HSPA6 is an ulcerative colitis susceptibility factor that is induced by cigarette smoke and protects intestinal epithelial cells by stabilizing anti-apoptotic Bcl-XL.

BACKGROUND: Cigarette smoking ameliorates ulcerative colitis (UC) and aggravates Crohn's disease (CD). Cigarette smoke suppresses inflammation-induced apoptosis in intestinal epithelial cells (DLD-1), which may explain its protective effect in UC. Here, we performed transcriptome profiling of cigarette smoke extract (CSE)-exposed DLD-1 and Jurkat cells (T-lymphocytes) and related this to UC susceptibility genes with protective functions in the intestinal epithelium. METHODS: CSE-regulated genes in DLD-1 and Jurkat cells were identified by Illumina microarrays and compared to genes in UC susceptibility loci. Colon biopsies were analyzed by immunohistochemistry for cell-specific expression of HSPA6. CSE-induced gene expression was analyzed by Q-PCR, Western blotting and immunofluorescence microscopy. Protein (HSPA6/Bcl-XL) interactions were analyzed by immunoprecipitation. RESULTS: CSE changed the expression of 536 and 2560 genes in DLD-1 and Jurkat cells, respectively. The "response to unfolded protein" was one of the most significantly affected gene sets with prominent induction (20.3-fold) of heat shock protein A6 (HSPA6). Six CSE-induced genes in DLD-1 cells were located in UC-susceptibility loci, including HSPA6 (rs1801274). HSPA6 is highly expressed in the human colonic epithelium. CSE caused a dose-dependent strong (>100-fold at 30% CSE for 6h), but transient induction of HSPA6 mRNA and protein in DLD-1 cells. HSPA6 co-immune precipitated with anti-apoptotic Bcl-XL, protein levels of which were increased while mRNA levels were unchanged. CONCLUSIONS: HSPA6 is a cigarette smoke-induced UC-susceptibility gene. The HSPA6 risk locus is associated with decreased HSPA6 expression. HSPA6 provides epithelial protection by stabilizing anti-apoptotic Bcl-XL, thereby contributing to the beneficial effect of cigarette smoking in UC.

The ATG16L1-T300A allele impairs clearance of pathosymbionts in the inflamed ileal mucosa of Crohn's disease patients.

OBJECTIVE: Crohn's disease (CD) is caused by a complex interplay among genetic, microbial and environmental factors. ATG16L1 is an important genetic factor involved in innate immunity, including autophagy and phagocytosis of microbial components from the gut. We investigated the effect of inflammation on the composition of microbiota in the ileal mucosa of CD patients in relation to the ATG16L1 risk status. DESIGN: Biopsies (n=35) were obtained from inflamed and non-inflamed regions of the terminal ileum of 11 CD patients homozygous for the ATG16L1 risk allele (ATG16L1-T300A) and 9 CD patients homozygous for the ATG16L1 protective allele (ATG16L1-T300). Biopsy DNA was extracted and the bacterial composition analysed by pyrosequencing. Intracellular survival rates of adherent-invasive Escherichia coli (AIEC) were analysed by determining colony forming units after exposure to monocytes isolated from healthy volunteers homozygous for the ATG16L1 risk or protective allele. RESULTS: Inflamed ileal tissue from patients homozygous for the ATG16L1 risk allele contained increased numbers of Fusobacteriaceae, whereas inflamed ileal tissue of patients homozygous for the ATG16L1 protective allele showed decreased numbers of Bacteroidaceae and Enterobacteriaceae and increased Lachnospiraceae. The ATG16L1 allele did not affect the bacterial composition in the non-inflamed ileal tissue. Monocytes homozygous for the ATG16L1 risk allele showed impaired killing of AIEC under inflammatory conditions compared with those homozygous for the ATG16L1 protective allele. CONCLUSIONS: CD patients homozygous for the ATG16L1-T300A risk allele show impaired clearance of pathosymbionts in ileal inflammation indicating that ATG16L1 is essential for effective elimination of pathosymbionts upon inflammation.

Mice defective in p53 nuclear localization signal 1 exhibit exencephaly.

p53 is a major suppressor of human malignancy. The protein levels and activity are tightly regulated in cells. Early experiments identified nuclear localization signal 1 (NLS1) as a regulator of p53 localization. We have generated mice bearing a mutation in p53 ( NLS1 ), designated p53 ( NLS1 ). Our experiments confirm a role for NLS1 in regulating p53 function. Murine embryonic fibroblasts generated from homozygous p53 ( NLS1 ) animals are partially defective in cell cycle arrest and do not respond to inhibitory signals from oncogenic Ras. In addition, p53-dependent apoptosis is abrogated in thymocytes. Contrary to predicted results, fibroblasts from homozygous p53 ( NLS1 ) animals have a greater rate of proliferation than p53-null cells. In addition, p53 ( NLS1 ) cells are more resistant to UV-induced death. Surprisingly, the homozygous p53 ( NLS1 ) animals exhibit embryonic and peri-natal lethality, with a significant portion of the animals developing exencephaly. Thus, p53 ( NLS1/NLS1 ) embryos exhibit a reduced viability relative to p53-null mice. These studies indicate that the NLS1 is a major regulator of p53 activity in vivo.

Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation.

The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only.

Suppression of p21Rac signaling and increased innate immunity mediate remission in Crohn's disease.

In inflammatory bowel disease (IBD), large areas of apparently healthy mucosa lie adjacent to ulcerated intestine. Knowledge of the mechanisms that maintain remission in an otherwise inflamed intestine could provide important clues to the pathogenesis of this disease and provide rationale for clinical treatment strategies. We used kinome profiling to generate comprehensive descriptions of signal transduction pathways in inflamed and noninflamed colonic mucosa in a cohort of IBD patients, and compared the results to non-IBD controls. We observed that p21Rac1 guanosine triphosphatase (GTPase) signaling was strongly suppressed in noninflamed colonic mucosa in IBD. This suppression was due to both reduced guanine nucleotide exchange factor activity and increased intrinsic GTPase activity. Pharmacological p21Rac1 inhibition correlated with clinical improvement in IBD, and mechanistically unrelated pharmacological p21Rac1 inhibitors increased innate immune functions such as phagocytosis, bacterial killing, and interleukin-8 production in healthy controls and patients. Thus, suppression of p21Rac activity assists innate immunity in bactericidal activity and may induce remission in IBD.

Role of defective autophagia and the intestinal flora in Crohn disease.

The precise mechanisms underlying the development of Crohn disease (CD) remain controversial, but sufficient data have been collected to suggest that an uncontrolled immune response within the intestinal mucosa leads to inflammation in a genetically susceptible host. Although lack of mucosal regulatory T cells causes colitis in humans and experimental rodents, patients with CD have more rather than less regulatory activity in the intestine, apparently excluding defects in tolerance as the cause of CD. Genome-wide association studies have identified many gene variants that confer susceptibility and which seem associated to diminished functioning of especially innate immunity. In apparent agreement, CD patients are impaired with respect to innate immune responses and controlling bacterial flora in the intestine. Furthermore, severe genetic deficiencies in innate immunity, like e.g., lack of NADP oxidase activity or diminished function of the Wiskott Aldrich syndrome protein are associated with colitis in mice and men, and are often mistakenly diagnosed as CD. Thus we favor the view that the primary defect in CD is a lack in innate immunity, causing second tier immunological defenses to combat otherwise easily controlled bacterial breaches of the mucosal barrier.