Altin Gjymishka

Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter.

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

MEK Signaling Is Required for Phosphorylation of eIF2α following Amino Acid Limitation of HepG2 Human Hepatoma Cells

The mammalian amino acid response (AAR) pathway is up-regulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2α (eukaryotic initiation factor 2α), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2α and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2α phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2α by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2α phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.

Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells.

The activated amino acid response (AAR) and unfolded protein response (UPR) stress signaling pathways converge at the phosphorylation of translation initiation factor eIF2α. This eIF2α modification suppresses global protein synthesis but enhances translation of selected mRNAs such as that for activating transcription factor 4 (ATF4). An ATF4 target gene, SNAT2 (system A sodium-dependent neutral amino acid transporter 2), contains a C/EBP-ATF site that binds ATF4 and triggers increased transcription during the AAR. However, the present studies show that despite increased ATF4 binding to the SNAT2 gene during UPR activation in HepG2 human hepatoma cells, transcription activity was not enhanced. Hyperacetylation of histone H3 and recruitment of the general transcription factors at the HepG2 SNAT2 promoter occurred in response to the AAR but not the UPR. In contrast, the UPR did enhance transcription from a plasmid-based reporter gene driven by a SNAT2 genomic fragment containing the C/EBP-ATF site. Simultaneous activation of the AAR and the UPR pathways revealed that the UPR actually suppressed the increased SNAT2 transcription by the AAR pathway, demonstrating that the UPR pathway generates a repressive signal that acts downstream of ATF4 binding.

Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway.

The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPbeta (CCAAT/enhancer-binding protein beta) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6alpha or ATF6beta expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.

Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor‐4–independent pathway

The FOXA (forkhead box A) proteins (FOXA1, FOXA2, and FOXA3) play a critical role in the development of the liver, and they also regulate metabolism in adult hepatic tissue. The liver responds to changes in nutrient availability by initiating a number of stress signaling pathways. The present studies demonstrated that in mouse dams fed a low‐protein diet hepatic expression of FOXA2 and FOXA3 messenger RNA, but not FOXA1, was induced. Conversely, fetal liver did not exhibit this regulation. Amino acid deprivation of HepG2 hepatoma cells also enhanced transcription from the FOXA2 and FOXA3 genes. In contrast, endoplasmic reticulum stress inhibited the expression of FOXA1, only slightly induced FOXA2, and had no effect on FOXA3. The FOXA2 and FOXA3 messenger RNA induction by amino acid deprivation did not require activating transcription factor 4, a critical component of the conventional amino acid response (AAR) pathway, but their induction was partially dependent on CCAAT/enhancer‐binding protein β. Simultaneous knockdown of both FOXA2 and FOXA3 by small interfering RNA did not affect the activation of other amino acid responsive genes, suggesting that the FOXA proteins are not required for the known AAR pathway. Collectively, the results document that the hepatic FOXA family of genes are differentially regulated by amino acid availability. (HEPATOLOGY 2009.)

Influence of host immunoregulatory genes, ER stress and gut microbiota on the shared pathogenesis of inflammatory bowel disease and Type 1 diabetes.

Inflammatory bowel disease (IBD) with its two distinct entities, Crohns disease and ulcerative colitis, and Type 1 diabetes mellitus (T1D) are autoimmune diseases. The prevalence of these diseases continues to rapidly rise in the industrialized world. Despite the identification of several genetic loci that are associated with both IBD and T1D, thus far, there is a paucity of epidemiological data to support a clinical overlap. In an effort to better understand the underlying pathogenic mechanisms of both IBD and T1D, this review summarizes the literature about these related autoimmune diseases, describes the most recent advances in their etiopathogenesis and emphasizes the genetic and nongenetic factors that exercise a differential influence. Genome-wide association studies have identified genetic loci with a role in immune response regulation that are linked to both IBD (particularly Crohns disease) and T1D. Some of these genetic loci (e.g., IL-18RAP) have a divergent role, conferring risk for one disease and protection for the other. Recent evidence highlights an important role of gut microbiota and cellular responses (e.g., endoplasmic reticulum stress) in the pathogenesis of both IBD and T1D.

Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex

It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

A disintegrin and metalloprotease with thrombospondin type I motif 7: a new protease for connective tissue growth factor in hepatic progenitor/oval cell niche.

Hepatic progenitor/oval cell (OC) activation occurs when hepatocyte proliferation is inhibited and is tightly associated with the fibrogenic response during severe liver damage. Connective tissue growth factor (CTGF) is important for OC activation and contributes to the pathogenesis of liver fibrosis. By using the Yeast Two-Hybrid approach, we identified a disintegrin and metalloproteinase with thrombospondin repeat 7 (ADAMTS7) as a CTGF binding protein. In vitro characterization demonstrated CTGF binding and processing by ADAMTS7. Moreover, Adamts7 mRNA was induced during OC activation, after the implantation of 2-acetylaminofluorene with partial hepatectomy in rats or on feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet in mice. X-Gal staining showed Adamts7 expression in hepatocyte nuclear factor 4α(+) hepatocytes and desmin(+) myofibroblasts surrounding reactive ducts in DDC-treated Adamts7(-/-) mice carrying a knocked-in LacZ gene. Adamts7 deficiency was associated with higher transcriptional levels of Ctgf and OC markers and enhanced OC proliferation compared to Adamts7(+/+) controls during DDC-induced liver injury. We also observed increased α-smooth muscle actin and procollagen type I mRNAs, large fibrotic areas in α-smooth muscle actin and Sirius red staining, and increased production of hepatic collagen by hydroxyproline measurement. These results suggest that ADAMTS7 is a new protease for CTGF protein and a novel regulator in the OC compartment, where its absence causes CTGF accumulation, leading to increased OC activation and biliary fibrosis.

Febrile pleuropericarditis, a potentially life-threatening adverse event of balsalazide – case report and literature review of the side effects of 5-aminosalicylates

Ulcerative colitis (UC) is an idiopathic chronic inflammatory disorder that affects the colonic mucosa. One class among the drugs used for its treatment is the 5-aminosalicylates (5-ASAs). While highly efficacious in treating mild-to-moderate UC, 5-ASAs are associated with rare but potentially life-threatening side effects such as pericarditis, myocarditis and pneumonitis. These adverse events appear to be caused by a hypersensitivity reaction and resolve after cessation of 5-ASA drugs. This article presents a case report of febrile pleuropericarditis in a UC patient treated with balsalazide, and provides a thorough literature review of the rare side effects of 5-ASAs, their incidence, clinical presentation, differential diagnosis and treatment. In conclusion, the clinicians should be aware that this type of adverse events to 5-ASA compounds can be easily overlooked but it has significant morbidity if not promptly diagnosed.

Suppression of islet homeostasis protein thwarts diabetes mellitus progression

During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.