The mRNA-binding protein IGF2BP1 maintains intestinal barrier function by up-regulating occludin expression

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate–induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate–induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis. 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Liang S. Wijeratne H.S. Marti S. Chau L. Giroux V. et al.Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1.EMBO Rep. 2019; 2010.15252/embr.201847074Crossref PubMed Scopus (3) Google Scholar). Together, these findings hint to the function of IGF2BP1 in maintaining intestinal homeostasis. For the current study, we generated mice that allow inducible knockout of IGF2BP1 specifically in the adult intestinal epithelium to study pathological processes independent of potential developmental defects. Here, we demonstrate that the ablation of Igf2bp1 in adult IECs leads to acute colitis in mice, and IGF2BP1 deficiency in IECs decreases occludin levels, resulting in a defective barrier function. These findings uncover an important function of IGF2BP1 in IECs to protect against colitis. To investigate the role of IGF2BP1 in intestinal homeostasis, we generated Villin-CreERT2-Igf2bp1fl/fl transgenic mice. In these mice, transient knockout of Igf2bp1 (hereafter, Igf2bp1IEC-Ind KO) is induced via tamoxifen. Immunoblotting with anti-IGF2BP1 antibody was performed to confirm the ablation of Igf2bp1 from intestinal epithelium cells of Igf2bp1IEC-Ind KO mice (Fig. 1A). Intriguingly, adult Igf2bp1IEC-Ind KO mice displayed loss in body weight compared with Igf2bp1fl/fl littermates (Fig. 1B). Next, we examined the gross appearance of intestine and length of colon to inspect colon shortening, which is considered as the indicator of inflammation. In our analysis, we did not observe any significant change in gross appearance of intestine and in colon length of Igf2bp1IEC-Ind KO mice (Fig. 1C). Histological analysis of small intestines revealed that all Igf2bp1IEC-Ind KO mice exhibited increased lamina propria cellularity with mild to moderate active enteritis and patchy to diffuse neutrophilic and lymphocytic infiltration, when compared with control mice (Fig. 1D and Fig. S1B). Likewise, the colons of Igf2bp1IEC-Ind KO mice displayed mild active colitis with increased lamina propria cellularity and mild to moderate patchy to diffuse neutrophilic and lymphocytic infiltration. At places, there were loss of crypts with abscess formation (Fig. 1D and Fig. S1A). Based on the hematoxylin/eosin (H&E)-stained sections, analyzed by the pathologist in blinded manner, the samples were scored on two major histomorphological criteria: (i) severity of inflammatory cell infiltrates and (ii) epithelial changes (cryptitis, crypt abscesses, and erosion) (Fig. 1E). These results illustrate the importance of epithelial IGF2BP1 for normal intestinal physiology. Next, we examined whether induced intestinal epithelial IGF2BP1 knockout affects sensitivity of mice to DSS, which is a toxic chemical that when delivered in the drinking water causes colitis by disrupting the intestinal epithelium. Here we performed chronic DSS treatment as it is the most relevant model for experimental colitis in mice. In the first cycle of this experiment, mice were treated with 2% DSS for 5 days, followed by intermittent periods of normal drinking water. This cycle was repeated two more times, and mice were then euthanized and examined. This experiment conferred 75% mortality in Igf2bp1IEC-Ind KO mice (Fig. 2A). We also observed significant loss in weight and increases in disease activity index (DAI) scores (32Rath E. Berger E. Messlik A. Nunes T. Liu B. Kim S.C. Hoogenraad N. Sans M. Sartor R.B. Haller D. Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation.Gut. 2012; 61 (21997551): 1269-127810.1136/gutjnl-2011-300767Crossref PubMed Scopus (83) Google Scholar) in Igf2bp1IEC-Ind KO mice when compared with control littermates (Fig. 2, B and C). As severe chronic inflammation is known to decrease colon lengths, we evaluated colons at the conclusion of the experiment and found significantly shorter colons in Igf2bp1IEC-Ind KO mice than in the control littermates (Fig. 2D). We have also analyzed the H&E-stained sections of these colons and observed that the colon sections from DSS-treated Igf2bp1IEC-Ind KO mice showed increased disruption of colonic mucosa along with crypt abscesses, loss of colonic surface epithelium and crypts, and diffuse infiltration of inflammatory cells when compared with control mice (Fig. 2E and Fig. S1C). These tissue sections were analyzed and histomorphologically scored based on established criteria (Fig. 2F) (33Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2 (17406617): 541-54610.1038/nprot.2007.41Crossref PubMed Scopus (1040) Google Scholar, 59Erben U. Loddenkemper C. Doerfel K. Spieckermann S. Haller D. Heimesaat M.M. 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To confirm these results, we used the FITC-dextran permeability assay, which is a classical method for evaluating the status of intestinal barrier function. Four hours after oral administration of FITC-dextran, we isolated serum and examined the level of FITC-dextran in the samples. Significantly higher levels of FITC-dextran were found in Igf2bp1IEC-Ind KO mice compared with Igf2bp1fl/fl littermates (Fig. 3B). These results indicate that IGF2BP1 is involved in the maintenance of intestinal barrier function, and its absence disrupted the barrier. A disrupted TJ is a major cause of intestine barrier dysfunction (34Catalioto R.M. Maggi C.A. Giuliani S. Intestinal epithelial barrier dysfunction in disease and possible therapeutical interventions.Curr. Med. Chem. 2011; 18 (21143118): 398-42610.2174/092986711794839179Crossref PubMed Scopus (95) Google Scholar), and this motivated us to investigate the levels of major TJ proteins in IECs of Igf2bp1IEC-Ind KO mice. We found decrease specifically in the levels of occludin TJ protein in the IECs from the intestines of Igf2bp1IEC-Ind KO mice, whereas other TJ proteins, CLAUDIN-1, CLAUDIN-2, ZO-1, and ZO-3, remained unaffected when compared with control littermates (Fig. 3C). Immunostaining analysis revealed significantly lower levels of occludin in colon IECs of Igf2bp1IEC-Ind KO mice when compared with control mice (Fig. 3D). These results suggest that IGF2BP1 affects the expression of occludin TJ protein and maintains intestine barrier function. To investigate the mechanism of regulation of occludin TJ expression by IGF2BP1, we chose the human CCD841-CoTr normal colon epithelial cell line with moderate expression of Igf2bp1. Knockdown of IGF2BP1 using shRNAs in this cell line resulted in significant inhibition of occludin expression (Fig. 4A). IGF2BP1 is an RNA-binding protein (RBP) that often stabilizes its mRNA targets (6Doyle G.A. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.Nucleic Acids Res. 1998; 26 (9801297): 5036-504410.1093/nar/26.22.5036Crossref PubMed Scopus (138) Google Scholar, 14Noubissi F.K. Elcheva I. Bhatia N. Shakoori A. Ougolkov A. Liu J. Minamoto T. Ross J. Fuchs S.Y. Spiegelman V.S. CRD-BP mediates stabilization of βTrCP1 and c-myc mRNA in response to β-catenin signalling.Nature. 2006; 441 (16778892): 898-90110.1038/nature04839Crossref PubMed Scopus (169) Google Scholar), and to evaluate whether IGF2BP1 binds to the mRNA of occludin directly, we performed the CLIP assay. In our CLIP analysis, we found enrichment of OCLN mRNA to a similar extent as bona fide IGF2BP1 targets, such as MYC and β-TRCP1 (Fig. 4B). To elucidate whether IGF2BP1 regulates the turnover of OCLN mRNAs, we performed an actinomycin D chase experiment in a pair of colorectal cell lines. The loss-of-function experiments (by knocking down IGF2BP1) were performed in Caco-2 cells characterized by high expression of IGF2BP1. The knockdown of IGF2BP1 and its effect on occludin expression were confirmed by immunoblotting (Fig. 4C). The actinomycin D chase experiments in Caco-2 cells showed that the knockdown of IGF2BP1 resulted in accelerated rate of OCLN mRNA degradation (Fig. 4D) but not of Claudin2 mRNA (Fig. S3A). The gain-of-function experiments (by overexpressing IGF2BP1) were performed in RKO cells that have negligible expression of endogenous IGF2BP1. Overexpression of IGF2BP1 protein in this cell line resulted in increased levels of occludin protein compared with control cells (Fig. 4E). Additional mRNA stability assays using these cells detected an increased t1/2 of OCLN mRNA upon overexpression of IGF2BP1 (Fig. 4F), whereas there was no significant change in t1/2 of Claudin2 mRNA (Fig. S3B). These results show that IGF2BP1 protein regulates occludin expression by directly interacting with OCLN mRNA and stabilizing it, establishing OCLN mRNA as a novel direct target of IGF2BP1. Occludin plays an important role in the formation of the tight-junction seal by regulating macromolecule flux across the barrier (23Turner J.R. Intestinal mucosal barrier function in health and disease.Nature reviews. Immunology. 2009; 9 (19855405): 799-80910.1038/nri2653Crossref PubMed Scopus (1850) Google Scholar, 26Cummins P.M. Occludin: one protein, many forms.Mol. Cell. Biol. 2012; 32 (22083955): 242-25010.1128/MCB.06029-11Crossref PubMed Scopus (210) Google Scholar). To determine whether disregulation of occludin contributes to altered barrier function induced by down-regulation of IGF2BP1, we analyzed the permeability of Caco-2 cells grown as a monolayer. As predicted, IGF2BP1 knockdown resulted in decreased occludin expression in Caco-2 cells (Figs. 4C and 5A). To analyze the barrier, we grew Caco-2, with or without knockdown of IGF2BP1, on transwell plates. We found that IGF2BP1 knockdown caused a significant decrease in trans-epithelial electrical resistance (TER), indicating increased permeability in the Caco-2 monolayer (Fig. 5B). The TER levels were completely restored after re-expressing occludin in IGF2BP1 knockdown cells (Fig. 5B). We had also found increase flux in IGF2BP1 knockdown cells while this flux was restored after re-expression of occludin in IGF2BP1 knockdown cells (Fig. 5C). Occludin knockout mice displayed retarded growth and chronically inflamed gastrointestinal tracts (35Saitou M. Furuse M. Sasaki H. Schulzke J.D. Fromm M. Takano H. Noda T. Tsukita S. Complex phenotype of mice lacking occludin, a component of tight junction strands.Molecular biology of the cell. 2000; 11 (11102513): 4131-414210.1091/mbc.11.12.4131Crossref PubMed Scopus (837) Google Scholar). 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