Positive association between blood glucose in physical examinations and self-reported fractures among middle-aged and elderly Japanese men: a prospective cohort study.

The association between blood glucose and fractures is not consistent across populations. Blood glucose was associated with fractures five years later in middle-aged and elderly men who underwent health examinations in Japan, respectively. Blood glucose-targeted fracture alerts are crucial for middle-aged and elderly individuals. OBJECTIVES: The evidence on blood glucose as a fracture risk marker has not been adequately examined in various populations, and there are no studies in middle-aged Japanese. We aimed to determine the association between blood glucose status and self-report fractures among middle-aged and elderly Japanese men. METHODS: The data from the Japan Multi-Institutional Collaborative Cohort (J-MICC) Okazaki Study were used. Hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) measured at baseline physical examinations were examined for association with fractures questioned five years later. Analyses were performed for the middle-aged and elderly respondents. RESULTS: The HbA1c was dichotomized into 290 (11.8%) with HbA1c ≥ 6.5% and 2165 (88.2%) with HbA1c < 6.5%. Compared to the group with an HbA1c < 6.5, the odds ratio for the risks of fracture among the group with an HbA1c ≥ 6.5% were 3.46 (95% confidence interval (CI), 1.75-6.84) in Model 1 (adjusted for age) and 3.60 (95% CI, 1.77-7.34) in Model 2 (adjusted for various confounding factors). These associations were also observed in both middle-aged and elderly generations, whereas no association was observed for FPG. CONCLUSIONS: Among Japanese men who have undergone physical examinations, those with an HbA1c of 6.5% or higher are at higher risk for fractures, and HbA1c-targeted fracture alerts are crucial for middle-aged and elderly individuals.

Genetic polymorphisms in CYP19A1 and ESR1 are associated with serum CK activity after prolonged running in men.

This study aimed to clarify 1) the influence of genetic polymorphisms in the cytochrome P450 aromatase gene (CYP19A1) on circulating estradiol levels in men and 2) whether estrogen-related genetic polymorphisms, such as the CYP19A1 rs936306 and estrogen receptor-α (ESR1) rs2234693 polymorphisms, predict exercise-induced serum creatine kinase (CK) activity, which is an index of skeletal muscle membrane disruption. Serum estradiol levels were examined in young men (n = 167). In a different cohort, serum CK activity was analyzed in a 2-day ultramarathon race: baseline, after the first day, and after the second day (114 males and 25 females). Genetic polymorphisms in CYP19A1 rs936306 C/T and ESR1 rs2234693 T/C were analyzed using the TaqMan SNP Genotyping Assay. Male subjects with the TT genotype of the CYP19A1 polymorphism exhibited significantly higher serum estradiol levels than the C allele carriers. Male runners had significantly higher postrace serum CK activity than female runners. The change in the CK activity during the ultramarathon race was significantly lower in male subjects with the CYP19A1 TT genotype than in those with the CC + CT genotypes and was correlated with the number of C alleles in ESR1 rs2234693 in male subjects. Furthermore, the genotype scores of these two polymorphisms were significantly correlated with changes in serum CK activity during race (r = -0.279, P = 0.003). The results of this study suggest that genetic polymorphisms in CYP19A1 rs936306 influence serum estradiol levels in men, and genetic polymorphisms in CYP19A1 and ESR1 are associated with serum CK activity in men.NEW & NOTEWORTHY Men with the TT genotype of the CYP19A1 polymorphism exhibited higher circulating estradiol levels than the TC + CC genotype. The TT genotype in the CYP19A1 polymorphism and the C allele of the ESR1 polymorphism, an allele increasing ESR1 expression, were associated with low serum CK activity after the ultramarathon. A combination of these polymorphisms was correlated with changes in the serum CK activity. Therefore, estrogen-related genetic polymorphisms partially predict exercise-induced muscle damage, that is, skeletal muscle membrane disruption.

Involvement of autophagy in exacerbation of eosinophilic airway inflammation in a murine model of obese asthma.

Obesity is a common comorbidity in patients with asthma, and obese asthma patients present the most refractory phenotype among patients with severe asthma. Similar to the observations in non-obese asthma patients, clinical studies have revealed heterogeneity in obese asthma patients, including the occurrences of T helper (Th)2-high and Th2-low phenotypes. However, the mechanisms underlying obesity-related asthma are not completely understood. Though macroautophagy/autophagy is involved in asthma and obesity, its role in obesity-associated asthma is unknown. We hypothesized that autophagy is involved in the pathogenesis of obese asthma. For our investigations, we used high-fat diet-induced Atg5 (autophagy related 5)-deficient mice and epithelial cell-specific atg5-/- (Scgb1a1/CCSP-atg5-/-) obesity-induced mice. House dust mite (HDM)-sensitized atg5-/- obese mice exhibited marked eosinophilic inflammation and airway hyper-reactivity (AHR), compared to wild-type (WT) obese mice. Analyses of atg5-/- obese mice showed increased levels of Th2 cells but not ILC2s together with elevated expression of Th2 cytokines in the lung. In response to the HDM challenge, activated epithelial autophagy was observed in lean but not obese WT mice. Epithelium-specific deletion of Atg5 induced eosinophilic inflammation in Scgb1a1/CCSP-atg5-/- obese mice, and genetic analyses of epithelial cells from HDM-immunized atg5-/- obesity-induced mice showed an elevated expression of thymic stromal lymphopoietin (TSLP) and IL33. Notably, HDM-sensitized atg5-/- mice developed TSLP- and IL33-dependent eosinophilic inflammation and AHR. Our results suggest that autophagy contributes to the exacerbation of eosinophilic inflammation in obese asthma. Modulations of autophagy may be a therapeutic target in obesity-associated asthma.Abbreviations: AHR: airway hyper-reactivity; BAL: bronchoalveolar lavage; Cdyn: dynamic compliance; BM: bone marrow; HDM: house dust mite; HFD: high-fat diet; ILC2s: type 2 innate lymphocyte cells; ROS: reactive oxygen species; RL: lung resistance; TSLP: thymic stromal lymphopoietin; TCC: total cell count; WT: wild type.

The Impact of Marital Transitions on Vegetable Intake in Middle-aged and Older Japanese Adults: A 5-year Longitudinal Study.

BACKGROUND: Marital transitions are associated with adverse health events, such as mortality and cardiovascular disease. Since marital transitions (eg, becoming widowed) are unavoidable life events, it is necessary to identify modifiable intermediate outcomes. Thus, we examined the association between marital transitions and vegetable intake among middle-aged and older Japanese adults. METHODS: This longitudinal study included Japanese adults aged 40-79 years who received an annual health checkup between 2007 and 2011 (baseline) and 5 years later (follow-up). Marital transitions were classified as whether and what type of transition occurred during the 5-year period and comprised five groups: consistently married, married to widowed, married to divorced, not married to married, and remained not married. Changes in total vegetable, green and yellow vegetable, and light-colored vegetable intake from baseline to follow-up were calculated using the Food Frequency Questionnaire. RESULTS: Data from 4,813 participants were analyzed (mean age, 59.4 years; 44.1% women). Regarding marital transitions, 3,960 participants were classified as "consistently married," 135 as "married to widowed," 40 as "married to divorced," 60 as "not married to married," and 529 as "remained not married." Multivariable linear regression analysis revealed that compared to consistently married, married to widowed was inversely associated with the change in total vegetable intake (ß = -16.64, SE = 7.68, P = 0.030) and light-colored vegetable intake (ß = -11.46, SE = 4.33, P = 0.008). CONCLUSION: Our findings suggest that being widowed could result in a reduced intake of vegetables. Hence, dietary counseling according to marital situation is necessary.

The Impact of Job Conditions on Health-Related Quality of Life among Working Japanese Older Adults: A Five-Year Longitudinal Study Using J-MICC Okazaki Study Data.

OBJECTIVES: The number of older adults who continue working after retirement is increasing in Japan. Little is known about how job conditions affect older adults' health. We examined the association between job conditions and health-related quality of life (HRQOL) during a five-year follow-up study. METHODS: This study included participants aged 65 years or older from the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area recruited at baseline between 2007 and 2011 and followed up five years later. Participants completed a self-reported questionnaire on the physical and mental health aspects of HRQOL (SF-8™), employment status, and job conditions (job satisfaction, skill use, and job suitability). RESULTS: Data of 1,146 men and 522 women were analyzed (mean age: 69.1 and 68.6 years, respectively). Generalized mixed linear regression analysis revealed that, compared to the not-working group, skill use was positively associated with mental health aspects among men (skill use × time: ß = 0.16, SE = 0.08, p < 0.05), while poor job satisfaction and job suitability were negatively associated with mental health aspects among women (job satisfaction, not satisfied × time: ß = -0.93, SE = 0.47, p < 0.05; job suitability, not suitable × time: ß = -1.06, SE = 0.50, p < 0.05). CONCLUSIONS: Regarding job conditions among older adults, skill use in men was marginally associated with mental health, and poor job satisfaction and suitability in women were negatively associated with mental health. Considering the job conditions of older workers is necessary to protect their mental health.

The Association between Family Caregiver Burden and Subjective Well-Being and the Moderating Effect of Social Participation among Japanese Adults: A Cross-Sectional Study.

We examined the association between family caregiver burden and subjective well-being with social participation's moderating effect among Japanese adults. Data were obtained from a cross-sectional survey by the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area between 2013 and 2017. Study participants included 5321 adults who visited the Public Health Center for annual health check-ups and answered a questionnaire regarding health status and lifestyle. Subjective well-being was assessed by a single item, out of 10 points, and analyzed with multivariable linear regression analysis models by subjective family caregiver burden ("none", "mild", "severe"), stratified by gender. Ultimately, 2857 men and 2223 women were included. Mean participant age (standard deviation) in years was 64.7 (10.4) for men and 61.3 (10.0) for women. Multivariable analysis revealed that, among women, higher caregiver burden was inversely associated with subjective well-being (p for trend < 0.001), and the interaction of severe caregiver burden and social participation on subjective well-being was positive and significant (p for interaction < 0.05). High family caregiver burden was inversely associated with subjective well-being among Japanese women, but moderated by the caregiver's social participation, suggesting the importance of community development that enables family caregivers' social participation to protect their subjective well-being.

Association Between Second-hand Smoke Exposure and Depressive Symptoms Among Japanese Adults: A Cross-sectional Study.

BACKGROUND: Second-hand smoke exposure has been associated with poor mental health. However, among Japanese adults, little is known about the association between second-hand smoking and depressive symptoms. We examined this association in a cross-sectional study among a Japanese general adult population sample. METHODS: Japanese adults were recruited from the Japan Multi-Institutional Collaborative Cohort Study in the Okazaki area between 2012 and 2017. Second-hand smoke exposure and smoking status were assessed using a self-administered questionnaire. Based on their frequency of exposure to second-hand smoke, non-smokers and smokers were categorized as "almost never," "sometimes," and "almost every day". Depressive symptoms were defined by a Kessler 6 score ≥5 points. We performed a multivariable Poisson regression analysis to obtain adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs) for depressive symptoms. RESULTS: Overall, 5,121 participants (4,547 non-smokers and 574 smokers) were included whose mean age was 63.6 (standard deviation [SD], 10.3) years for non-smokers and 59.33 (SD, 10.2) years for smokers. The association between second-hand smoking and depressive symptoms was significant among non-smokers, but not among smokers. Among non-smokers, PRs compared with "almost never" were 1.25 (95% CI, 1.09-1.42) for "sometimes" and 1.41 (95% CI, 1.09-1.84) for "almost every day" (P for trend <0.001); among smokers, PRs compared with "almost never" were 1.30 (95% CI, 0.82-2.06) for "sometimes" and 1.44 (95% CI, 0.90-2.33) for "almost every day" (P for trend = 0.144). CONCLUSIONS: Second-hand smoking and depressive symptoms were associated among non-smokers. Our findings indicate the importance of tobacco smoke control for mental health.

Handling missing data in an FFQ: multiple imputation and nutrient intake estimates.

OBJECTIVE: We aimed to examine missing data in FFQ and to assess the effects on estimating dietary intake by comparing between multiple imputation and zero imputation. DESIGN: We used data from the Okazaki Japan Multi-Institutional Collaborative Cohort (J-MICC) study. A self-administered questionnaire including an FFQ was implemented at baseline (FFQ1) and 5-year follow-up (FFQ2). Missing values in FFQ2 were replaced by corresponding FFQ1 values, multiple imputation and zero imputation. SETTING: A methodological sub-study of the Okazaki J-MICC study.ParticipantsOf a total of 7585 men and women aged 35-79 years at baseline, we analysed data for 5120 participants who answered all items in FFQ1 and at least 50% of items in FFQ2. RESULTS: Among 5120 participants, the proportion of missing data was 3·7%. The increasing number of missing food items in FFQ2 varied with personal characteristics. Missing food items not eaten often in FFQ2 were likely to represent zero intake in FFQ1. Most food items showed that the observed proportion of zero intake was likely to be similar to the probability that the missing value is zero intake. Compared with FFQ1 values, multiple imputation had smaller differences of total energy and nutrient estimates, except for alcohol, than zero imputation. CONCLUSIONS: Our results indicate that missing values due to zero intake, namely missing not at random, in FFQ can be predicted reasonably well from observed data. Multiple imputation performed better than zero imputation for most nutrients and may be applied to FFQ data when missing is low.

Physical Activity Earlier in Life Is Inversely Associated With Insulin Resistance Among Adults in Japan.

BACKGROUND: It is known that physical activity affects glucose metabolism. However, there have been no reports on the influence of physical activity earlier in life on subsequent glucose metabolism. Therefore, we analyzed the influence of physical activity in earlier decades of life on insulin resistance in middle aged and older residents in Japan. METHODS: The subjects were 6,883 residents of Okazaki City between the ages of 40 and 79 years who underwent physical examinations at the Okazaki City Medical Association Public Health Center from April 2007 through August 2011. They gave informed consent for participation in the study. Data on individual characteristics were collected via a questionnaire and from the health examination records. Fasting blood glucose and insulin levels were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). HOMA-IR >1.6 was considered to indicate insulin resistance for the purpose of logistic regression models. RESULTS: The study sample included 3,683 men and 3,200 women for whom complete information was available. For those who exercised regularly throughout their teens to their 30s-40s, the odds ratio for having insulin resistance was 0.75 (95% confidence interval [CI], 0.58-0.96) for men and 0.76 (95% CI, 0.58-0.99) for women after adjusting for other variables, including age, body mass index, and present physical activity. A linear trend was also observed in both men and women. CONCLUSIONS: Subjects who have exercised regularly in the early decades of life are less likely to have insulin resistance later in life.

LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis.

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on Ltbp2, the gene encoding latent transforming growth factor-ß (TGF-ß) binding protein-2 (LTBP2), because of the signal similarity to Acta2, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-ß1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = -0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026-1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.

Changes in blood biochemical markers before, during, and after a 2-day ultramarathon.

We studied changes in blood markers of 18 nonprofessional, middle-aged runners of a 2-day, 130 km ultramarathon. Blood was sampled at baseline, after the goals on the first and second day, and at three time points (1, 3, and 5/6 days) after the race. Blood indices showed three patterns. First pattern indices showed essentially no changes after the two goals and after the race, including red blood cell indices, gamma-glutamyl transferase, and tumor necrosis factor-α. Second pattern markers, including the majority of indices, were elevated during the race (and also after the race for some parameters) and then returned to baseline afterward, including hemolysis/red blood cell destruction markers (indirect bilirubin) and an iron reservoir index (ferritin), muscle damage parameters (uric acid, creatine kinase, lactate dehydrogenase, and aspartate aminotransferase), renal function markers (creatinine and blood urea nitrogen), liver injury index (alanine aminotransferase), lipid metabolism indices (free fatty acid), reactive oxygen species and inflammation parameters (white blood cells, interleukin-6, and C-reactive protein), and energy production and catecholamines (adrenaline, noradrenaline, and dopamine). Third pattern index of a lipid metabolism marker - triglyceride - decreased during the race periods and started returning to baseline from then onward. Some hormonal markers such as insulin, leptin, and adiponectin showed unique patterns. These findings appeared informative for nonprofessional athletes to know about an optimal physical activity level, duration, and total exercise for elevating physical performance and monitoring physical/mental conditioning as well as for prevention of overtraining and physical injuries.

Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation.

Herein, we report the characterization of Limb expression 1-like, (LIX1L), a putative RNA-binding protein (RBP) containing a double-stranded RNA binding motif, which is highly expressed in various cancer tissues. Analysis of MALDI-TOF/TOF mass spectrometry and RNA immunoprecipitation-sequencing of interacting proteins and the microRNAs (miRNAs) bound to LIX1L revealed that LIX1L interacts with proteins (RIOK1, nucleolin and PABPC4) and miRNAs (has-miRNA-520a-5p, -300, -216b, -326, -190a, -548b-3p, -7-5p and -1296) in HEK-293 cells. Moreover, the reduction of phosphorylated Tyr(136) (pTyr(136)) in LIX1L through the homeodomain peptide, PY136, inhibited LIX1L-induced cell proliferation in vitro, and PY136 inhibited MKN45 cell proliferation in vivo. We also determined the miRNA-targeted genes and showed that was apoptosis induced through the reduction of pTyr(136). Moreover, ROS1, HCK, ABL1, ABL2, JAK3, LCK and TYR03 were identified as candidate kinases responsible for the phosphorylation of Tyr(136) of LIX1L. These data provide novel insights into the biological significance of LIX1L, suggesting that this protein might be an RBP, with implications for therapeutic approaches for targeting LIX1L in LIX1L-expressing cancer cells.

Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of THR176 in GATA-binding protein 2.

A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

Cell line differences in replication timing of human glutamate receptor genes and other large genes associated with neural disease.

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (> 200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.

Single-cell time-of-flight secondary ion mass spectrometry reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells.

Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.

Fbw7 targets GATA3 through cyclin-dependent kinase 2-dependent proteolysis and contributes to regulation of T-cell development.

Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4(+) and CD8(+) cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages.

Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences.

BACKGROUND: Myofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions. RESULTS: We demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts. CONCLUSIONS: This is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

JmjC-domain containing histone demethylase 1B-mediated p15(Ink4b) suppression promotes the proliferation of leukemic progenitor cells through modulation of cell cycle progression in acute myeloid leukemia.

The histone demethylase JHDM1B has been implicated in cell cycle regulation and tumorigenesis. In addition, it has been reported that JHDM1B is highly expressed in various human tumors, including leukemias. However, it is not clearly understood how JHDM1B contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of JHDM1B in AML cells. In AML cell lines and AML-derived ALDH(hi) (high aldehyde dehydrogenase activity)/CD34(+) cells, the levels of JHDM1B mRNA were significantly higher than in normal ALDH(hi) /CD34(+) cells. Reduction of JHDM1B expression in AML cells inhibited cell proliferation compared to control cells, through induction of G1 cell cycle arrest, an increase in the p15(Ink4b) mRNA and protein expression. JHDM1B mRNA was overexpressed in all 133 AML clinical specimens tested (n = 22, 57, 34, and 20 for M1, 2, 4, and 5 subtypes respectively). Compared to normal ALDH(hi) /CD34(+) cells, JHDM1B gene expression was 1.57- to 1.87-fold higher in AML-derived ALDH(hi) /CD34(+) cells. Moreover, the JHDM1B protein was more strongly expressed in AML-derived ALDH(hi) /CD34(+) cells from compared to normal ALDH(hi) /CD34(+) cells. In addition, depletion of JHDM1B reduced colony formation of AML-derived ALDH(hi) /CD34(+) cells due to induction of p15(Ink4b) expression through direct binding to p15(Ink4b) promoter and loss of demethylation of H3K36me2. In summary, we found that JHDM1B mRNA is predominantly expressed in AML-derived ALDH(hi) /CD34(+) cells, and that aberrant expression of JHDM1B induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of JHDM1B expression represents an attractive target for AML therapy.

Mouse CD11bhigh lung dendritic cells have more potent capability to induce IgA than CD103+ lung dendritic cells in vitro.

Lung dendritic cells (LDCs) are primary antigen-presenting cells that develop IgA-producing plasma cells in the lung through class switch recombination (CSR) in naive B cells. Recently, the major LDC subsets were found to comprise CD103(-)CD11b(high) LDCs (CD11b(high) LDCs) and CD103(+)CD11b(low or negative) LDCs (CD103(+) LDCs), but their abilities to induce IgA production have not been defined. Under T cell-dependent (T-D) and T cell-independent (T-ID) conditions, we compared the abilities of these two LDC populations to induce IgA. CD11b(high) or CD103(+) LDCs obtained from BALB/c mice were cocultured with naive IgD(+) B cells in the presence of LPS, with or without anti-CD40 monoclonal antibody (mAb) (i.e., T-D and T-ID coculture conditions, respectively). Under both T-D and T-ID conditions, CD11b(high) LDCs induced significantly greater amounts of IgA production, together with a significantly higher mRNA expression of activation-induced cytidine deaminase, than did CD103(+) LDCs. However, the protein expression of a proliferation-inducing ligand, B cell-activating factor of the tumor necrosis family, or retinaldehyde dehydrogenase-1 did not differ between the two LDC subsets. CD11b(high) LDCs displayed a significantly greater capacity to secrete IL-6 and IL-10 in response to LPS, with or without anti-CD40 mAb. Moreover, the IgA production induced by CD11b(high) LDCs in T-D coculture was attenuated by neutralizing both IL-6 and IL-10. These findings suggest that, of the two major LDCs, CD11b(high) LDCs more efficiently induce IgA than do CD103(+) LDCs, possibly through their potent capacity to produce IgA-inducing cytokines.

Down-regulation of Thanatos-associated protein 11 by BCR-ABL promotes CML cell proliferation through c-Myc expression.

Bcr-Abl activates various signaling pathways in chronic myelogenous leukemia (CML) cells. The proliferation of Bcr-Abl transformed cells is promoted by c-Myc through the activation of Akt, JAK2 and NF-κB. However, the mechanism by which c-Myc regulates CML cell proliferation is unclear. In our study, we investigated the role of Thanatos-associated protein 11 (THAP11), which inhibits c-Myc transcription, in CML cell lines and in hematopoietic progenitor cells derived from CML patients. The induction of THAP11 expression by Abl kinase inhibitors in CML cell lines and in CML-derived hematopoietic progenitor cells resulted in the suppression of c-Myc. In addition, over-expression of THAP11 inhibited CML cell proliferation. In colony forming cells derived from CML-aldehyde dehydrogenase (ALDH)(hi) /CD34(+) cells, treatment with Abl kinase inhibitors and siRNA depletion of Bcr-Abl induced THAP11 expression and reduced c-Myc expression, resulting in inhibited colony formation. Moreover, overexpression of THAP11 significantly decreased the colony numbers, and also inhibited the expression of c-myc target genes such as Cyclin D1, ODC and induced the expression of p21(Cip1) . The depletion of THAP11 inhibited JAK2 or STAT5 inactivation-mediated c-Myc reduction in ALDH(hi) /CD34(+) CML cells. Thus, the induced THAP11 might be one of transcriptional regulators of c-Myc expression in CML cell. Therefore, the induction of THAP11 has a potential possibility as a target for the inhibition of CML cell proliferation.