Amika Singla

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

The COMMD1 protein, implicated in copper homeostasis, is found to regulate endosomal sorting of the copper transporter ATP7A through a novel protein complex containing CCDC22, CCDC93, and C16orf62, which link COMMD1 to the WASH complex.

Enteropathogenic Escherichia coli Infection Inhibits Intestinal Serotonin Transporter Function and Expression

BACKGROUND & AIMS Serotonin transporter (SERT) plays a critical role in regulating serotonin (5-hydroxytryptamine [5-HT]) availability in the gut. Elevated 5-HT levels are associated with diarrheal conditions such as irritable bowel syndrome and enteric infections. Whether alteration in SERT activity contributes to the pathophysiology of diarrhea induced by the food-borne pathogen enteropathogenic Escherichia coli (EPEC) is not known. The present studies examined the effects of EPEC infection on SERT activity and expression in intestinal epithelial cells and elucidated the underlying mechanisms. METHODS Caco-2 cells as a model of human intestinal epithelia and EPEC-infected C57BL/6J mouse model of infection were utilized. SERT activity was measured as Na(+) and Cl(-) dependent (3)[H] 5-HT uptake. SERT expression was measured by real-time quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence studies. RESULTS Infection of Caco-2 cells with EPEC for 30-120 minutes decreased apical SERT activity (P < .001) in a type 3 secretion system dependent manner and via involvement of protein tyrosine phosphatases. EPEC infection decreased V(max) of the transporter; whereas cell surface biotinylation studies revealed no alteration in the cellular or plasma membrane content of SERT in Caco-2 cells. EPEC infection of mice (24 hours) reduced SERT immunostaining with a corresponding decrease in SERT messenger RNA levels, 5-HT uptake, and mucosal 5-HT content in the small intestine. CONCLUSIONS Our results demonstrate inhibition of SERT by EPEC and define the mechanisms underlying these effects. These data may aid in the development of a novel pharmacotherapy to modulate the serotonergic system in treatment of infectious diarrheal diseases.

Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-γ

Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical Cl(-)/OH(-) (HCO(3)(-)) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-gamma (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-gamma on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-gamma in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-gamma on DRA promoter (p--1183/+114) activity. Deletion analysis indicated that the IFN-gamma response element is located within the -1183 to -790 region, and sequence analysis of this region revealed the presence of potential gamma-activated site (GAS), a binding site (-933/-925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-gamma. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-gamma in the human intestine.

Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical Cl/OH exchange

Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical Cl(-)/OH(-) exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and Cl(-)/OH(-) exchange activity was measured as DIDS-sensitive (36)Cl(-) uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical Cl(-)/OH(-) exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 muM) significantly stimulated Cl(-)/OH(-) exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of Cl(-)/OH(-) exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical Cl(-)/OH(-) exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in Cl(-)/OH(-) exchange may contribute to the antidiarrheal effects of LPA.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Differential expression and activity of the cellular energy regulators GAPDH and NDPK underlie reactive oxygen species–induced damage in the mouse liver and may contribute to human liver disease progression.

SREBP2 mediates the modulation of intestinal NPC1L1 expression by curcumin

Curcumin, the major phenolic compound in the spice turmeric, exhibits numerous biological effects, including lowering plasma cholesterol and preventing diet-induced hypercholesterolemia. The mechanisms underlying the hypocholesterolemic effect of curcumin are not fully understood. In this regard, intestinal Niemann-Pick C1-like 1 (NPC1L1) cholesterol transporter, the molecular target of intestinal cholesterol absorption inhibitor ezetimibe, plays an essential role in the maintenance of cholesterol homeostasis. The current studies were designed to investigate the effect of curcumin on NPC1L1 function, expression, and promoter activity in intestinal Caco-2 monolayers. NPC1L1 function was evaluated by the measurement of ezetimibe-sensitive [(3)H]cholesterol esterification. Relative abundance of NPC1L1 mRNA and protein was evaluated by real-time PCR and Western blotting, respectively. Luciferase assays were used to measure NPC1L1 promoter activity. Our results showed that curcumin significantly inhibited ezetimibe-sensitive cholesterol esterification in a dose-dependent manner with a maximum decrease (by 52% compared with control) occurring at 50 μM concentration. Curcumin treatment of Caco-2 monolayers also significantly decreased NPC1L1 mRNA and protein expression. Similarly, the promoter activity of the NPC1L1 gene was inhibited significantly (55%) by 50 μM curcumin. The decrease in NPC1L1 promoter activity by curcumin was associated with a reduction in the expression and the DNA-binding activity of the sterol response element-binding protein 2 (SREBP2) transcription factor. Furthermore, the overexpression of active SREBP2 protected NPC1L1 from the inhibitory effect of curcumin. Our studies demonstrate that curcumin directly modulates intestinal NPC1L1 expression via transcriptional regulation and the involvement of SREBP2 transcription factor.

Oxidative stress, Nrf2 and keratin up‐regulation associate with Mallory‐Denk body formation in mouse erythropoietic protoporphyria

Mallory‐Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fechm1Pas mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC‐induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug‐induced MDBs. In 13‐ and 20‐week‐old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13‐week‐old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase‐2 and keratin overexpression, with a greater keratin 8 (K8)‐to‐keratin 18 (K18) ratio, which are critical for drug‐induced MDB formation. In 20‐week‐old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short‐term (3‐week) DDC feeding markedly induced MDB formation in 20‐week‐old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative‐stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up‐regulation. Nrf2 knockdown in HepG2 cells down‐regulated K8, but not K18. Conclusion: Fch/fch mice develop age‐associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2‐mediated increase in K8, promotes MDB formation. (Hepatology 2012;56:322–331)

Structural and mechanistic insights into regulation of the retromer coat by TBC1d5

Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29–VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.

LPA stimulates intestinal DRA gene transcription via LPA2 receptor, PI3K/AKT, and c-Fos-dependent pathway

DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl(-) absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl(-)/HCO(3)(-)(OH(-)) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl(-)/HCO(3)(-) exchange activity in Caco-2 cells. LPA treatment (50-100 μM) of Caco-2 cells significantly stimulated DRA mRNA levels and DRA promoter activity (-1183/+114). This increase in DRA promoter activity involved the LPA2 receptor and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Progressive deletions from -1183/+114 to -790/+114 abrogated the stimulatory effects of LPA, indicating that the -1183/-790 promoter region harbors LPA response elements. Utilizing EMSA and mutational studies, our results showed that LPA induced the DRA promoter activity in a c-Fos-dependent manner. LPA also increased the protein expression of c-Fos and c-Jun in Caco-2 cells. Furthermore, overexpression of c-Fos but not c-Jun enhanced the DRA promoter activity. This increase in DRA transcription in response to LPA indicates that LPA may act as an antidiarrheal agent and could be exploited for the treatment of diarrhea associated with inflammatory or infectious diseases of the gut.

Lamin aggregation is an early sensor of porphyria-induced liver injury

Summary Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.