cell migration in two RCC cell lines, Caki1 and 769P. To further investigate the role of TRIM44 in RCC, we performed integrated microarray analysis in Caki1 and 769P cells and explored the data in the Oncomine database. Interestingly, FRK was identified as a promising candidate target gene of TRIM44, which was downregulated in RCC compared with normal renal tissues. We found that cell proliferation was inhibited by TRIM44 knockdown and then recovered by siFRK treatment. Taken together, the present study revealed the association between high expression of TRIM44 and poor prognosis in RCC patients and that TRIM44 promotes cell proliferation by regulating FRK., Based on the results of immunohistochemistry and functional experiments, TRIM44 promotes cell proliferation and migration by inhibiting FRK in renal cell carcinoma.

Vitamin K is a fat-soluble vitamin that was originally found as an essential factor for blood coagulation. With the discovery of its role as a co-factor for γ-glutamyl carboxylase (GGCX), its function for blood coagulation was understood as the activation of several blood coagulation factors by their γ-carboxylation. Over the last two decades, other modes of vitamin K actions have been discovered, such as the regulation of transcription by activating the steroid and xenobiotic receptor (SXR), physical association to 17β-Hydroxysteroid dehydrogenase type 4 (17β-HSD4), covalent modification of Bcl-2 antagonist killer 1 (Bak), and the modulation of protein kinase A (PKA) activity. In addition, several epidemiological studies have revealed that vitamin K status is associated with some aging-related diseases including osteoporosis, osteoarthritis, and sarcopenia. Clinical studies on single nucleotide polymorphisms of GGCX suggested an association between higher GGCX activity and bone protective effect, while recent findings using conditional knockout mice implied that a contribution in protective effect for bone loss by GGCX in osteoblastic lineage was unclear. GGCX in other cell lineages or in other tissues might play a protective role for osteoporosis. Meanwhile, animal experiments by our groups among others revealed that SXR, a putative receptor for vitamin K, could be important in the bone metabolism. In terms of the cartilage protective effect of vitamin K, both GGCX- and SXR-dependent mechanisms have been suggested. In clinical studies on osteoarthritis, the γ-carboxylation of matrix Gla protein (MGP) and gla-rich protein (GRP) may have a protective role for the disease. It is also suggested that SXR signaling has protective role for cartilage by inducing family with sequence similarity 20a (Fam20a) expression in chondrocytes. In the case of sarcopenia, a high vitamin K status in plasma was associated with muscle strength, large muscle mass, and high physical performance in some observational studies. However, the basic studies explaining the effects of vitamin K on muscular tissue are limited. Further research on vitamin K will clarify new biological mechanisms which contribute to human longevity and health through the prevention and treatment of aging-related musculoskeletal disorders.

A 76-year-old man visited our hospital with complaints of appetite loss and diarrhea. Abdominal computed tomography (CT)showed a large transverse colon tumor at the splenic flexure and a gastrocolic fistula. Upper gastrointestinal series and gastroscopy demonstrated a type 2 tumor in the transverse colon and a gastrocolic fistula as the scope was inserted into the transverse colon. Colonoscopy showed a type 2 tumor of the rectum. Based on the diagnosis of advanced transverse colon cancer with gastrocolic fistula and synchronous rectal cancer, a one-stage curative operation was performed. Pathologically, both cancers were well-differentiated adenocarcinomas, but the transvers colon cancer was partially mucinous. Lymph node metastasis was absent. Gastrocolic fistula complicating colon cancer is rare, to our knowledge, with only 29 cases reported in Japan. A curative operation was performed in 73%of these cases, including ours, and lymph node metastasis was observed in only 22%. This suggests that colon cancer with a gastrocolic fistula might undergo less lymph node metastasis despite increased invasion depth, and that a curative operation for the colon cancer and gastrocolic fistula should be considered.

Vitamin K is a fat-soluble vitamin that is necessary for blood coagulation. In addition, it has bone-protective effects. Vitamin K functions as a cofactor of γ-glutamyl carboxylase (GGCX), which activates its substrates by carboxylation. These substrates are found throughout the body and examples include hepatic blood coagulation factors. Furthermore, vitamin K functions as a ligand of the nuclear receptor known as steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR). We have previously reported on the bone-protective role of SXR/PXR signaling by demonstrating that systemic Pxr-knockout mice displayed osteopenia. Because systemic Ggcx-knockout mice die shortly after birth from severe hemorrhage, the GGCX-mediated effect of vitamin K on bone metabolism has been difficult to evaluate. In this work, we utilized Ggcx-floxed mice to generate osteoblast-specific GGCX-deficient (GgcxΔobl/Δobl) mice by crossing them with Col1-Cre mice. The bone mineral density (BMD) of GgcxΔobl/Δobl mice was significantly higher than that of control Col1-Cre (Ggcx+/+) mice. Histomorphometrical analysis of trabecular bones in the proximal tibia showed increased osteoid volume and a higher rate of bone formation in GgcxΔobl/Δobl mice. Histomorphometrical analysis of cortical bones revealed a thicker cortical width and a higher rate of bone formation in GgcxΔobl/Δobl mice. Electron microscopic examination revealed disassembly of mineralized nodules and aberrant calcification of collagen fibers in GgcxΔobl/Δobl mice. The mechanical properties of bones from GgcxΔobl/Δobl mice tended to be stronger than those from control Ggcx+/+ mice. These results suggest that GGCX in osteoblasts functions to prevent abnormal mineralization in bone formation, although this function may not be a prerequisite for the bone-protective effect of vitamin K. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.

Objective: In August 2012, an outbreak of enterohaemorrhagic Escherichia coli (EHEC) O157 infection was investigated by the City of Sapporo and Hokkaido Prefectural Government. The initial notification reported an illness affecting 94 residents of 10 private nursing homes distributed across multiple areas of Hokkaido, the northernmost island of Japan; at this time three cases were confirmed as EHEC O157 infection. The objectives of the investigation were to identify the source of infection and recommend control measures to prevent further illness. Methods: A suspected case was defined as a resident of one of the private nursing homes in Hokkaido who had at least one of the following gastrointestinal symptoms: diarrhoea, bloody stool, abdominal pain or vomiting between 10 July and 10 September 2012. Cases were confirmed by the presence of Shiga toxin 1- and 2-producing EHEC O157 in stool samples of suspected cases. We conducted an epidemiological analysis and an environmental investigation. Results: We identified 54 confirmed and 53 suspected cases in 12 private nursing homes including five fatalities. Of the 107 cases, 102 (95%) had consumed pickles, all of which had been manufactured at the same facility. EHEC O157 isolates from two pickle samples, 11 cases and two staff members of the processing company were indistinguishable. The company that produced the pickles used inadequate techniques to wash and sanitize the vegetables. Discussion: Contaminated pickles were the likely source of this outbreak. We recommended that the processing company improve their methods of washing and sanitizing raw vegetables. As a result of this outbreak, the sanitation requirements for processing pickles were revised.

Many of the tripartite motif (TRIM) proteins function as E3 ubiquitin ligases and are assumed to be involved in various events, including oncogenesis. In regard to tripartite motif-containing 44 (TRIM44), which is an atypical TRIM family protein lacking the RING finger domain, its pathophysiological significance in breast cancer remains unknown. We performed an immunohistochemical study of TRIM44 protein in clinical breast cancer tissues from 129 patients. The pathophysiological role of TRIM44 in breast cancer was assessed by modulating TRIM44 expression in MCF-7 and MDA-MB-231 breast cancer cells. TRIM44 strong immunoreactivity was significantly associated with nuclear grade (p = 0.033), distant disease-free survival (p = 0.031) and overall survival (p = 0.027). Multivariate analysis revealed that the TRIM44 status was an independent prognostic factor for distant disease-free survival (p = 0.005) and overall survival (p = 0.002) of patients. siRNA-mediated TRIM44 knockdown significantly decreased the proliferation of MCF-7 and MDA-MB-231 cells and inhibited the migration of MDA-MB-231 cells. Microarray analysis and qRT–PCR showed that TRIM44 knockdown upregulated CDK19 and downregulated MMP1 in MDA-MB-231 cells. Notably, TRIM44 knockdown impaired nuclear factor-kappa B (NF-κB)-mediated transcriptional activity stimulated by tumor necrosis factor α (TNFα). Moreover, TRIM44 knockdown substantially attenuated the TNFα-dependent phosphorylation of the p65 subunit of NF-κB and IκBα in both MCF-7 and MDA-MB-231 cells. TRIM44 would play a role in the progression of breast cancer by promoting cell proliferation and migration, as well as by enhancing NF-κB signaling.

Vitamin K-dependent γ-glutamyl carboxylase (GGCX) is an enzyme that catalyzes the conversion of glutamic acid to gamma-carboxyglutamic acid in substrate proteins. Among GGCX target proteins, recent evidence indicates that osteocalcin regulates insulin sensitivity and secretion. However, the precise contribution of GGCX to glucose metabolism remains to be clarified. To address this question, we generated osteoblast-specific Ggcx-deficient (i.e., conditional knockout [cKO]) mice using collagen type 1 α1 (Col1)-Cre mice. Ggcx cKO mice exhibited altered metabolism compared with their controls; serum glucose levels could be maintained with low amounts of insulin, and the weight of white adipose tissue (WAT) significantly decreased in Ggcx cKO mice. Our findings suggest that GGCX expressed in osteoblasts is critical for the maintenance of blood glucose and WAT.

Vitamin K is a fat-soluble vitamin, which is involved in blood coagulation mediated by maintaining the activity of coagulation factors in the liver. Vitamin K also has extrahepatic actions and has been shown to prevent bone fractures in clinical studies. In addition, epidemiological studies suggest that a lack of vitamin K is associated with several geriatric diseases, including osteoporosis, osteoarthritis, dementia and arteriosclerosis. It has also been shown that vitamin K contributes to the prevention and treatment of some kinds of malignancies. Recently, we discovered a novel role for vitamin K as a ligand of the nuclear receptor, steroid and xenobiotic receptor (SXR), and its murine ortholog, pregnane X receptor (PXR). In addition to its established roles as a cofactor of γ-glutamyl carboxylase (GGCX) in mediating post-transcriptional modifications, vitamin K has a different mode of action mediated by transcriptional regulation of SXR/PXR target genes. Analysis of bone tissue from PXR-deficient mice showed that the bone protective effects of vitamin K are partially mediated by SXR/PXR-dependent signaling. The discoveries of a novel mode of vitamin K action have opened up new possibilities that vitamin K might be useful for prevention or treatment of a variety of diseases that affect the geriatric population. Geriatr Gerontol Int 2014; 14: 1–7.

Steroid and Xenobiotic Receptor (SXR) belongs to nuclear receptor superfamily. It was shown that secondary bile acids such as lithocholic acid and several chemical compounds such as rifampicin could be ligands for this receptor. Recently, we have demonstrated that vitamin K2 also serves as a ligand for SXR and activation of SXR by vitamin K2 suppressed proliferation and motility of hepatocellular carcinoma (HCC) cells. To analyze function of SXR in HCC cells, we overexpressed exogenous SXR double-tagged with FLAG and HA in a HCC cell line, HepG2 cells, and purified SXR-binding molecules by immunoprecipitation from the nuclear extracts of these cells. Several binding molecules were identified by TOF-MS analyses. One of the SXR-binding molecules was a transcription factor PROX1. We confirmed the interaction of PROX1 and SXR in HEK293 cells. Then, we have shown that AF2 domain of SXR is necessary for binding with PROX1. We further demonstrated that PROX1 negatively regulated the transcriptional activity of SXR by promoter analyses of SXR target gene. These results suggest that PROX1 could negatively regulate SXR signals in some tumor cells, such as HCC cells, where both SXR and PROX1 are expressed.

Glucocorticoids exert their function by regulating glucocorticoid-responsive genes through interaction with glucocorticoid receptor alpha (GRalpha), a nuclear receptor. Glucocorticoids also affect bone metabolism; this is evidenced by the fact that GRalpha is expressed in several kinds of cells in bone tissue, including osteoblasts, osteocytes, osteoclasts, mononuclear cells in bone marrow, and hypertrophic chondrocytes. Glucocorticoids are known to induce osteoblastic differentiation and bone formation. However, this effect of glucocorticoids on bone tissue is still controversial since long-term use of glucocorticoids results in osteoporosis in vivo. To identify glucocorticoid-regulated genes in human osteoblastic cells, SaOS2 cells were treated with dexamethasone (10(-8) M) for 6 hours, and were then subjected to microarray analysis. Genes such as C/EBPdelta, DUSP1, Per1 and TRIM63 were found to be induced by dexamethasone. The induction of mRNAs of these genes by dexamethasone (10(-8) M, 10(-7) M, and 10(-6) M) was confirmed by quantitative real-time polymerase chain reaction (PCR). TRIM63, also called muscle-specific ring finger protein 1 (MuRF1), was reported to be an E3 ubiquitin ligase expressed mainly in muscular tissue. SaOS2 cells overexpressing exogenous TRIM63 showed increased expression of an osteoblastic differentiation marker gene, alkaline phosphatase, with reduced proliferation. These results suggest that TRIM63 is a candidate for genes mediating the glucocorticoid-induced promotion of osteoblastic differentiation.

Glucocorticoid-induced osteoporosis is a clinical problem in patients under chronic steroid therapy. To delineate the action of glucocorticoids on osteoblasts, we performed microarray analysis using rat primary osteoblasts and identified several glucocorticoid target genes. We validated the dexamethasone-induced upregulation of CCAAT/ enhancer-binding protein delta (C/EBP ), endothelial PAS-domain protein 1 (EPAS1), matrix Gla protein (MGP), and nerve growth factor inducible-B (NGFI-B) expression by quantitative real time-polymerase chain reaction (qPCR). EPAS1 overexpression inhibited, whereas dominant-negative EPAS1 overexpression enhanced the upregulation of osteoblastic marker genes and the mineralization in ST2 mesenchymal cells under the simulated osteoblastogenesis conditions. These results suggest that glucocorticoids could inhibit osteoblastic differentiation by regulating its target genes, as exemplified by EPAS1.

Estrogen receptor α (ERα) is a nuclear receptor that functions as a ligand-activated transcription factor. Besides its genomic action in nuclei, ERα could exert nongenomic actions at the plasma membrane. To investigate the mechanism underlying the nongenomic action of ERα in breast cancer cells, we generated a construct of membrane-targeted ERα (memER), an expression vector of ERα without the nuclear localizing signal and including instead the membrane-targeting sequence of Src kinase. MemER was stably expressed in human breast cancer MCF-7 cells. Cell migration test and tumorigenic assay in nude mice revealed that the in vitro motility and the in vivo proliferation activity of MCF-7 cells expressing memER were significantly enhanced compared with those of vector-transfected cells. Interestingly, the acetylation level of tubulin in memER-overexpressing cells was lower than that in control cells. We found that histone deacetylase (HDAC) 6 translocated to the plasma membrane shortly after estrogen stimulation, and rapid tubulin deacetylation subsequently occurred. We also showed that memER associated with HDAC6 in a ligand-dependent manner. Although tamoxifen is known for its antagonistic role in the ERα genomic action in MCF-7 cells, the agent showed an agonistic function in the memER-HDAC6 association and tubulin deacetylation. These findings suggest that ERα ligand dependently forms a complex with HDAC6 and tubulin at the plasma membrane. Estrogen-dependent tubulin deacetylation could provide new evidence for the nongenomic action of estrogen, which potentially contributes to the aggressiveness of ERα-positive breast cancer cells. [Cancer Res 2009;69(7):2935–40]

To acquire information on signal alteration corresponding to the changes in metastatic potential, we analysed protein tyrosine phosphorylation of low- and high-metastatic human osteosarcoma HuO9 sublines, which were recently established as the first metastatic model of human osteosarcoma. Tyrosine phosphorylation of proteins around 60, 70, and 120-130 kDa was enhanced in high-metastatic sublines. Among these proteins, the protein around 70 kDa, which was most remarkably phosphorylated, was identified as paxillin, a scaffold protein in integrin signaling. Activity of Src family kinase correlated well with metastatic potential, and a Src family kinase inhibitor, PP2, not only abolished tyrosine phosphorylation of paxillin but also impaired the motility of high-metastatic sublines. The expression of paxillin was also elevated in high-metastatic sublines, and knocking down of paxillin expression by RNAi method resulted in attenuated motility of high-metastatic cells. We also demonstrated that the phosphorylated form of paxillin is essential for the migration-promoting effect in human osteosarcoma. These findings suggest that enhanced activity of Src family kinases and overexpression of paxillin synergistically contribute to the high metastatic potential of human osteosarcoma through the hyperphosphorylation of paxillin.

Vitamin K is well known as a critical blood coagulation factor. In addition, epidemiological studies suggest that reduced vitamin K intake is associated with several geriatric diseases, such as osteoporosis, osteoarthritis, dementia, and arteriosclerosis. Indeed, the therapeutic benefits of vitamin K have been demonstrated in osteoporosis patients in several clinical studies. It has also been proposed that vitamin K may contribute to the prevention and treatment of some types of malignancies. Vitamin K functions as a co-factor of γ-glutamyl carboxylase (GGCX) and regulates the activity of vitamin K-dependent proteins expressed in various tissues via posttranscriptional modifications. As an additional mechanism of vitamin K action, we revealed that vitamin K activates the Steroid and Xenobiotic receptor (SXR), a member of nuclear receptor superfamily, leading to changes in gene transcription. Analysis of the bones of pregnane X receptor (PXR)-deficient mice demonstrated that the bone protective effects of vitamin K are partially mediated by SXR/PXR-dependent signaling. In certain malignancies, vitamin K exerts therapeutic activity in an SXR/PXR-dependent manner. Another distinct function of vitamin K involves apoptotic induction of leukemia cells, which may be attributed to the covalent association of vitamin K2 epoxide with pro-apoptotic protein Bak. The progress of vitamin K research has opened up new possibilities that vitamin K can be useful for prevention and treatment of a variety of diseases.

© 2015 Azuma et al. Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.

Vitamin K is a fat-soluble vitamin that is necessary for blood coagulation. In addition, it has bone-protective effects. Vitamin K functions as a cofactor of γ-glutamyl carboxylase (GGCX), which activates its substrates by carboxylation. These substrates are found throughout the body and examples include hepatic blood coagulation factors. Furthermore, vitamin K functions as a ligand of the nuclear receptor known as steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR). We have previously reported on the bone-protective role of SXR/PXR signaling by demonstrating that systemic Pxr-knockout mice displayed osteopenia. Because systemic Ggcx-knockout mice die shortly after birth from severe hemorrhage, the GGCX-mediated effect of vitamin K on bone metabolism has been difficult to evaluate. In this work, we utilized Ggcx-floxed mice to generate osteoblast-specific GGCX-deficient (Ggcx(Δobl/Δobl)) mice by crossing them with Col1-Cre mice. The bone mineral density (BMD) of Ggcx(Δobl/Δobl) mice was significantly higher than that of control Col1-Cre (Ggcx(+/+)) mice. Histomorphometrical analysis of trabecular bones in the proximal tibia showed increased osteoid volume and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Histomorphometrical analysis of cortical bones revealed a thicker cortical width and a higher rate of bone formation in Ggcx(Δobl/Δobl) mice. Electron microscopic examination revealed disassembly of mineralized nodules and aberrant calcification of collagen fibers in Ggcx(Δobl/Δobl) mice. The mechanical properties of bones from Ggcx(Δobl/Δobl) mice tended to be stronger than those from control Ggcx(+/+) mice. These results suggest that GGCX in osteoblasts functions to prevent abnormal mineralization in bone formation, although this function may not be a prerequisite for the bone-protective effect of vitamin K.

Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span. Azuma K, Tsukui T, Ikeda K, Shiba S, Nakagawa K, et al. PLOS ONE. 2014. 9(2) doi:10.1371/journal.pone.0088643, Vitamin K is a fat-soluble vitamin that plays important roles in blood coagulation and bone metabolism. One of its functions is as a co-factor for γ-glutamyl carboxylase (Ggcx). Conventional knockout of Ggcx causes death shortly after birth in homozygous mice. We created Ggcx-floxed mice by inserting loxP sequences at the sites flanking exon 6 of Ggcx. By mating these mice with albumin-Cre mice, we generated Ggcx-deficient mice specifically in hepatocytes (GgcxΔliver/Δliver mice). In contrast to conventional Ggcx knockout mice, GgcxΔliver/Δliver mice had very low activity of Ggcx in the liver and survived several weeks after birth. Furthermore, compared with heterozygous mice (Ggcx+/Δliver), GgcxΔliver/Δliver mice had shorter life spans. Ggcx Δliver/Δliver mice displayed bleeding diathesis, which was accompanied by decreased activity of coagulation factors II and IX. Ggcx-floxed mice can prove useful in examining Ggcx functions in vivo.

Vitamin K is a fat-soluble vitamin, which is involved in blood coagulation mediated by maintaining the activity of coagulation factors in the liver. Vitamin K also has extrahepatic actions and has been shown to prevent bone fractures in clinical studies. In addition, epidemiological studies suggest that a lack of vitamin K is associated with several geriatric diseases, including osteoporosis, osteoarthritis, dementia and arteriosclerosis. It has also been shown that vitamin K contributes to the prevention and treatment of some kinds of malignancies. Recently, we discovered a novel role for vitamin K as a ligand of the nuclear receptor, steroid and xenobiotic receptor (SXR), and its murine ortholog, pregnane X receptor (PXR). In addition to its established roles as a cofactor of γ-glutamyl carboxylase (GGCX) in mediating post-transcriptional modifications, vitamin K has a different mode of action mediated by transcriptional regulation of SXR/PXR target genes. Analysis of bone tissue from PXR-deficient mice showed that the bone protective effects of vitamin K are partially mediated by SXR/PXR-dependent signaling. The discoveries of a novel mode of vitamin K action have opened up new possibilities that vitamin K might be useful for prevention or treatment of a variety of diseases that affect the geriatric population.

Estrogen affects transcriptional status by activating its corresponding nuclear receptor, the estrogen receptor (ER). It can also induce rapid cellular reactions within a few minutes, and this feature cannot be explained by the transcription-mediated effects of estrogen. The latter mechanisms are called ‘non-genomic actions’ of estrogen. In contrast, the former classic modes of action came to be called ‘genomic actions’. One of the recent developments of research on estrogen was the substantiation of the non-genomic actions of estrogen; these were initially observed and reported as intriguing phenomena more than 40 years ago. The interacting molecules as well as the biological significance of non-genomic actions have now been shown. In the field of genomic actions, invention and spread of new technologies, including high-throughput sequencers, promoted a comprehensive view of estrogen-mediated transcriptional regulation.

The steroid and xenobiotic receptor (SXR) regulates cytochrome P450 (CYP) enzymes, which are key inactivators of testosterone in the liver and prostate. In the present study, we investigated SXR expression in human prostate tissues. We determined SXR immunoreactivity using an anti-SXR antibody in benign (n = 78) and cancerous (n = 106) tissues obtained by radical prostatectomy. Stained slides were evaluated for the proportion and staining intensity of immunoreactive cells. Total immunoreactivity (IR) scores (range: 0–8) were calculated as the sum of the proportion and intensity scores. Associations between the clinicopathological features of the patients, SXR status, and CYP3A4 immunoreactivity were analyzed. Western blot analyses validated the specificity of the anti-SXR antibody in 293T cells transfected with pcDNA–FLAG–SXR. Positive (IR score: ≥ 2) nuclear SXR staining was observed in 91% (71/78) of benign foci and 47% (50/106) of cancerous lesions. Immunoreactivity scores were significantly lower in the cancerous lesions than in the benign foci (P

Steroid and Xenobiotic Receptor (SXR) belongs to nuclear receptor superfamily. It was shown that secondary bile acids such as lithocholic acid and several chemical compounds such as rifampicin could be ligands for this receptor. Recently, we have demonstrated that vitamin K2 also serves as a ligand for SXR and activation of SXR by vitamin K2 suppressed proliferation and motility of hepatocellular carcinoma (HCC) cells. To analyze function of SXR in HCC cells, we overexpressed exogenous SXR double-tagged with FLAG and HA in a HCC cell line, HepG2 cells, and purified SXR-binding molecules by immunoprecipitation from the nuclear extracts of these cells. Several binding molecules were identified by TOF-MS analyses. One of the SXR-binding molecules was a transcription factor PROX1. We confirmed the interaction of PROX1 and SXR in HEK293 cells. Then, we have shown that AF2 domain of SXR is necessary for binding with PROX1. We further demonstrated that PROX1 negatively regulated the transcriptional activity of SXR by promoter analyses of SXR target gene. These results suggest that PROX1 could negatively regulate SXR signals in some tumor cells, such as HCC cells, where both SXR and PROX1 are expressed.

The steroid and xenobiotic receptor (SXR) and its murine ortholog pregnane X receptor (PXR) are nuclear receptors that are expressed mainly in the liver and intestine where they function as xenobiotic sensors. In addition to its role as a xenobiotic sensor, previous studies in our laboratories and elsewhere have identified a role for SXR/PXR as a mediator of bone homeostasis. Here, we report that systemic deletion of PXR results in marked osteopenia with mechanical fragility in female mice as young as 4 months old. Bone mineral density (BMD) of PXR knockout (PXRKO) mice was significantly decreased compared with the BMD of wild-type (WT) mice. Micro-computed tomography analysis of femoral trabecular bones revealed that the three-dimensional bone volume fraction of PXRKO mice was markedly reduced compared with that of WT mice. Histomorphometrical analysis of the trabecular bones in the proximal tibia showed a remarkable reduction in bone mass in PXRKO mice. As for bone turnover of the trabecular bones, bone formation is reduced, whereas bone resorption is enhanced in PXRKO mice. Histomorphometrical analysis of femoral cortical bones revealed a larger cortical area in WT mice than that in PXRKO mice. WT mice had a thicker cortical width than PXRKO mice. Three-point bending test revealed that these morphological phenotypes actually caused mechanical fragility. Lastly, serum levels of phosphate, calcium, and alkaline phosphatase were unchanged in PXRKO mice compared with WT. Consistent with our previous results, we conclude that SXR/PXR promotes bone formation and suppresses bone resorption thus cementing a role for SXR/PXR as a key regulator of bone homeostasis.

Insufficient vitamin K nutrition is one of the risks for bone fracture. Vitamin K is clinically applied to osteoporosis treatment in Japan and Asian countries, as the administration has preventive effects on bone fracture. Recent studies have revealed that vitamin K functions as a ligand for Steroid and Xenobiotic Receptor (SXR), as well as a cofactor for gamma-carboxylase. In osteoblastic cells, several SXR responsive genes have been identified, including tsukushi, matrilin-2, CD14, and Msx2. Working together with gamma-carboxylated bone proteins, these SXR targets will function in the vitamin K-mediated bone protection. It has been also suggested that vitamin K could prevent or treat the hepatocellular carcinoma (HCC) in some clinical studies. SXR may also contribute to the vitamin K-dependent reduction of HCC.

Diagnosis and treatment of osteoporosis are evolving from BMD (bone mineral density) dependent manner to bone quality conscious manner. However, clinical methods to evaluate bone quality of diabetic patients have not been established yet. Therefore, many potentially osteoporotic patients with diabetes are not recognized as high fracture risk patients under the current therapeutic guideline of osteoporosis. On the other hand, some drugs for osteoporosis affect pathophysiology of diabetes. In the future, it is possible that drugs for osteoporosis are chosen according to the diabetic state of each patient.