Genetic evidence for involvement of ß2-adrenergic receptor in brown adipose tissue thermogenesis in humans.

BACKGROUND: Sympathetic activation of brown adipose tissue (BAT) thermogenesis can ameliorate obesity and related metabolic abnormalities. However, crucial subtypes of the ß-adrenergic receptor (AR), as well as effects of its genetic variants on functions of BAT, remains unclear in humans. We conducted association analyses of genes encoding ß-ARs and BAT activity in human adults. METHODS: Single nucleotide polymorphisms (SNPs) in ß1-, ß2-, and ß3-AR genes (ADRB1, ADRB2, and ADRB3) were tested for the association with BAT activity under mild cold exposure (19 °C, 2 h) in 399 healthy Japanese adults. BAT activity was measured using fluorodeoxyglucose-positron emission tomography and computed tomography (FDG-PET/CT). To validate the results, we assessed the effects of SNPs in the two independent populations comprising 277 healthy East Asian adults using near-infrared time-resolved spectroscopy (NIRTRS) or infrared thermography (IRT). Effects of SNPs on physiological responses to intensive cold exposure were tested in 42 healthy Japanese adult males using an artificial climate chamber. RESULTS: We found a significant association between a functional SNP (rs1042718) in ADRB2 and BAT activity assessed with FDG-PET/CT (p < 0.001). This SNP also showed an association with cold-induced thermogenesis in the population subset. Furthermore, the association was replicated in the two other independent populations; BAT activity was evaluated by NIRTRS or IRT (p < 0.05). This SNP did not show associations with oxygen consumption and cold-induced thermogenesis under intensive cold exposure, suggesting the irrelevance of shivering thermogenesis. The SNPs of ADRB1 and ADRB3 were not associated with these BAT-related traits. CONCLUSIONS: The present study supports the importance of ß2-AR in the sympathetic regulation of BAT thermogenesis in humans. The present collection of DNA samples is the largest to which information on the donor's BAT activity has been assigned and can serve as a reference for further in-depth understanding of human BAT function.

Effects of bathing-induced changes in body temperature on sleep.

BACKGROUND: Passive body heating before sleep is well known to lead to improved sleep. However, the effects of the degree of change in body temperature by bathing on sleep quality are unclear. The present study aimed to clarify the effects on sleep of bathing-induced changes in body temperature. METHODS: Twenty-three healthy males and females in their 20 s to 50 s bathed in their homes 1.5-2 h before bedtime under three bathing conditions: showering only; short bathing in a bathtub; and long bathing in a bathtub. Sublingual and skin temperatures and thermal sensation before and after bathing, sleep indices such as sleep onset latency, time in bed, sleep efficiency, and wake after sleep onset, all of which were evaluated using an actimeter, and subjective evaluations of sleep were compared among conditions. RESULTS: Sublingual temperature just after bathing was significantly higher with long bathing than with other conditions, and the fall in sublingual temperature from after bathing to before sleep was significantly larger with long bathing than with short bathing. Sleep onset latency by actimeter was significantly reduced with long bathing compared to showering. In addition, subjective evaluations of falling asleep and sleep quality were better with long bathing than with showering or short bathing. CONCLUSIONS: In conclusion, bathing conditions that produce a 0.9 °C increase in sublingual temperature appear effective for falling asleep and sleep quality, because core temperature shows a greater drop to before sleep than those producing an increase of about 0.3 °C increase in sublingual temperature.

Endocrine, inflammatory and immune responses and individual differences in acute hypobaric hypoxia in lowlanders.

When lowlanders are exposed to environments inducing hypobaric hypoxia (HH) such as high mountains, hemodynamic changes occur to maintain oxygen levels in the body. However, changes to other physiological functions under such conditions have yet to be clarified. This study investigated changes in endocrine, inflammatory and immune parameters and individual differences during acute HH exposure using a climatic chamber (75 min of exposure to conditions mimicking 3500 m) in healthy lowlanders. Aldosterone and cortisol were significantly decreased and interleukin (IL)-6, IL-8 and white blood cell (WBC) counts were significantly increased after HH. Lower peripheral oxygen saturation (SpO2) was associated with higher IL-6 and WBC counts, and higher IL-8 was associated with higher cortisol. These findings suggest that endocrine, inflammatory and immune responses are evoked even with a short 75-min exposure to HH and individuals with lower SpO2 seemed to show more pronounced responses. Our results provide basic data for understanding the physiological responses and interactions of homeostatic systems during acute HH.

Autonomic thermoregulatory responses and subjective thermal perceptions upon the initiation of thermal behavior among resting humans in hot and humid environment.

BACKGROUND: While thermoregulatory behavior is critical for maintaining homeostasis, our knowledge of behavioral thermoeffectors in humid heat is limited compared to the control of autonomic thermoeffectors. The predictions that the frequency and duration of intensified humid heat events are expected to increase in the coming years underline this shortcoming. Therefore, this study aims to elucidate the activation of autonomic thermoregulatory responses and subjective thermal perceptions upon deciding to initiate thermal behavior in a hot and humid environment. METHODS: Ten young male adults participated in an experimental trial where local cooling was permitted at any time during the behavioral assessment during passive exposure to humid heat. The air temperature and relative humidity were kept at 33[Formula: see text]C and 80[Formula: see text], respectively. Skin temperatures, core body temperature (T[Formula: see text]), and skin blood flow (forearm, upper arm, and upper back) were obtained 120 s preceding thermal behavior. Local sweat rate (forearm and upper arm) and subjective thermal perceptions (neck and whole-body) upon thermal behavior initiation were also recorded. RESULTS: Mean skin temperature ([Formula: see text]) and T[Formula: see text] increased prior to thermal behavior initiation (p [Formula: see text] 0.002; p [Formula: see text] 0.001). An increase in mean body temperature ([Formula: see text]) was also observed (p < 0.001). However, the initiation of thermal behavior is not preceded by an increase in skin blood flow (p [Formula: see text] 0.154) and local sweat rate (p [Formula: see text] 0.169). An increase in thermal discomfort and skin wetness perception was observed (p [Formula: see text] 0.048; p [Formula: see text] 0.048), while thermal sensation did not differ from the baseline (p [Formula: see text] 0.357). CONCLUSION: These findings suggest that when given the opportunity to behaviorally thermoregulate in a hot and humid environment, changes in skin blood flow and sweat rate are not required for thermal behavior to be initiated in resting humans. Moreover, an increase in [Formula: see text] and T[Formula: see text], which appears to cause an increase in thermal discomfort, precedes thermal behavior. In addition, an increase in [Formula: see text] leading up to thermal behavior initiation was observed, suggesting that changes in [Formula: see text] rather than [Formula: see text] and T[Formula: see text] alone mediate thermal behavior in humid heat. Collectively, the results of this study appear to support the hypothesis that the temporal recruitment of autonomic thermoeffectors follows an orderly manner based on their physiological cost.

Upregulation of cathepsin L gene under mild cold conditions in young Japanese male adults.

BACKGROUND: Physiological thermoregulatory systems in humans have been a key factor for adaptation to local environments after their exodus from Africa, particularly, to cold environments outside Africa. Recent studies using high-throughput sequencing have identified various genes responsible for cold adaptation. However, the molecular mechanisms underlying initial thermoregulation in response to acute cold exposure remain unclear. Therefore, we investigated transcriptional profiles of six young Japanese male adults exposed to acute cold stress. METHODS: In a climatic chamber, the air temperature was maintained at 28°C for 65 min and was then gradually decreased to 19°C for 70 min. Saliva samples were obtained from the subjects at 28°C before and after 19°C cold exposure and were used for RNA sequencing. RESULTS: In the cold exposure experiment, expression levels of 14 genes were significantly changed [false discovery rate (FDR) < 0.05] although the degree of transcriptional changes was not high due to experimental conditions or blunted transcriptional reaction in saliva to cold stress. As a result, differential gene expression analyses detected the cathepsin L (CTSL) gene to be significantly upregulated, with FDR < 0.05 and log2 fold change value > 1; thus, this gene was identified as a differentially expressed gene. Given that the cathepsin L protein is related to invasion of the novel coronavirus (SARS-CoV-2), mild cold stress might alter the susceptibility to coronavirus disease-19 in humans. The gene ontology enrichment analysis for 14 genes with FDR < 0.05 suggested that immune-related molecules could be activated by mild cold stress. CONCLUSIONS: The results obtained from this study indicate that CTSL expression levels can be altered by acute mild cold stress.

Transcriptomic Changes in Young Japanese Males After Exposure to Acute Hypobaric Hypoxia.

After the genomic era, the development of high-throughput sequencing technologies has allowed us to advance our understanding of genetic variants responsible for adaptation to high altitude in humans. However, transcriptomic characteristics associated with phenotypic plasticity conferring tolerance to acute hypobaric hypoxic stress remain unclear. To elucidate the effects of hypobaric hypoxic stress on transcriptional variability, we aimed to describe transcriptomic profiles in response to acute hypobaric hypoxia in humans. In a hypobaric hypoxic chamber, young Japanese males were exposed to a barometric pressure of 493 mmHg (hypobaric hypoxia) for 75 min after resting for 30 min at the pressure of 760 mmHg (normobaric normoxia) at 28°C. Saliva samples of the subjects were collected before and after hypobaric hypoxia exposure, to be used for RNA sequencing. Differential gene expression analysis identified 30 significantly upregulated genes and some of these genes may be involved in biological processes influencing hematological or immunological responses to hypobaric hypoxic stress. We also confirmed the absence of any significant transcriptional fluctuations in the analysis of basal transcriptomic profiles under no-stimulus conditions, suggesting that the 30 genes were actually upregulated by hypobaric hypoxia exposure. In conclusion, our findings showed that the transcriptional profiles of Japanese individuals can be rapidly changed as a result of acute hypobaric hypoxia, and this change may influence the phenotypic plasticity of lowland individuals for acclimatization to a hypobaric hypoxic environment. Therefore, the results obtained in this study shed light on the transcriptional mechanisms underlying high-altitude acclimatization in humans.

Effects of acute hypobaric hypoxia on thermoregulatory and circulatory responses during cold air exposure.

BACKGROUND: The thermoregulatory responses during simultaneous exposure to hypoxia and cold are not well understood owing to the opposite reactions of vasomotor tone in these two environments. Therefore, the purpose of this study was to investigate the influences of hypobaric hypoxia on various thermoregulatory responses, including skin blood flow (SkBF) during cold exposure. METHODS: Ten subjects participated in two experimental conditions: normobaric normoxia with cold (NC, barometric pressure (PB) = 760 mmHg) and hypobaric hypoxia with cold (HC, PB = 493 mmHg). The air temperature was maintained at 28 °C for 65 min and gradually decreased to 19 °C for both conditions. The total duration of the experiment was 135 min. RESULTS: The saturation of percutaneous oxygen (SpO2) was maintained at 98-99% in NC condition, but decreased to around 84% in HC condition. The rectal and mean skin temperatures showed no significant differences between the conditions; however, the forehead temperature was higher in HC condition than in NC condition. The pulse rate increased in HC condition, and there was a strong negative relationship between SpO2 and pulse rate (r = - 0.860, p = 0.013). SkBF and blood pressure showed no significant differences between the two conditions. CONCLUSION: These results suggest that hypobaric hypoxia during cold exposure did not alter the overall thermoregulatory responses. However, hypobaric hypoxia did affect pulse rate regardless of cold exposure.

Multiorgan contribution to non-shivering and shivering thermogenesis and vascular responses during gradual cold exposure in humans.

PURPOSE: Human brown adipose tissue (BAT) is known to be a significant thermoeffector in non-shivering thermogenesis (NST), albeit with individual variations in the BAT activity. We hypothesized that humans with less BAT would have more contribution from the skeletal muscle (SM) to NST or earlier shivering onset and greater vasoconstriction to compensate for less BAT-mediated thermogenesis. METHODS: Eighteen males participated in this study. Their BAT activity and detectable volume were investigated. A gradual cold exposure was conducted for inducing NST at 18.6 °C and initiating shivering at 11.6 °C. The energy expenditure, electromyograph of the pectoralis major, skin blood flow, and rectal (Tre) and skin temperatures were evaluated. RESULTS: BAT volume significantly correlated with the change in metabolic heat production during mild cold phase relative to baseline (NST; r = 0.562, P < 0.05), but not with shivering initiation phase (NST+ ST). SM mass correlated with baseline metabolic heat production (Mbase; r = 0.839, P < 0.01) but not with NST or NST + ST. A positive correlation was noted between BAT volume and Tre at the end of the 18.6 °C exposure period (r = 0.586, P < 0.05), which positively correlated with shivering onset time (r = 0.553, P < 0.05). The skin blood flow, mean skin temperature, and forearm and finger skin temperature difference at the end of the 18.6 °C exposure period did not correlate with NST or BAT volume. CONCLUSION: BAT volume positively correlated with NST. Notably, lower Tre in individuals with less BAT volume induced earlier shivering onset for offsetting the less NST. Whereas, no correlation between metabolic and vasomotor responses was observed.

Productivity and physiological responses during exposure to varying air temperatures and clothing conditions.

This study assessed the effects of clothing and air temperature combinations on workplace productivity and physiological response. Ten male Japanese subjects were exposed to six combinations of clothing (0.3 clo and 0.9 clo) and air temperature (16°C, 26°C, and 36°C) during which cognitive performance (Bourdon and calculation tests), manual motor performance (finger-tapping test), and physiological responses (heart rate, blood pressure, and skin and oral temperatures) were measured. Both cold exposure and lower clothing levels likely increased the Bourdon test performance. Calculation test performance tended to be affected by exposure to cold or neutral temperatures at the beginning of the test. Cold exposure undermined manual motor performance (especially when combined with fewer clothing items) while heat exposure significantly increased heart rate. Both cold exposure and higher clothing level during heat exposure increased blood pressure. Body temperature, particularly mean skin temperature, increased with higher air temperature and was significantly influenced by clothing insulation during cold exposure. These results provide novel evidence for the effects of clothing and air temperature (particularly cold) on human productivity and physiological responses in humans.

Non-visual effects of diurnal exposure to an artificial skylight, including nocturnal melatonin suppression.

BACKGROUND: Recently, more consideration is being given to the beneficial effects of lighting on the maintenance and promotion of the health and well-being of office occupants in built environments. A new lighting technology using Rayleigh scattering has made it possible to simulate a blue sky. However, to date, no studies have examined the possible beneficial effects of such artificial skylights. The aims of this study were to examine the non-visual effects of artificial skylights and conventional fluorescent lights in a simulated office environment and to clarify the feature effects of the artificial skylights. METHODS: Participants were 10 healthy male adults. Non-visual effects were evaluated based on brain arousal levels (α-wave ratio and contingent negative variation [CNV]), autonomic nervous activity (heart rate variability [HRV]), work performance, and subjective responses during daytime exposure to either an artificial skylight or fluorescent lights, as well as nocturnal melatonin secretion. RESULTS: Subjective evaluations of both room lighting-related "natural" and "attractive" items and the "connected to nature" item were significantly higher with the skylight than with the fluorescent lights. Cortical arousal levels obtained from the early component of the CNV amplitude were significantly lower with the skylight than with the fluorescent lights, whereas α-wave ratio and work performance were similar between the two light sources. The HRV evaluation showed that sympathetic nerve tone was lower and parasympathetic nerve tone was higher, both significantly, for the skylight than for the fluorescent lights during daytime. Nocturnal melatonin secretion was significantly greater before and during light exposure at night under the daytime skylight than under the fluorescent lights. CONCLUSIONS: Our results suggest that artificial skylights have some advantages over conventional fluorescent lights in maintaining ordinary work performance during daytime with less psychological and physiological stress. The findings also suggest that the artificial skylights would enable built environments to maintain long-term comfort and productivity.

Energy cost and lower leg muscle activities during erect bipedal locomotion under hyperoxia.

BACKGROUND: Energy cost of transport per unit distance (CoT) against speed shows U-shaped fashion in walking and linear fashion in running, indicating that there exists a specific walking speed minimizing the CoT, being defined as economical speed (ES). Another specific gait speed is the intersection speed between both fashions, being called energetically optimal transition speed (EOTS). We measured the ES, EOTS, and muscle activities during walking and running at the EOTS under hyperoxia (40% fraction of inspired oxygen) on the level and uphill gradients (+ 5%). METHODS: Oxygen consumption [Formula: see text] and carbon dioxide output [Formula: see text] were measured to calculate the CoT values at eight walking speeds (2.4-7.3 km h-1) and four running speeds (7.3-9.4 km h- 1) in 17 young males. Electromyography was recorded from gastrocnemius medialis, gastrocnemius lateralis (GL), and tibialis anterior (TA) to evaluate muscle activities. Mean power frequency (MPF) was obtained to compare motor unit recruitment patterns between walking and running. RESULTS: [Formula: see text], [Formula: see text], and CoT values were lower under hyperoxia than normoxia at faster walking speeds and any running speeds. A faster ES on the uphill gradient and slower EOTS on both gradients were observed under hyperoxia than normoxia. GL and TA activities became lower when switching from walking to running at the EOTS under both FiO2 conditions on both gradients, so did the MPF in the TA. CONCLUSIONS: ES and EOTS were influenced by reduced metabolic demands induced by hyperoxia. GL and TA activities in association with a lower shift of motor unit recruitment patterns in the TA would be related to the gait selection when walking or running at the EOTS. TRIAL REGISTRATION: UMIN000017690 ( R000020501 ). Registered May 26, 2015, before the first trial.

Relationship between maximum oxygen uptake and peripheral vasoconstriction in a cold environment.

BACKGROUND: Various individual characteristics affect environmental adaptability of a human. The present study evaluates the relationship between physical fitness and peripheral vasoconstriction in a cold environment. METHODS: Seven healthy male students (aged 22.0 years) participated in this study. Cold exposure tests consisted of supine rest for 60 min at 28 °C followed by 90 min at 10 °C. Rectal and skin temperatures at seven sites, oxygen consumption, and the diameter of a finger vein were measured during the experiment. Metabolic heat production, skin heat conductance, and the rate of vasoconstriction were calculated. Individual maximum oxygen consumption, a direct index of aerobic fitness, was measured on the day following the cold exposure test. RESULTS: Decreases in temperature of the hand negatively correlated with the changes in rectal temperature. Maximum oxygen consumption and the rate of vasoconstriction are positively correlated. Furthermore, pairs of the following three factors are also significantly correlated: rate of metabolic heat production, skin heat conductance, and the rate of vasoconstriction. CONCLUSION: The results of this study suggested that the capacity for peripheral vasoconstriction can be improved by physical exercise. Furthermore, when exposed to a cold environment, fitter individuals could maintain metabolic heat production at the resting metabolic level of a thermoneutral condition, as they correspondingly lost less heat.

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability.

BACKGROUND: Recent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted. METHODS: With an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome. RESULTS: Unsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3' end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and ß-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01). CONCLUSIONS: Somatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Synthesis of lead-free piezoelectric powders by ultrasonic-assisted hydrothermal method and properties of sintered (K0.48Na0.52)NBO3 ceramics.

(K,Na)NbO3 ceramics have attracted much attention as lead-free piezoelectric materials with high piezoelectric properties. High-quality (K,Na)NbO3 ceramics can be sintered using KNbO3 and NaNbO3 powders synthesized by a hydrothermal method. In this study, to enhance the quality factor of the ceramics, high-power ultrasonic irradiation was employed during the hydrothermal method, which led to a reduction in the particle size of the resultant powders.

Comparison of cardiovascular response to sinusoidal and constant lower body negative pressure with reference to very mild whole-body heating.

BACKGROUND: The purpose of the present study was to compare sinusoidal versus constant lower body negative pressure (LBNP) with reference to very mild whole-body heating. Sinusoidal LBNP has a periodic load component (PLC) and a constant load component (CLC) of orthostatic stress, whereas constant LBNP has only a CLC. We tested two sinusoidal patterns (30-s and 180-s periods with 25 mmHg amplitude) of LBNP and a constant LBNP with -25 mmHg in 12 adult male subjects. RESULTS: Although the CLC of all three LBNP conditions were configured with -25 mmHg, the mean arterial pressure (MAP) results showed a significantly large decrease from baseline in the 30-s period condition (P <0.01). In contrast, the other cardiovascular indices (heart rate (HR), stroke volume (SV), cardiac output (CO), basal thoracic impedance (Z(0)), total peripheral resistance (TPR), the natural logarithmic of the HF component (lnHF), and LF/HF (ln(LF/HF))) of heart rate variability (HRV) showed relatively small variations from baseline in the 30-s period condition (P <0.01). The result of the gain and phase of transfer function at the sinusoidal period of LBNP showed that the very mild whole-body heating augmented the orthostatic responses. CONCLUSION: These results revealed that the effect of the CLC of LBNP on cardiovascular adjustability was attenuated by the addition of the PLC to LBNP. Based on the results of suppressed HRV response from baseline in the 30-s period condition, we suggest that the attenuation may be caused by the suppression of the vagal responsiveness to LBNP.

The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals.

Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers.

Aberrant methylation of the Pleckstrin and Sec7 domain-containing gene is implicated in ulcerative colitis-associated carcinogenesis through its inhibitory effect on apoptosis.

The Pleckstrin and Sec7 domain-containing (PSD) gene, which regulates skeletal rearrangements, has been found to be more frequently methylated both in ulcerative colitis (UC)-associated colorectal cancer tissues (5 of 7; 71.4%) and matched normal epithelia (4 of 7; 57.1%) compared to non-neoplastic UC epithelia (6 of 22; 27.3%) and sporadic colorectal cancer tissues (6 of 32; 18.8%). The levels of PSD mRNA were positively correlated with the methylation status of PSD, as shown by both MSP and bisulfite sequencing. To determine the potential role of PSD silencing in the mechanisms underlying UC-associated carcinogenesis, the levels of senescence, proliferation and apoptosis were evaluated in a normal human fibroblast cell line (NHDF) in which 93% of PSD expression was knocked down by a small-interfering RNA (si-RNA). Although there were no significant differences in the levels of senescence and proliferation caused by PSD knockdown, the level of apoptosis was significantly decreased by PSD knockdown (5.3% in siControl-treated cells vs. 0.67% in siPSD-treated cells, p=0.0001). In addition, reactive oxygen species inducers accelerated apoptosis in NHDF and a neutrophil-like cell line, which was significantly reduced by PSD knockdown. To verify the effect of PSD methylation in tissue sections including 21 samples from UC patients with or without tumors, we elucidated PSD promoting accumulation of filamentous-actin (F-actin) and apoptosis by immunohistochemistry and TUNEL assay, respectively. Both levels of accumulation of F-actin and apoptosis were significantly decreased in specimens from UC patients with PSD methylation compared to those without PSD methylation (F-actin: 0.69±0.86 with vs. 1.57±0.51 without, p=0.0031, apoptotic index: 0.31±0.63 with vs. 1.0±0.88 without, p=0.0277). In conclusion, our results indicate that PSD methylation plays a significant role in the mechanisms underlying UC-associated carcinogenesis through its inhibitory effect on apoptosis in the interaction between colorectal mucosa and neutrophils.

Aberrant methylation of PSD disturbs Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis in ulcerative colitis-associated carcinogenesis.

We previously reported that the Pleckstrin and Sec7 domain-containing (PSD) gene is preferentially methylated in patients with ulcerative colitis (UC) who developed colorectal cancer (CRC), and is implicated in UC-associated carcinogenesis through its inhibition of apoptosis. This study aimed to determine the potential effect of PSD methylation on its downstream molecule, Ras-related C3 botulinum toxin substrate 1 (Rac1), which governs neutrophil chemotaxis and apoptosis signaling. PSD was knocked down in a normal human fibroblast cell line (HNDF) and a neutrophil-like cell line (HL-60). Both NHDF and HL-60 cells exhibited numerous filamentous-actin (F-actin) rich membrane extensions, resulting in the activation of Rac1; this activation was hampered by PSD silencing. Lipopolysaccharide, a reactive oxygen species (ROS) inducer, stimulated NHDF cells to release ROS and activated caspase­3/7 in the presence of neutrophils, which was inhibited by PSD knockdown. Migration assays demonstrated that chemotaxis of HL-60 cells was affected by PSD silencing in NHDF cells. Tissue sections from 6 UC patients with CRC and 15 UC patients without CRC were examined. To verify Rac1-mediated chemotaxis in tissue sections, we evaluated the grade of neutrophil infiltration by histological assessment and assessed F-actin and PSD expression by immunohistochemistry. Neutrophil infiltration, F-actin and PSD expression were significantly decreased in specimens from UC patients with PSD methylation compared with those without. Decreased levels of F-actin expression were observed in colorectal mucosa, as well as in infiltrating cells with PSD methylation. PSD expression was preferentially inhibited in colorectal mucosa by PSD methylation, whereas PSD expression was rarely observed in infiltrating cells, regardless of PSD methylation status. These data indicate that aberrant methylation of PSD occurs in UC-associated colorectal mucosa, enabling circumvention of Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis, and thus plays a pivotal role in the mechanisms underlying UC-associated carcinogenesis.

Identification of a potent epigenetic biomarker for resistance to camptothecin and poor outcome to irinotecan-based chemotherapy in colon cancer.

Drug resistance remains a major obstacle to successful cancer treatment. Genome-wide comprehensive analysis identified a novel gene, glucocorticoid-induced protein-coding gene (DEXI), which was frequently methylated in colorectal (CRC; 36 of 73 patients; 49%) and gastric (28 of 89 patients; 31%) cancer patients. Here, we show that DEXI methylation is implicated in mechanisms facilitating resistance to camptothecin (CPT) via inhibition of apoptosis. Silencing of DEXI by siRNA significantly reduced CPT-induced apoptosis in a fibroblast cell line (1/6-fold; p<0.01) originally expressing endogenous DEXI. Restored expression of DEXI by 5-aza-2'-deoxycytidine (DAC) significantly enhanced susceptibility to CPT (3-fold; p<0.01) in a colon cancer cell line originally suppressing endogenous DEXI due to almost complete methylation. Exogenous induction of DEXI confirmed that DEXI per se contributed to enhanced susceptibility to CPT. 5-Fluorouracil (5-FU) did not exhibit these synergistic effects by DEXI restoration. Further, to estimate the clinical usefulness of DEXI methylation status as biomarker for drug resistance to irinotecan (CPT-11), 16 CRC patients who underwent FOLFIRI (5-FU + CPT-11) therapy because they were refractory to FOLFOX (5-FU + oxaliplatin) were analyzed. Significantly poor response and outcome were observed in 8 CRC patients harboring DEXI methylation. In 8 CRC patients harboring DEXI methylation disease control rate, progression-free survival and overall survival were 25.0%, 2 and 11.8 months, respectively, whereas in 8 CRC patients without DEXI methylation they were 62.5%, 5.3 and 15 months, respectively (p<0.01). These significant differences were not observed in patients undergoing treatment with FOLFOX. In conclusion, silencing of DEXI leads to resistance, but restored expression enhances susceptibility to CPT in vitro and DEXI methylation results in poor response and outcome to CPT-11-based chemotherapy, suggesting that DEXI is a potent therapeutic target and an epigenetic biomarker for the selection of patients more likely to benefit from CPT-11-based chemotherapy.