Impaired Intestinal Farnesoid X Receptor Signaling in Cystic Fibrosis Mice.

BACKGROUND & AIMS: The bile acid (BA)-activated farnesoid X receptor (FXR) controls hepatic BA synthesis and cell proliferation via the intestinal hormone fibroblast growth factor 19. Because cystic fibrosis (CF) is associated with intestinal dysbiosis, anomalous BA handling, and biliary cirrhosis, we investigated FXR signaling in CF. METHODS: Intestinal and hepatic expression of FXR target genes and inflammation markers was assessed in Cftr null mice and controls. Localization of the apical sodium-dependent BA transporter was assessed, and BAs in gastrointestinal tissues were analyzed. The CF microbiota was characterized and FXR signaling was investigated in intestinal tissue and organoids. RESULTS: Ileal murine fibroblast growth factor 19 ortholog (Fgf15) expression was strongly reduced in CF mice, compared with controls. Luminal BA levels and localization of apical sodium-dependent BA transporter was not affected, and BAs induced Fgf15 up to normal levels in CF ileum, ex vivo, and CF organoids. CF mice showed a dysbiosis that was associated with a marked up-regulation of genes involved in host-microbe interactions, including those involved in mucin glycosylation, antimicrobial defense, and Toll-like receptor signaling. Antibiotic treatment reversed the up-regulation of inflammatory markers and restored intestinal FXR signaling in CF mice. Conversely, FXR-dependent gene induction in ileal tissue and organoids was repressed by bacterial lipopolysaccharide and proinflammatory cytokines, respectively. Loss of intestinal FXR activity was associated with a markedly blunted hepatic trophic response to oral BA supplementation, and with impaired repression of Cyp7a1, the gene encoding the rate-limiting enzyme in BA synthesis. CONCLUSIONS: In CF mice, the gut microbiota represses intestinal FXR activity, and, consequently, FXR-dependent hepatic cell proliferation and feedback control of BA synthesis.

Transcriptome analysis of the distal small intestine of Cftr null mice.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CFTR anion channel. Loss of CFTR function in pancreatic, biliary and intestinal epithelia, severely affects gastrointestinal function. Transcriptome analysis indicated the activation of an innate and adaptive immune response in the distal small intestine of Cftr null mice. Inflammation was associated with differential regulation of numerous genes involved in the transport and metabolism of nutrients and, particularly, lipids, that are targeted by ligand-dependent nuclear receptors and/or HNF4α. Among the most strongly down-regulated genes are the FXR targets Fgf15 and Nr0b2, the PPARα target Pdk4, and the PXR target Ces2a, whereas expression of the CF modifier gene Slc6a14 was strongly increased. Most changes in gene expression were reversed by bacterial containment. Our data suggest that the gut microbiota has a pervasive effect on gene expression in CF mice, affecting enterocyte maturation, lipid metabolism, and nutrient absorption in CF.

Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage.

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair.

DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.

Cystic fibrosis: toward personalized therapies.

Cystic fibrosis (CF), the most common, life-threatening monogenetic disease in Caucasians, is caused by mutations in the CFTR gene, encoding a cAMP- and cGMP-regulated epithelial chloride channel. Symptomatic therapies treating end-organ manifestations have increased the life expectancy of CF patients toward a mean of 40 years. The recent development of CFTR-targeted drugs that emerged from high-throughput screening and are capable of correcting the basic defect promises to transform the therapeutic landscape from a trial-and-error prescription to personalized medicine. This stratified approach is tailored to a specific functional class of mutations in CFTR, but can be refined further to an individual level by exploiting recent advances in ex vivo drug testing methods. These tests range from CFTR functional measurements in rectal biopsies donated by a CF patient to the use of patient-derived intestinal or pulmonary organoids. Such organoids may serve as an inexhaustible source of epithelial cells that can be stored in biobanks and allow medium- to high-throughput screening of CFTR activators, correctors and potentiators on the basis of a simple microscopic assay monitoring organoid swelling. Thus the recent breakthrough in stem cell biology allowing the culturing of mini-organs from individual patients is not only relevant for future stem cell therapy, but may also allow the preclinical testing of new drugs or combinations that are optimally suited for an individual patient.

RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling.

Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.