Decreased TESK1-mediated cofilin 1 phosphorylation in the jejunum of IBS-D patients may explain increased female predisposition to epithelial dysfunction

Disturbed intestinal epithelial barrier and mucosal micro-inflammation characterize irritable bowel syndrome (IBS). Despite intensive research demonstrating ovarian hormones modulation of IBS severity, there is still limited knowledge on the mechanisms underlying female predominance in this disorder. Our aim was to identify molecular pathways involved in epithelial barrier dysfunction and female predominance in diarrhea-predominant IBS (IBS-D) patients. Total RNA and protein were obtained from jejunal mucosal biopsies from healthy controls and IBS-D patients meeting the Rome III criteria. IBS severity was recorded based on validated questionnaires. Gene and protein expression profiles were obtained and data integrated to explore biological and molecular functions. Results were validated by western blot. Tight junction signaling, mitochondrial dysfunction, regulation of actin-based motility by Rho, and cytoskeleton signaling were differentially expressed in IBS-D. Decreased TESK1-dependent cofilin 1 phosphorylation (pCFL1) was confirmed in IBS-D, which negatively correlated with bowel movements only in female participants. In conclusion, deregulation of cytoskeleton dynamics through TESK1/CFL1 pathway underlies epithelial intestinal dysfunction in the small bowel mucosa of IBS-D, particularly in female patients. Further understanding of the mechanisms involving sex-mediated regulation of mucosal epithelial integrity may have significant preventive, diagnostic, and therapeutic implications for IBS.

acetone-trichloroacetic acid precipitation (2D-CleanUp kit; GE Healthcare, Maryland, USA). The protein pellets were resuspended in the DIGE labeling buffer (8 M urea, 4% [wt/vol] 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 30 mM Tris [pH 8.0]), and the protein concentration was determined using the Bio-Rad RCDC protein assay as described by the manufacturer (Bio-Rad, California, USA). The protein concentration in the samples was then adjusted to 2 mg/mL by addition of DIGE labeling buffer. Fifty micrograms of each sample was labeled with either Cy3 or Cy5 cyanine dye (GE Healthcare), following general procedures.
Two-dimension electrophoresis was performed using GE Healthcare reagents and equipment.
First-dimension isoelectric focusing was performed on immobilized pH gradient strips (24 cm; pH 3 to 10 or 4 to 7) using an Ettan IPGphor system. Samples were applied via cup loading near the basic ends of the strips, which were previously rehydrated overnight in 450 μL of rehydration buffer (8 M urea, 4% [wt/vol]  a rocking platform. Second-dimension SDS-polyacrylamide gel electrophoresis was run by overlaying the strips on 12.5% isocratic Laemmli gels (24 by 20 cm), cast in low-fluorescence glass plates, on an Ettan DALT VI system. Gels were run at 20°C and at a constant power of 2.5 W per gel for 30 min, followed by 17 W per gel until the bromophenol blue tracking front had run off the bottoms of the gels (about 5 h).
Fluorescence images of the gels were acquired on a Typhoon 9400 scanner (GE Healthcare).
Cy3 and Cy5 images were scanned at 532 nm excitation/580 nm emission and 633 nm excitation/670 nm emission, respectively, at a 100 μm resolution. Image analysis and determination of significant alterations in protein abundances were performed automatically with the DeCyder V. 5.0 software (GE Healthcare).

Protein identification and data analysis
Protein spots of interest were excised from the gel by using an automated Spot Picker (GE Healthcare). In-gel trypsin digestion was performed essentially as previously described[1], using autolysis-stabilized trypsin (Promega, Wisconsin, USA). Tryptic digests were purified using ZipTip microtiter plates (Millipore, Carrigtwohill, Ireland).
Matrix-assisted laser desorption ionization mass spectrometric analysis of tryptic peptides was performed on an Ultraflex time-of-flight/time-of-flight (TOF-TOF) instrument (Bruker, Bremen, Germany). Samples were prepared using α-cyano-4-hydroxycinnamic acid as a matrix on anchor chip targets (Bruker). Identification of the proteins was carried out by peptide mass fingerprint data and/or by TOF-TOF PSD. Database searches were performed using the Mascot algorithm (Matrix Science, London, UK). Identified proteins where then submitted to Ingenuity Pathway Analysis (IPA) Software 7.0 as previously described [2].

Western blot
Jejunal biopsies were homogenized using FastPrep (MP Biomedicals) in TRIzol. Protein extractions from the organic phase of TRIzol were performed following the manufacturer instructions (ThermoFisher Scientific). Protein quantifications were performed using Quick Start™ Bradford Protein Assay (Biorad). Equal amounts of protein were separated by NuPAGE® Novex® 4-12% Bis-Tris Protein Gels (ThermoFisher Scientific). Primary antibodies and dilutions used are listed in supplementary table 1. Peroxidase-conjugated secondary antibody and the chemiluminescence detection system SuperSignal West Femto (ThermoFisher Scientific) were used to detect bound antibodies. All blots were probed with mouse antihuman β-actin as a protein loading control. Image acquisition was performed with a LAS-3000 Imaging System From Fuji. Densitometric comparison was carried out on the same immunoblot using the ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/).

Proteomic core analysis
The list of proteins obtained in profiling experiments was overlaid onto a global molecular network developed from information contained in the IPA knowledge base (IPKB). For network analysis, IPA computed a score (P-score=-log10(P-value)) according to the fit of the set of supplied genes and a list of biological functions stored in the IPKB. The score takes into account the number of proteins in the network and the size of the network to approximate how relevant this network is to the original list of genes and allows the networks to be prioritized for further studies. A score >3 (P<0.001) indicates a >99.9% confidence that a network was not generated by chance alone. The network identified is presented as a graph indicating the molecular relationships between proteins. Moreover, networks are preferentially enriched for proteins with the most extensive interactions, and for which interactions are specific with the other proteins in the network (rather than proteins that are promiscuous, those that interact with a broad selection of proteins throughout IPKB).
The functional analysis identified the biological functions and the canonical signaling pathways that were most significant to the input data set. The significance of the association between the input data set and the functions or pathways was determined based on two parameters: (1) a ratio of the number of proteins from the data set that map to the function/pathway divided by the total number of proteins that map to the function/pathway and (2)