A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

RADAR: differential analysis of MeRIP-seq data with a random effect model.

Epitranscriptome profiling using MeRIP-seq is a powerful technique for in vivo functional studies of reversible RNA modifications. We develop RADAR, a comprehensive analytical tool for detecting differentially methylated loci in MeRIP-seq data. RADAR enables accurate identification of altered methylation sites by accommodating variability of pre-immunoprecipitation expression level and post-immunoprecipitation count using different strategies. In addition, it is compatible with complex study design when covariates need to be incorporated in the analysis. Through simulation and real dataset analyses, we show that RADAR leads to more accurate and reproducible differential methylation analysis results than alternatives, which is available at https://github.com/scottzijiezhang/RADAR.

Acute Deletion of METTL14 in ß-Cells of Adult Mice Results in Glucose Intolerance.

N6-Methyladenosine (m6A) is the most common and abundant mRNA modification that involves regulating the RNA metabolism. However, the role of m6A in regulating the ß-cell function is unclear. Methyltransferase-like 14 (METTL14) is a key component of the m6A methyltransferase complex. To define the role of m6A in regulating the ß-cell function, we generated ß-cell METTL14-specific knockout (ßKO) mice by tamoxifen administration. Acute deletion of Mettl14 in ß-cells results in glucose intolerance as a result of a reduction in insulin secretion in ß-cells even though ß-cell mass is increased, which is related to increased ß-cell proliferation. To define the molecular mechanism, we performed RNA sequencing to detect the gene expression in ßKO islets. The genes responsible for endoplasmic reticulum stress, such as Ire1α, were among the top upregulated genes. Both mRNA and protein levels of IRE1α and spliced X-box protein binding 1 (sXBP-1) were increased in ßKO islets. The protein levels of proinsulin and insulin were decreased in ßKO islets. These results suggest that acute METTL14 deficiency in ß-cells induces glucose intolerance by increasing the IRE1α/sXBP-1 pathway.

METTL14 is essential for ß-cell survival and insulin secretion.

Defects in the development, maintenance or expansion of ß-cell mass can result in impaired glucose metabolism and diabetes. N6-methyladenosine affects mRNA stability and translation efficiency, and impacts cell differentiation and stress response. To determine if there is a role for m6A in ß-cells, we investigated the effect of Mettl14, a key component of the m6A methyltransferase complex, on ß-cell survival and function using rat insulin-2 promoter-Cre-mediated deletion of Mettl14 mouse line (ßKO). We found that ßKO mice with normal chow exhibited glucose intolerance, lower levels of glucose-stimulated insulin secretion, increased ß-cell death and decreased ß-cell mass. In addition, HFD-fed ßKO mice developed glucose intolerance, decreased ß-cell mass and proliferation, exhibited lower body weight, increased adipose tissue mass, and enhanced insulin sensitivity due to enhanced AKT signaling and decreased gluconeogenesis in the liver. HFD-fed ßKO mice also showed a decrease in de novo lipogenesis, and an increase in lipolysis in the liver. RNA sequencing in islets revealed that Mettl14 deficiency in ß-cells altered mRNA expression levels of some genes related to cell death and inflammation. Together, we showed that Mettl14 in ß-cells plays a key role in ß-cell survival, insulin secretion and glucose homeostasis.

Deficiency of VCP-Interacting Membrane Selenoprotein (VIMP) Leads to G1 Cell Cycle Arrest and Cell Death in MIN6 Insulinoma Cells.

BACKGROUND/AIMS: VCP-interacting membrane selenoprotein (VIMP), an ER resident selenoprotein, is highly expressed in ß-cells, however, the role of VIMP in ß-cells has not been characterized. In this study, we studied the relationship between VIMP deficiency and ß-cell survival in MIN6 insulinoma cells. METHODS: To determine the role of VIMP in ß-cells, lentiviral VIMP shRNAs were used to knock down (KD) expression of VIMP in MIN6 cells. Cell death was quantified by propidium iodide (PI) staining followed by flow cytometric analyses using a FACS Caliber and FlowJo software. Cell apoptosis and proliferation were determined by TUNEL assay and Ki67 staining, respectively. Cell cycle was analyzed after PI staining. RESULTS: The results show that 1) VIMP suppression induces ß-cell apoptosis, which is associated with a decrease in Bcl-xL, and the ß-cell apoptosis induced by VIMP suppression can be inhibited by overexpression of Bcl-xL; 2) VIMP knockdown (KD) decreases cell proliferation and G1 cell cycle arrest by accumulating p27 and decreasing E2F1; 3) VIMP KD suppresses unfolded protein response (UPR) activation by regulating the IRE1α and PERK pathways; 4) VIMP KD increases insulin secretion. CONCLUSION: These results suggest that VIMP may function as a novel regulator to modulate ß-cell survival, proliferation, cell cycle, UPR and insulin secretion in MIN6 cells.

Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma.

Reactive oxygen species (ROS) play important roles in follicular development and survival. Granulosa cell death is associated with increased ROS, but the mechanism of granulosa cell death induced by ROS is not clear. In order to define the molecular link between ROS and granulosa cell death, COV434, human granulosa tumor cells, were treated with H2O2. Compared to control cells, H2O2 induced granulosa cell death in a dose- and time-dependent manner. H2O2 induced an increase in Bax, Bak and Puma, and a decrease in anti-apoptotic molecules such as Bcl-2, Bcl-xL and Mcl-1. Both knockdown of Puma and overexpression of Bcl-xL could inhibit H2O2-induced granulosa cell death. These results suggest that suppression of Puma and overexpression of anti-apoptotic Bcl-2 family members could improve granulosa cell survival. To explore the mechanisms responsible for these findings, ROS in granulosa cells treatment with H2O2 were measured. The results showed that ROS was increased in a H2O2 dose- and time-dependent manner at the earlier time point. In addition, H2O2 induced an increase in Nrf2 and phosphorylation of JNK and p53. SP600125, an inhibitor of JNK, inhibits H2O2-induced phosphorylation of JNK and p53, and granulosa cell death. Antioxidant N-acetylcysteine (NAC) dose-dependently prevents H2O2-induced granulosa cell death. Furthermore, NAC also prevents phosphorylation of JNK and p53 induced by H2O2. Taken together, these data suggest that H2O2 regulates cell death in granulosa cells via the ROS-JNK-p53 pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be useful for future clinical applications.

Pancreatic ß-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak.

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in ß-cell death leading to reduced ß-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic ß-cell death. In Pdx1 haploinsufficient mice, Bax ablation in ß-cells increased ß-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates ß-cell apoptosis in Pdx1-deficient diabetes.

An interconnected hierarchical model of cell death regulation by the BCL-2 family.

Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX-BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid(-/-)Bim(-/-)Puma(-/-)Noxa(-/-) and Bax(-/-)Bak(-/-) cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and 'BH3-exposed' monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.

BH3-only molecule Bim mediates ß-cell death in IRS2 deficiency.

Irs2-deficient mice develop type 2-like diabetes due to a reduction in ß-cell mass and a failure of pancreatic islets to undergo compensatory hyperplasia in response to insulin resistance. In order to define the molecular mechanisms, we knocked down Irs2 gene expression in mouse MIN6 insulinoma cells. Insulin receptor substrate 2 (IRS2) suppression induced apoptotic cell death, which was associated with an increase in expression of the BH3-only molecule Bim. Knockdown (KD) of Bim reduced apoptotic ß-cell death induced by IRS2 suppression. In Irs2-deficient mice, Bim ablation restored ß-cell mass, decreased the number of TUNEL-positive cells, and restored normal glucose tolerance after glucose challenge. FoxO1 mediates Bim upregulation induced by IRS2 suppression, and FoxO1 KD partially inhibits ß-cell death induced by IRS2 suppression. These results suggest that Bim plays an important role in mediating the increase in ß-cell apoptosis and the reduction in ß-cell mass that occurs in IRS2-deficient diabetes.

Role of BH3-only molecules Bim and Puma in ß-cell death in Pdx1 deficiency.

Mutations in pancreatic duodenal homeobox-1 (PDX1) are associated with diabetes in humans. Pdx1-haploinsufficient mice develop diabetes due to an increase in ß-cell death leading to reduced ß-cell mass. For definition of the molecular link between Pdx1 deficiency and ß-cell death, Pdx1-haploinsufficient mice in which the genes for the BH3-only molecules Bim and Puma had been ablated were studied on a high-fat diet. Compared with Pdx1(+/-) mice, animals haploinsufficient for both Pdx1 and Bim or Puma genes showed improved glucose tolerance, enhanced ß-cell mass, and reduction in the number of TUNEL-positive cells in islets. These results suggest that Bim and Puma ablation improves ß-cell survival in Pdx1(+/-) mice. For exploration of the mechanisms responsible for these findings, Pdx1 gene expression was knocked down in mouse MIN6 insulinoma cells resulting in apoptotic cell death that was found to be associated with increased expression of BH3-only molecules Bim and Puma. If the upregulation of Bim and Puma that occurs during Pdx1 suppression was prevented, apoptotic ß-cell death was reduced in vitro. These results suggest that Bim and Puma play an important role in ß-cell apoptosis in Pdx1-deficient diabetes.

NF-κB­inducing kinase (NIK) promotes hyperglycemia and glucose intolerance in obesity by augmenting glucagon action.

The canonical inhibitor of nuclear factor κB kinase subunit ß (IKK-ß)­nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB1) pathway has been well documented to promote insulin resistance; however, the noncanonical NF-κB­inducing kinase (NIK)­NF-κB2 pathway is not well understood in obesity. Additionally, the contribution of counter-regulatory hormones, particularly glucagon, to hyperglycemia in obesity is unclear. Here we show that NIK promotes glucagon responses in obesity. Hepatic NIK was abnormally activated in mice with dietary or genetic obesity. Systemic deletion of Map3k14, encoding NIK, resulted in reduced glucagon responses and hepatic glucose production (HGP). Obesity is associated with high glucagon responses, and liver-specific inhibition of NIK led to lower glucagon responses and HGP and protected against hyperglycemia and glucose intolerance in obese mice. Conversely, hepatocyte-specific overexpression of NIK resulted in higher glucagon responses and HGP. In isolated mouse livers and primary hepatocytes, NIK also promoted glucagon action and glucose production, at least in part by increasing cAMP response element-binding (CREB) stability. Therefore, overactivation of liver NIK in obesity promotes hyperglycemia and glucose intolerance by increasing the hyperglycemic response to glucagon and other factors that activate CREB.

BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program.

Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are associated with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.

Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis.

While activation of BAX/BAK by BH3-only molecules (BH3s) is essential for mitochondrial apoptosis, the underlying mechanisms remain unsettled. Here we demonstrate that BAX undergoes stepwise structural reorganization leading to mitochondrial targeting and homo-oligomerization. The alpha1 helix of BAX keeps the alpha9 helix engaged in the dimerization pocket, rendering BAX as a monomer in cytosol. The activator BH3s, tBID/BIM/PUMA, attack and expose the alpha1 helix of BAX, resulting in secondary disengagement of the alpha9 helix and thereby mitochondrial insertion. Activator BH3s remain associated with the N-terminally exposed BAX through the BH1 domain to drive homo-oligomerization. BAK, an integral mitochondrial membrane protein, has bypassed the first activation step, explaining why its killing kinetics are faster than those of BAX. Furthermore, death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis. Accordingly, deficiency of Bim/Puma impedes ER stress-induced BAX/BAK activation and apoptosis. Our study provides mechanistic insights regarding the spatiotemporal execution of BAX/BAK-governed cell death.

The p53-cathepsin axis cooperates with ROS to activate programmed necrotic death upon DNA damage.

Three forms of cell death have been described: apoptosis, autophagic cell death, and necrosis. Although genetic and biochemical studies have formulated a detailed blueprint concerning the apoptotic network, necrosis is generally perceived as a passive cellular demise resulted from unmanageable physical damages. Here, we conclude an active de novo genetic program underlying DNA damage-induced necrosis, thus assigning necrotic cell death as a form of "programmed cell death." Cells deficient of the essential mitochondrial apoptotic effectors, BAX and BAK, ultimately succumbed to DNA damage, exhibiting signature necrotic characteristics. Importantly, this genotoxic stress-triggered necrosis was abrogated when either transcription or translation was inhibited. We pinpointed the p53-cathepsin axis as the quintessential framework underlying necrotic cell death. p53 induces cathepsin Q that cooperates with reactive oxygen species (ROS) to execute necrosis. Moreover, we presented the in vivo evidence of p53-activated necrosis in tumor allografts. Current study lays the foundation for future experimental and therapeutic discoveries aimed at "programmed necrotic death."

Bax and Bak do not exhibit functional redundancy in mediating radiation-induced endothelial apoptosis in the intestinal mucosa.

PURPOSE: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. METHODS AND MATERIALS: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. RESULTS: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. CONCLUSIONS: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.

Differential role of SH2-B and APS in regulating energy and glucose homeostasis.

SH2-B and APS, two members of a pleckstrin homology and SH2 domain-containing adaptor family, promote both insulin and leptin signaling in a similar fashion in cultured cells. In addition, APS mediates insulin-stimulated activation of the c-Cbl/CAP/TC10 pathway in cultured adipocytes. Here we characterized genetically modified mice lacking SH2-B, APS, or both to determine the physiological roles of these two proteins in animals. Disruption of the SH2-B gene resulted in obesity, hyperglycemia, hyperinsulinemia, and glucose intolerance. Conversely, deletion of the APS gene did not alter adiposity, energy balance, and glucose metabolism. Energy intake, energy expenditure, fat content, body weight, and plasma insulin, leptin, glucose, and lipid levels were similar between APS(-/-) and WT littermates fed either normal chow or a high-fat diet. Moreover, deletion of APS failed to alter insulin and glucose tolerance. APS(-/-)/SH2-B(-/-) double knockout mice also developed energy imbalance, obesity, hyperleptinemia, hyperinsulinemia, hyperglycemia, and glucose intolerance; however, plasma leptin and insulin levels were significantly lower in APS(-/-)/SH2-B(-/-) than in SH2-B(-/-) mice. These results suggest that SH2-B, but not APS, is a key positive regulator of energy and glucose metabolism in mice.

Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice.

Leptin regulates energy balance and body weight by activating its receptor LEPRb and multiple downstream signaling pathways, including the STAT3 and the IRS2/PI 3-kinase pathways, in the hypothalamus. Leptin stimulates activation of LEPRb-associated JAK2, which initiates cell signaling. Here we identified SH2-B, a JAK2-interacting protein, as a key regulator of leptin sensitivity, energy balance, and body weight. SH2-B homozygous null mice were severely hyperphagic and obese and developed a metabolic syndrome characterized by hyperleptinemia, hyperinsulinemia, hyperlipidemia, hepatic steatosis, and hyperglycemia. The expression of hypothalamic orexigenic NPY and AgRP was increased in SH2-B(-/-) mice. Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hypothalamic STAT3 and IRS2 were significantly impaired in SH2-B(-/-) mice. Moreover, overexpression of SH2-B counteracted PTP1B-mediated inhibition of leptin signaling in cultured cells. Our data suggest that SH2-B is an endogenous enhancer of leptin sensitivity and required for maintaining normal energy metabolism and body weight in mice.

Effect of polysaccharide sulfate 916 on the production of nitric oxide in ECV304 cells induced by cytokines and H(2)O(2).

OBJECTIVE: To investigate the effect of polysaccharide sulfate 916 (PS916) on the production of nitric oxide (NO) in ECV304 cells induced by tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta) and H(2)O(2) in vitro. METHODS: Production of NO in ECV304 cells was measured by the Griess method and the proliferation of cells was tested by the MTT method. The activity of NO synthase was detected spectrophotometrically. RESULTS: Production of NO in ECV304 cells decreased after treatment with 40 ng/ml IL-1 beta and 40 ng/ml TNF alpha, but increased in the presence of H(2)O(2) 0.1 mmol/L. PS916 significantly enhanced NO production in ECV304 cells in a dose-dependent manner in the TNF alpha and IL-1 beta treated groups and decreased it in the H(2)O(2) treated group. Proliferation of ECV304 cells was inhibited by TNFalpha and H(2)O(2) and no effect was found in the IL-1 beta treated group. PS916 increased the proliferation of cells treated with TNFalpha and H(2)O(2) dose-dependently. In vitro, PS916 has no effect on the activity of NO synthase. CONCLUSION: PS916 has a protective effect on ECV304 cells exposed to IL-1 beta, TNF alpha and H(2)O(2).

Polysaccharide sulfate 916 inhibits neutrophil-endothelial adhesion.

OBJECTIVE: To study the effect of polysaccharide sulfate 916 (PS916) on neutrophil-endothelial cell adhesion. METHODS: Cell adhesion was evaluated by testing neutrophil myeloperoxidase activity. Expression of adhesion molecule in human umbilical vein endothelial cell (HUVEC) was measured by ELISA. The neutrophil activation rate induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was tested by nitroblue tetrazolium (NBT) reduction. RESULTS: Tumor necrosis factor alpha (TNFalpha, 50 - 800 U/ml) increased the adherence of neutrophil to TNFalpha-stimulated HUVEC in a concentration and time dependent manner. PS916 (0.01 - 1.0 mg/ml) dose-dependently inhibited the adherence of neutrophils to TNFalpha-stimulated HUVEC. fMLP increased the activation rate of neutrophils independent of concentration. PS916 also inhibited the adherence of fMLP-activated neutrophils to HUVEC. Moreover, PS916 inhibited adhesion molecule expression in TNFalpha-stimulated HUVEC. CONCLUSIONS: PS916 inhibited neutrophil-endothelial adhesion. The mechanism of its action was partially related to suppressing the expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1).