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Previously, we showed that augmented O-linked N-acetylglucosaminylation (O-GlcNAcylation) mitigates cardiac remodeling in O-GlcNAc transferase-transgenic (Ogt-Tg) mice exposed to acute (2-week) intermittent hypoxia (IH) by suppressing nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF-κB) via the O-GlcNAcylation of glycogen synthase kinase 3 beta (GSK-3β) and NF-κB p65. Because this effect is time dependent, we exposed Ogt-Tg mice to IH for 4 weeks (IH4W) in the present study. O-GlcNAcylation was significantly enhanced in Ogt-Tg mice vs. wild-type (WT) mice exposed to normoxia and IH4W. Total O-GlcNAcylation levels were significantly increased in WT and Ogt-Tg mice after IH4W vs. normoxia. After IH4W, Ogt-Tg mice displayed significantly exacerbated signs of cardiac hypertrophy and fibrosis in the right ventricles (RVs) but not the left ventricles (LVs). Echocardiography revealed IH4W-induced right ventricular dysfunction. Phosphorylated GSK-3β levels were increased in Ogt-Tg mice vs. WT mice after IH4W, whereas phosphorylated NF-κB p65 levels were unaffected. Mitophagy, which is associated with cardiac dysfunction, was increased in the RVs of Ogt-Tg mice after IH4W. Furthermore, the levels of phosphorylated dynamin-related protein 1 (p-Drp1) were significantly increased, and the expression of mitofusin-2 (MFN2) was significantly decreased. In human embryonic kidney cells, mitochondrial uncoupler-induced mitochondrial dysfunction was accelerated in Ogt-overexpressing cells. In addition to increasing the levels of phosphorylated Smad2, IH4W promoted cardiac fibrosis in the RVs of Ogt-Tg mice. Thus, augmented O-GlcNAcylation may aggravate IH4W-induced right ventricular dysfunction and remodeling by promoting hypertrophy, mitophagy, and fibrosis via GSK-3β inactivation, an increased p-Drp-1/MFN2 ratio, and Smad2 activation, respectively.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621. These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Reversible post-translational modification of serine and threonine residues by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation has been indicated to regulate the activities of a number of different proteins. Augmented O-GlcNAcylation contributes to the etiologies of type 2 diabetes mellitus (T2DM) and cancer. Moreover, diabetic conditions increase the risk of colorectal cancer. However, the effect of O-GlcNAcylation in patients with colorectal cancer and concurrent T2DM has not been elucidated. The current study evaluated the level of O-GlcNAcylation in patients with colorectal cancer with or without T2DM. Notably, O-GlcNAcylation levels were significantly higher in tissues from patients with T2DM compared with those in patients without T2DM, and higher in cancer tissues compared with corresponding adjacent tissues. O-GlcNAcylation and cancer stage were more strongly correlated in cancer tissues from patients with T2DM compared with those from patients without T2DM. Additionally, distant metastasis was significantly correlated with O-GlcNAcylation in cancer tissues from patients with T2DM. β-catenin levels in colorectal cancer tissues were the highest in patients with advanced-stage cancer and concurrent T2DM. In SW480 human colon cancer cells, thiamet G (TMG) treatment and OGA silencing, which increased O-GlcNAcylation, significantly increased β-catenin and SNAIL in high-glucose, but not during normal-glucose conditions. These data suggest that O-GlcNAcylation is closely associated with distant metastasis, most likely through upregulation of the β-catenin/SNAIL signaling pathway in colorectal cancer patients with T2DM.

Atopic dermatitis (AD) is the foremost non-fatal skin-related disease that affects all age groups. Despite the growing prevalence of AD in low- and middle-income countries, its physiological consequences remain overlooked in countries like Bangladesh. Therefore, we aim to assess and characterize the influence of AD on the health-related quality of life (HRQoL) in Bangladeshi patients. A cross-sectional study comprising 184 eligible adults (83 men and 101 women, mean age, 33.46 ± 15.44 years) was conducted at the dermatology outpatient department of Shaheed Suhrawardy Medical College Hospital (a tertiary hospital in Dhaka, Bangladesh). AD was determined using the UK Working Party criteria. A structured questionnaire, Eczema Area and Severity Index (EASI), and Dermatology Life Quality Index (DLQI) were administered to obtain information on patient characteristics, AD severity, and HRQoL. The mean DLQI score for the entire sample was 11.29 ± 5.27 (range, 1–26), and 51.60% reported the disease greatly affected their lives. Bivariate analysis revealed significant differences in self-rated health measures of DLQI scores in terms of self-reported AD severity, overall health, and the EASI. In multivariable regression models adjusted for patient characteristics, the self-perceived severe AD group reported significantly higher DLQI scores (coefficient = 2.72, 95% confidence interval (CI) = 0.38–5.05, p = 0.022) than the mild group. Concurrently, we observed a substantial increase in the DLQI scores among patients with moderate and severe EASI scores (coefficient = 1.96, 95% CI = 0.08–3.92, p &lt, 0.05 and coefficient = 4.35, 95% CI = 1.98–6.72, p &lt, 0.001, respectively) than in those with mild EASI scores, suggesting that HRQoL was markedly influenced by greater AD severity. These findings highlight the need for a more patient-centric approach to the management of AD in order to alleviate patient suffering and, thereby, improve HRQoL.

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment–mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Type 2 diabetes mellitus (T2DM) has been reported to be associated with cardiac remodeling. Although O-GlcNAcylation is known to be elevated in diabetic and ischemic hearts, the effects of O-GlcNAcylation on cardiac remodeling induced by intermittent hypoxia (IH), such as sleep apnea syndrome (SAS), remain unknown. To evaluate the effects, we induced IH in wild-type (WT) and transgenic O-GlcNAc transferase (Ogt-Tg) mice. Two weeks of IH increased O-GlcNAcylation in the heart tissues of both strains of mice, whereas O-GlcNAcylation in Ogt-Tg mice was significantly higher than that in WT mice under both normoxic and IH conditions. WT mice exhibited cardiac remodeling after IH, whereas cardiac remodeling was significantly attenuated in Ogt-Tg mice. Oxidative stress and apoptosis increased after IH in both strains of mice, whereas the rate of increase in these processes in Ogt-Tg mice was significantly lower than that in WT mice. To examine the mechanism of cardiac remodeling attenuation in Ogt-Tg mice after IH, the effects of O-GlcNAcylation on the activities of the master regulators nuclear factor of activated T cells (NFAT) and NF-κB were determined. The O-GlcNAcylation of GSK-3β, a negative regulator of NFAT, was significantly increased in Ogt-Tg mice, whereas the phosphorylation of GSK-3β was reciprocally reduced. The same result was observed for NF-κB p65. An in vitro reporter assay showed that the augmentation of O-GlcNAcylation by an O-GlcNAcase inhibitor suppressed NFAT and NF-κB promoter activity. These data suggest that augmented O-GlcNAcylation mitigates IH-induced cardiac remodeling by suppressing NFAT and NF-κB activities through the O-GlcNAcylation of GSK-3β and NF-κB p65.

To explore impact of enhancing social advancement of females in Japan, this study determined the effects of the dual-income household rate on suicide mortalities disaggregated by attributes of gender, age, and motives between 2009 and 2017 in Japan. This study analysed impact of dual-income household rate, other household-related factors (savings, liabilities and yearly incomes per household, minors and elderly rate per household), and social/employment factors (complete unemployment rate, employment rate, temporary male and female employment rates and certification rate of long-term care insurance) on suicide mortalities disaggregated by attributes of gender, age, and motives using hierarchical linear-regression model. Dual-income household rate was significantly/negatively related to suicide mortality of the working-age female population, but significantly/positively related to that of the elderly female population. Suicide mortalities of the working-age male population and the elderly male population were significantly/positively related to dual-income household rate. Male suicide mortalities caused by family-, health-, economy- and employment-related motives were significantly/positively related to dual-income household rate, however, the dual-income household rate was significantly/positively related to female suicide mortalities caused by family-, health-, economy- and school-related motives, but significantly/negatively related to suicide mortalities caused by romance-related motives. Dual-income households suppress social-isolation and develop economical/psychological independence of females, leading to reduced suicide mortality in working-age females. However, elderly and school-age populations, who are supported by the working-age female, suffer from isolation. Working-age males also suffer from inability to adapt from the traditional concept of work–life and work–family balances to the novel work–family balance concept adapted to dual-income households. These results suggest occurrence of new social/family problems in the 21st century due to vulnerability of traditional Japanese culture and life–working–family balance concepts as well as novel sociofamilial disturbances induced by declining birth rate and ageing population in Japan.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca(2+) entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser(621) and Thr(626) were O-GlcNAcylated and that Thr(626) was O-GlcNAcylated in the steady state but Ser(621) was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser(621). Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser(621). These data suggest that both decrease in O-GlcNAcylation at Thr(626) and increase in O-GlcNAcylation at Ser(621) in STIM1 lead to impairment of SOCE activity through decrease in Ser(621) phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Colon cancer prevalence is high worldwide. O-GlcNAcylation has been associated with tumor growth in various tissues, including the colon; however, its link to carcinogenesis is not fully understood. We investigated the association of O-GlcNAcylation with colon carcinogenesis using a 1,2-dimethylhydrazine/dextran sodium sulfate-induced colon carcinogenesis model in wild type and O-GlcNAc transferase-transgenic (Ogt-Tg) mice. The incidence of colon cancer was significantly lower in Ogt-Tg than in wild type mice. The colonic length was not shortened in Ogt-Tg mice, and NF-κB p65 phosphorylation was strongly suppressed, indicating that reduction of inflammation might be related to the alleviation of colon carcinogenesis. Dextran sodium sulfate-induced acute colitis mice were used to evaluate the effect of O-GlcNAcylation on inflammation at the maximal inflammation period. In Ogt-Tg mice, NF-κB p65 phosphorylation and interleukin-1β mRNA expression were suppressed. Histochemical staining demonstrated shedding of colon epithelial cells in wild type mice a few days after dextran sodium sulfate treatment, whereas they remained essentially intact in Ogt-Tg mice. There were no significant differences on histochemical staining in the remaining epithelia between groups. These data suggest that O-GlcNAcylation could prevent colon carcinogenesis through reducing acute maximum inflammation, suggesting modulation of O-GlcNAcylation as a novel therapeutic option.

Increasing incidence of various cancers has been reported in diabetic patients. O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins at serine/threonine residues (O-GlcNAcylation) is an essential post-translational modification that is upregulated in diabetic patients and has been implicated in tumor growth. However, the mechanisms by which O-GlcNAcylation promotes tumor growth remain unclear. Given that AMP-activated kinase (AMPK) has been thought to play important roles in suppressing tumor growth, we evaluated the involvement of AMPK O-GlcNAcylation on the growth of LoVo cells, a human colon cancer cell line. Results revealed that treatment with Thiamet G (TMG), an inhibitor of O-GlcNAc hydrolase, increased both anchorage-dependent and -independent growth of the cells. O-GlcNAc transferase overexpression also increased the growth. These treatments increased AMPK O-GlcNAcylation in a dose-dependent manner, which led to reduced AMPK phosphorylation and mTOR activation. Chemical inhibition or activation of AMPK led to increased or decreased growth, respectively, which was consistent with the data with genetic inhibition of AMPK. In addition, TMG-mediated acceleration of tumor growth was abolished by both chemical and genetic inhibition of AMPK. To examine the effects of AMPK O-GlcNAcylation in vivo, the LoVo cells were s.c. transplanted onto the backs of BALB/c-nu/nu mice. Injection of TMG promoted the growth and enhanced O-GlcNAcylation of the tumors of the mice. Consistent with in vitro data, AMPK O-GlcNAcylation was increased, which reduced AMPK phosphorylation and resulted in activation of mTOR. Collectively, the higher colon cancer risk of diabetic patients could be due to O-GlcNAcylation-mediated AMPK inactivation and subsequent activation of mTOR.

Reversible post-translational modification of serine and threonine residues by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation has been indicated to regulate the activities of a number of different proteins. Augmented O-GlcNAcylation contributes to the etiologies of type 2 diabetes mellitus (T2DM) and cancer. Moreover, diabetic conditions increase the risk of colorectal cancer. However, the effect of O-GlcNAcylation in patients with colorectal cancer and concurrent T2DM has not been elucidated. The current study evaluated the level of O-GlcNAcylation in patients with colorectal cancer with or without T2DM. Notably, O-GlcNAcylation levels were significantly higher in tissues from patients with T2DM compared with those in patients without T2DM, and higher in cancer tissues compared with corresponding adjacent tissues. O-GlcNAcylation and cancer stage were more strongly correlated in cancer tissues from patients with T2DM compared with those from patients without T2DM. Additionally, distant metastasis was significantly correlated with O-GlcNAcylation in cancer tissues from patients with T2DM. β-catenin levels in colorectal cancer tissues were the highest in patients with advanced-stage cancer and concurrent T2DM. In SW480 human colon cancer cells, thiamet G (TMG) treatment and OGA silencing, which increased O-GlcNAcylation, significantly increased β-catenin and SNAIL in high-glucose, but not during normal-glucose conditions. These data suggest that O-GlcNAcylation is closely associated with distant metastasis, most likely through upregulation of the β-catenin/SNAIL signaling pathway in colorectal cancer patients with T2DM.

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na(+)/H(+) exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment–mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na(+)/H(+) exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Type 2 diabetes mellitus (T

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2 (NRG2), is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4, and transactivates ErbB2. NTAK/NRG2 is structurally homologous to NRG1. The biological function of NTAK/NRG2 still remains unknown, especially in the nervous system, whereas NRG1 is known to be essential for nervous system function. In the present study, we examined the functions of NTAK/NRG2 secreted from astrocytes to neurons. NTAK/NRG2 was expressed in both neurons and astrocytes, as evidenced by immunohistochemical staining and RT-PCR methods. The conditioned medium (CM) from astrocytes promoted survival and neurite outgrowth of neurons. The CM stimulated phosphorylation of ErbB3 in neurons. When phosphorylation of ErbB3 was blocked by AZD8931, an ErbB3 inhibitor, neuronal survival and neurite outgrowth were reduced. Conversely, canertinib, an ErbB4 inhibitor, did not affect survival or neurite outgrowth of neurons. Survival and neurite outgrowth of neurons were lower in CM of NTAK/NRG2-knockdown astrocytes than in the CM of control astrocytes, whereas the CM of NRG1-knockdown astrocytes had little effect on survival and neurite outgrowth. The present study demonstrated that NTAK/NRG2 secreted from astrocytes bound to ErbB3 on neurons, and promoted neuronal survival and neurite extension in vitro.

Phospholamban (PLN) regulates cardiac type sarco (endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) via Ser(16)-phosphorylation. During heart failure, PLN expression is downregulated with SERCA2a; however, the mechanism of its regulation is not fully understood. Phosphorylation triggers protein degradation and because PLN phosphorylation is upregulated in failing hearts, we examined whether PLN is degraded by Ser(16)-phosphorylation. Cells overexpressing PLN exhibited its degradation post isoproterenol (Iso), forskolin, or 3-isobutyl-1-methylxanthine (IBMX) addition. Moreover, this degradation was inhibited by a cAMP-dependent protein kinase (PKA) inhibitor--H89. Co-immunoprecipitation revealed that Lys(3) of PLN was oligo-ubiquitinated when ubiquitin was overexpressed, and was degraded by Iso treatment. However, when co-expressed with SERCA2a, oligo-ubiquitinated PLN at Lys(3) was not degraded by Iso treatment. In failing hearts from 16 week-old TgPLN(R9C) mice, oligo-ubiquitinated PLN levels increased and PLN expression was downregulated. Furthermore, SERCA2a mRNA levels in TgPLN(R9C) mice hearts were lower than that in wild type mice; however, PLN mRNA levels showed no changes. In another heart failure model, MG132 treatment reversed PLN degradation. These data suggest that PLN is, at least partially, oligo-ubiquitinated at Lys(3) and degraded through Ser(16)-phosphorylation-mediated poly-ubiquitination during heart failure.

Introduction The purpose of this study was to evaluate whether cryopreserved (frozen) adipose-derived regenerative cells (ADRCs) have a therapeutic effect on burn wound healing as well as freshly isolated (fresh) ADRCs. Methods Full thickness burns were created on dorsum of nude mice and burn wound was excised. The wound was covered by artificial dermis with; (i) fresh ADRCs, (ii) frozen ADRCs, and (iii) PBS (control). The assessment for wound healing was performed by morphological, histopathological and immunohistochemical analyses. Results In vivo analyses exhibited the significant therapeutic effect of frozen ADRCs on burn wound healing up to the similar or higher level of fresh ADRCs. There were significant differences of wound closure, epithelized tissue thickness, and neovascularization between the treatment groups and control group. Although there was no significant difference of therapeutic efficacy between fresh ADRC group and frozen ADRC group, frozen ADRCs improved burn wound healing process in dermal regeneration with increased great type I collagen synthesis compared with fresh ADRCs. Conclusions These findings indicate that frozen ADRCs allow us to apply not only quickly but also for multiple times, and the cryopreserved ADRCs could therefore be useful for the treatment of burn wounds in clinical settings., Highlights • Frozen ADRCs had a therapeutic effect on burn wounds as well as fresh ADRCs. • Frozen ADRCs promoted tissue regeneration by paracrine factors. • Frozen ADRCs could be useful for the treatment of burn wounds in clinical setting.

Non-steroidal anti-inflammatory drug (NSAID)-induced epithelial cell damage occurs not only in the stomach but also in the intestines and colon. Although several studies have investigated the related mechanism underlying lower gastrointestinal tract injury, the details of this mechanism are still unclear. Since it was reported that protein degradation might play an important role, herein, we focused on one of the major ubiquitin E3 ligases, the von Hippel-Lindau protein (pVHL). To understand whether pVHL is involved in the observed cell damage, we examined whether indomethacin (IM) treatment affects pVHL expression in the rat IEC6 intestinal epithelial cell line. We showed that pVHL was upregulated after IM treatment through increased oxidative stress. pVHL siRNA prevented cell injury after IM treatment. Furthermore, the collagen I and HIF-1α protein band intensities were both decreased after IM treatment, whereas MG132 reversed the proteins' downregulation, indicating that the IM treatment-induced downregulation was due to the degradation through pVHL-mediated polyubiquitination. Co-immunoprecipitation showed that pVHL interacted with both collagen I and HIF-1α. The degradation of collagen I and HIF-1α after IM treatment was reversed by siVHL or a Mn-SOD mimetic, Mn(III)TMPyP. The expression of collagen I and HIF-1α was correlated with pVHL expression level, whereas only HIF-1α, not collagen I, was upregulated after the treatment of a prolyl hydroxylase inhibitor, CoCl2. The effect of pVHL on the intestinal epithelium after IM treatment was also tested in vivo. Western blot analyses were used to test whether pVHL's protein expression level might increase after oral administration of IM to mice, and which showed that IM upregulated pVHL expression and degraded collagen I and HIF-1α, consistent with the data obtained in IEC6 cells. These data suggested that intestinal epithelial cells were injured after IM treatment through the pVHL overexpression-induced degradation of collagen I or HIF-1α. Therefore, pVHL might be a molecular target for IM-induced intestinal epithelial cell injury.

Hypoxia-inducible factor-1 (HIF-1) is one of the most promising pharmacological targets for all types of cancer, including ovarian cancer. Ovarian clear cell carcinoma (OCCC) has poor prognosis because of its insensitivity to chemotherapy. To elucidate the characteristics of this troublesome cancer, we examined HIF-1α expression under normoxia or hypoxia in various ovarian cancer cell lines. HIF-1α was highly expressed under normoxia only in RMG-1, an OCCC cell line. To examine whether HIF-1 is involved in the tumorigenesis of RMG-1 cells, we established HIF-1α-silenced cells, RMG-1HKD. The proliferation rate of RMG-1HKD cells was faster than that of RMG-1 cells. Furthermore, the activity of MEK/ERK in the Ras pathway increased in RMG-1HKD cells, whereas that of mTOR in the PI3K pathway did not change. Activation of the Ras pathway was attributable to the increase in phosphorylated MEK via PP2A inactivation. To confirm the crosstalk between the PI3K and Ras pathways in vivo, RMG-1 or RMG-1HKD cells were transplanted into the skin of nude mice with rapamycin (an inhibitor of mTOR), PD98059 (an inhibitor of MEK), or both. RMG-1HKD cells showed higher sensitivity to PD98059 than that observed in RMD-1 cells, whereas the combination therapy resulted in synergistic inhibition of both cells. These findings suggest that inhibition of HIF-1, a downstream target of mTOR in the PI3K pathway, activates the Ras pathway on account of the increase in MEK phosphorylation via PP2A inactivation, and the crosstalk between the 2 pathways could be applied in the combination therapy for HIF-1-overexpressing cancers such as OCCC.

P4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2−/− mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.

Erythropoietin (Epo) is a potent inducer of erythropoiesis that is mainly produced in the kidney. Epo is expressed not only in the normal kidney, but also in renal cell carcinomas (RCCs). The aim of the present study was to gain insights into the roles of Epo and its receptor (EpoR) in RCC cells. The study used two RCC cell lines, Caki-1 and SKRC44, in which Epo and EpoR are known to be highly expressed. The proliferation rate and expression level of hypoxia-inducible factor-1α (HIF-1α) were measured prior to and following Epo treatment and under normoxic and hypoxic conditions. To examine whether HIF-1α or Epo were involved in cellular proliferation during hypoxia, these proteins were knocked down using small interfering RNA (siRNA) in Caki-1 and SKRC44 cells. The results demonstrated that Epo enhanced the proliferation of the Caki-1 and SKRC44 cells. HIF-1α expression was increased upon the induction of hypoxia in the Caki-1 cells, but remained unaffected in the SKRC44 cells. The proliferation rate was increased under hypoxic conditions in the Caki-1 cells, but was decreased in the SKRC44 cells. Under hypoxic conditions, the proliferation of the Caki-1 cells was significantly reduced by the knock-down of HIF-1α or Epo, while the proliferation of the SKRC44 cells was significantly suppressed by the knock-down of Epo, but not HIF-1α. In conclusion, these data suggest that the induction of Epo may accelerate the proliferation of the RCC cell lines in either a HIF-1α-dependent or -independent manner.

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause epithelial cell damage in the stomach, intestine, and colon. NSAIDs are reported to induce autophagy and apoptosis in intestinal epithelial cells; however, their role in cell damage is poorly understood. To examine the role of autophagy in cell damage, we used autophagy-related gene Atg5-conditional knockout mice, in which the Atg5 gene is only knocked out in intestinal epithelial cells. In an indomethacin (IM)-induced gastrointestinal ulcer mouse model, intestinal epithelium damage was reduced in Atg5-conditional knockout mice compared with wild-type mice. IM-induced damage in IEC6 rat intestinal epithelial cells was reduced when Atg5 was silenced (IEC6shAtg5 cells). Western blot analyses indicated that IM-induced apoptosis decreased, and the potent, oxidative stress-related extracellular signal-regulated kinase (ERK)/nuclear factor-erythroid2-like2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway was upregulated in IEC6shAtg5 cells. An experiment using a reactive oxygen species (ROS)-sensitive fluorescent dye in IEC6shAtg5 cells revealed that the amount of ROS at the baseline and the rate of increase after IM treatment were lower than in intact IEC6 cells. The mitochondrial membrane potential at the baseline and the reduction rate in IM-treated IEC6shAtg5 cells were lower than in intact IEC6 cells, indicating that autophagy deficiency increased ROS production caused by mitochondrial disturbance. Furthermore, MnTMPyP, a manganese-superoxide dismutase mimetic, significantly inhibited IM-induced autophagy and subsequent apoptosis as well as activation of the ERK/Nrf2/HO-1 pathway. These data suggest that autophagy deficiency and subsequent activation of the ERK/Nrf2/HO-1 pathway diminished IM-induced, apoptosis-mediated intestinal epithelial cell damage, and genetic analyses of single nucleotide polymorphisms in autophagy-related genes could predict NSAID-induced intestinal injury.

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Alpha1,6-fucosyltransferase (Fut8) plays important roles in physiological and pathological conditions. Fut8-deficient (Fut8-/-) mice exhibit growth retardation, earlier postnatal death, and emphysema-like phenotype. To investigate the underlying molecular mechanism by which growth retardation occurs, we examined the mRNA expression levels of Fut8-/- embryos (18.5 days postcoitum [dpc]) using a cDNA microarray. The DNA microarray and real-time polymerase chain reaction (PCR) analysis showed that a group of genes, including trypsinogens 4, 7, 8, 11, 16, and 20, were down-regulated in Fut8-/- embryos. Consistently, the expression of trypsinogen proteins was found to be lower in Fut8-/- mice in the duodenum, small intestine, and pancreas. Trypsin, an active form of trypsinogen, regulates cell growth through a G-protein-coupled receptor, the proteinase-activated receptor 2 (PAR-2). In a cell culture system, a Fut8 knockdown mouse pancreatic acinar cell carcinoma, TGP49-Fut8-KDs, showed decreased growth rate, similar to that seen in Fut8-/- mice, and the decreased growth rate was rescued by the application of the PAR-2-activating peptide (SLIGRL-NH2). Moreover, epidermal growth factor (EGF)-induced receptor phosphorylation was attenuated in TGP49-Fut8-KDs, which was highly associated with a reduction of trypsinogens mRNA levels. The addition of exogenous EGF recovered c-fos, c-jun, and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, the EGF-induced up-regulation of c-fos and c-jun mRNA expression was significantly blocked by the protein kinase C (PKC) inhibitor. Our findings clearly demonstrate a relationship between Fut8 and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathway in controlling cell growth and that the EGFR-trypsin-PAR-2 pathway is suppressed in TGP49-Fut8-KDs as well as in Fut8-/- mice.

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and α1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

Miyagawa S, Nakatsu S, Hazama K, Nakagawa T, Kondo A, Matsunami K, Yamamoto A, Yamada J, Miyazawa T, Shirakura R. A novel strategy for preventing PERV transmission to human cells by remodeling the viral envelope glycoprotein. Xenotransplantation 2006; 13: 258–263. © Blackwell Munksgaard, 2006 Abstract: Background: Porcine endogenous retrovirus (PERV) released from pig cells is a main problem associated with clinical xenotransplantation. In a previous study, we demonstrated that the high mannose type of N-glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of reducing the high mannose type of N-glycan on PERV infectivity. Methods: Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV-B) to produce PEC(Z)/PB. The PEC(Z)/PBs were next transfected with the α 1,2 mannosidase Ib (Man Ib), N-acetylglucosaminyltransferase I (GnT-I) or α-mannosidase II (Man II) gene in order to reduce the levels of high mannose type of N-glycan. HEK293 cells were inoculated with the PERV in each of the culture supernatants. The inoculated cells were histochemically stained and the LacZ-positive cells were counted. Results: In experiment I, PERV transmission from the PEC(Z)/PB with GnT-I or Man II to HEK 293 cells was significantly reduced in comparison with control PEC(Z)/PB, while the PEC(Z)/PB with Man Ib was not. However, in experiment II, PERV transmission from the PEC(Z)/PB with ManIb to HEK 293 cells was also significantly reduced in comparison with control PEC(Z)/PB. Conclusion: The transfection of these genes to pig cells is effective in reducing the susceptibility of human cells to PERV infection. The results suggest that this represents a potentially useful strategy for further decreasing the likelihood of PERV infections.

In early pregnancy, placental trophoblast cells rapidly grow and invade into maternal uterine tissue. N-Acetylglucosaminyltransferase V (GnT-V) and its product, beta1-6-GlcNAc branching glycan, are known to correlate with tumor invasion and metastasis. Since the placentation process resembles invasion of cancer cells, we examined the expression of beta1-6-GlcNAc branching glycan and GnT-V in human placenta. Placentas derived from the first trimester contained a larger amount of beta1-6-GlcNAc branching glycan, detected by leukoagglutinating phytohemagglutinin lectin blotting, than those at term. Immunohistochemical study revealed that beta1-6-GlcNAc branching glycans and GnT-V protein were localized in the trophoblast layer. Both protein expression and the enzyme activity of GnT-V in first trimester placentas were higher than those at term. These results suggest that GnT-V would contribute to placentation in the early phase of pregnancy, possibly regulating the process of invasion of trophoblast cells.

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.

Fucosylated alpha-fetoprotein (AFP) is a more specific biomarker for hepatocellular carcinoma (HCC) than AFP. However, the mechanisms underlying the increase in fucosylated AFP in sera of HCC patients remain largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of hepatic glycoproteins into bile and that the fucosylation-based sorting machinery might be disrupted in the liver bearing HCC. In this study, we investigated the selective secretion of fucosylated AFP into bile canaliculus (BC) structures of the human hepatoma cell line HepG2. The proportion of fucosylated AFP in BC structures was higher than that in the medium, as judged by lectin affinity electrophoresis. Suppression of fucosylation by the double knock-down of GDP-mannose-4,6-dehydratase and the human homologue of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which contribute to the synthesis of GDP-fucose, a donor substrate for fucosyltransferases, did not decrease the proportion of fucosylated AFP in BC structures but decreased this proportion in conditioned medium. Furthermore, increased AFP fucosylation was observed in medium, but not in BC structures, upon adding free fucose. These results suggest that saturation of fucosylated AFP in BC structures is accompanied by its increase in conditioned medium, probably leading to increased fucosylated AFP in sera of HCC patients.

Since heparin-binding epidermal growth factor (HB-EGF) is a member of the EGF family and binds to EGF receptor, we tested recombinant HB-EGF for its ability to stimulate human keratinocyte proliferation. The effect of HB-EGF on human keratinocytes was dependent on the cell density. HB-EGF optimally increased the cell number by 1.8-fold at 1.0 ng/ml for a 4-day incubation period under subconfluent culture. In contrast, under confluent culture, 10 ng/ml HB-EGF optimally increased the DNA synthesis 2.1-fold. To examine the production of HB-EGF by human keratinocytes, the analysis of human keratinocyte-conditioned medium was undertaken by a combination of heparin affinity column chromatography, EGF receptor-stimulating assay, immunoblotting, and neutralization. Heparin column chromatography fractionated three activities, peaks 1, 2, and 3, which contained immunoreactive 30- and 27-, 19-, and 14.5-kDa bands, respectively. The anti-HB-EGF-blocking antibody neutralized the activities of peaks 2 and 3 by 38 and 22%, respectively, but did not neutralize the activity of peak 1 at all. The antibody reduced the cell growth by 37% for a 4-day incubation period. Northern blot analysis detected a 2.5-kilobase transcript of HB-EGF. The addition of 1 ng/ml HB-EGF optimally increased the levels of HB-EGF mRNA 5.4-fold at 1 h and TGF-alpha mRNA 3.1-fold at 3 h. Interestingly, the addition of TGF-alpha at 1 ng/ml to keratinocyte cultures enhanced the level of HB-EGF mRNA 10.2-fold at 6 h. 1 ng/ml EGF also increased HB-EGF mRNA levels 10.9-fold at 1 h. These results suggest that HB-EGF is an autocrine growth factor for human keratinocytes, and HB-EGF and TGF-alpha act not only by an autoinductive mechanism but also by mutual amplification.

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

6I,6IV-Di-O-[α- l -fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β- d -glucopyranosyl]-cyclomaltoheptaose (βCD) {6I,6IV-di-O-[α- l -Fuc-(1→6)-β- d -GlcNAc]-βCD (5)} and 6-O-[α- l -fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β- d -glucopyranosyl]-βCD {6-O-[α- l -Fuc-(1→6)-β- d -GlcNAc]-βCD (6)} were chemically synthesized using the corresponding authentic compounds, bis(2,3-di-O-acetyl)-pentakis(2,3,6-tri-O-acetyl)-βCD as the glycosyl acceptor and 2,3,4-tri-O-benzyl-α- l -fucopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)- d -glucopyranosyl trichloroacetimidate as the fuco-glucosaminyl donor. NMR confirmed that α- l -Fuc-(1→6)- d -GlcNAc was bonded by β-linking to the βCD ring. To evaluate biological efficiency, the biological activities of the new branched βCDs were examined. The cell detachment activity of 5 was lower than that of 6 in real-time cell sensing (RT-CES) assay, indicating that 5 has lower toxicity. In SPR analysis, 5 had a higher special binding with AAL, a fucose-recognizing lectin. These results suggest that 5 could be an efficient drug carrier directed at cells expressing fucose-binding proteins.

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.

The N-glycan structures of the Lens culinaris agglutinin (LCA)-reactive fraction of alpha-fetoprotein (AFP-L3), a tumor marker of hepatocellular carcinomas (HCC), were analyzed in relationship to glycosyltransferases and LCA-affinity electrophoresis. Using HPLC and MALDI-TOF MS, we determined the N-glycan structures of AFP from HCC cell lines, and demonstrated they were affected by N-acetylglucosaminyltransferase III and fucosyltransferase VIII, but not by N-acetylglucosaminyltransferase V. Moreover, we identified the N-glycan structures of AFP in HCC patients.

Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohn's disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.

Mice with a targeted gene disruption of Fut8 (Fut8(-/-)) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4, alpha4beta1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8(-/-) mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8(-/-) pre-B cells. Indeed, the frequency of preBCR(+)CD79b(low) cells in bone marrow pre-B cells in Fut8(-/-) was much lower than that in Fut8(+/+) cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.

Changes in the levels of fucosylation regulate the biological phenotype of cancer cells and a specific fucosylation, such as fucosylated alpha-fetoprotein (AFP-L3) has been clinically used as a tumor marker for hepatocellular carcinoma (HCC). However, detailed molecular mechanisms that explain the increased fucosylation in HCC remain unknown despite 10 years of study by these researchers. Fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter (GDP-Fuc Tr), and synthetic enzymes of GDP-fucose, such as GDP-mannose 4, 6-dehydratase (GMD), GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase (FX), and GDP-fucose pyrophosphorylase. In this study, the expression of fucosylation-related genes in HCC tissues was studied and it was found that GDP-Fuc Tr is a key factor for increases in fucosylation. A real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed significant increases in GDP-Fuc Tr and FX expression in HCC, and levels of the GMD protein were upregulated by posttranslational modification in HCC tissues. In vitro cell experiments showed that the level of GDP-Fuc Tr was the most significantly correlated with the level of cellular fucosylation and the overexpression of GDP-Fuc Tr dramatically increased fucosylation in Hep3B cells. The importance of GDP-Fuc Tr in the increase of fucosylation was also confirmed with immunohistochemical analyses. These findings suggest that the upregulation of GDP-Fuc Tr plays a pivotal role in increased fucosylation in HCC and represents an attractive target for new treatments and diagnosis for HCC.