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. 2014 Nov 1;25(21):3308-18.
doi: 10.1091/mbc.E14-03-0853. Epub 2014 Aug 27.

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Lan Liu  1 , Eleni Christodoulou-Vafeiadou  2 , Jaladanki N Rao  1 , Tongtong Zou  1 , Lan Xiao  1 , Hee Kyoung Chung  1 , Hong Yang  1 , Myriam Gorospe  3 , Dimitris Kontoyiannis  2 , Jian-Ying Wang  4
Affiliations

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Lan Liu et al. Mol Biol Cell. .

Abstract

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor-related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3'-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.

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Figures

FIGURE 1:
FIGURE 1:
Characterization of intestinal epithelium-specific HuR deletion (IE-HuR−/−) mice. (A) PCR analysis of genomic DNA from small intestinal mucosa indicates floxed, HuR deletion, and Cre bands in HuRfl/fl, IE-HuR−/−, and IE-HuR+/− mice. (B) Levels of HuR mRNA (a) and protein (b) in small (S) intestinal (left) and colonic (right) mucosa as measured by RT-PCR and Western blotting analyses. (C) Immunohistochemical staining of HuR, shown as in dark brown in small intestinal (a) and colonic (b) mucosa. Scale bar, 50 μm.
FIGURE 2:
FIGURE 2:
HuR deletion in IECs inhibits small intestinal mucosal growth. (A) Body weights of age- and sex-matched littermate and IE-HuR−/− mice. Values are the means ± SEM (n = 10). (B) Comparison of gastrointestinal gross morphology in littermates and IE-HuR−/− mice. (C) Photomicrographs of hematoxylin and eosin stain staining of the small intestine. Scale bar, 50 μm. (D) Changes in the length of villi (left) and crypt (right) of the mucosa described in C (n = 6). *p < 0.05 compared with littermates. (E) Proliferating cells in small intestinal crypts as measured by BrdU labeling. BrdU (1 h postinjection, S phase), green. (F) Expression of PCNA and Ki67 proteins in the small intestinal mucosa.
FIGURE 3:
FIGURE 3:
HuR deletion in IECs alters the regenerative potential of crypt progenitors. (A) Histograms depicting the labeling indices of cells at the S phase in resting (untreated) or S-phase descendants in the regenerating mucosa at different times (hours) in littermate (control) IEC-HuR−/− (IEC-KO) mice postirradiation as measured by BrdU detection. Values are shown as means ± SEM (n = 4). *p < 0.05. (B) Representative photomicrographs of paraffin-embedded small intestine. Sections stained with hematoxylin (blue) and anti-BrdU (brown). (C) Villus/crypt ratio of small intestinal mucosa postregeneration. Data were derived from 12 random villi and/or crypts from three mice per group/per time point; **p < 0.001 compared with littermates.
FIGURE 4:
FIGURE 4:
HuR deficiency correlates with reduction in LRP6 levels. (A) Changes in expression of LRP6, Fzd7, p65, E-cad, and Smad7 proteins in small intestinal mucosa in littermates and IE-HuR−/− mice. (B) Immunohistochemical staining of LRP6 in mucosal tissues. Scale bar, 50 μm. (C) Lrp6 mRNA levels in the intestinal mucosa as measured by RT-PCR (left) and qPCR (right) analyses. Values are the means ± SEM (n = 6). *p < 0.05 compared with littermates. (D) Levels of Fzd7, p65, E-cad, and Smad7 mRNAs as measured by qPCR analysis. *p < 0.05 compared with littermates.
FIGURE 5:
FIGURE 5:
HuR binds the 3′-UTR of Lrp6 mRNA. (A) Association of endogenous HuR with endogenous Lrp6 mRNA in IEC-6 cells as measured by RIP/qPCR analysis using either anti-HuR antibody (Ab) or control IgG: (a) Lrp6 mRNAs in HuR IP as measured by RT-PCR (left) and qPCR (right) analyses; and (b) levels of total input mRNAs. Values are the means ± SEM from triplicate samples. *p < 0.05 compared with IgG IP. (B) HuR immunoblots using the pull-down materials by biotinylated transcripts of Lrp6 5′-UTR, CR, and 3′-UTR. Left, schematic representation of various biotinylated Lrp6 transcripts. (C) Association of HuR with the Lrp6 mRNA in small intestinal mucosa in littermates and IE-HuR−/− mice as measured by RIP/qPCR analysis. Values are the means ± SEM (n = 5). *p < 0.05 compared with littermates.
FIGURE 6:
FIGURE 6:
HuR silencing destabilizes Lrp6 mRNA and represses its translation. (A) Immunoblots of HuR and LRP6 in cells transfected with siHuR or C-siRNA for 48 h. (B) Levels of Lrp6 mRNA as measured by RT-PCR (top) and qPCR (bottom) analyses in cells treated as described in A. Values are the means ± SEM (n = 3). *p < 0.05 compared with C-siRNA. (C) Half-life of the Lrp6 mRNA in cells described in A. (D) Newly synthesized LRP6 protein as measured by l-azidohomoalaine (AHA) incorporation assays. (E) Distributions of Lrp6 (top) and Gapdh (bottom) mRNAs in each gradient fraction of polysomal profiles prepared from cells described in A. (F) Levels of reporter activities as measured by analysis of Lrp6 5′-UTR, CR, and 3′-UTR luciferase reporters after HuR silencing. Top, schematic of plasmids of different chimeric firefly luciferase Lrp6 reporters. *p < 0.05 compared with C-siRNA.
FIGURE 7:
FIGURE 7:
Silencing HuR or LRP6 inhibits cell proliferation in stable Wnt3a-transfected IEC-6 cells. (A) Western blot analysis of the levels of Wnt3a protein (a), Wnt/β-catenin signaling activity as measured by using the TOPFLASH reporter assay (b) and cell growth as examined by MTT assay (c) in stable Wnt3a-transfected cells. Values are the means ± SEM (n = 6). *p < 0.05 compared with vector. (B) Immunoblots of HuR and LRP6 after transfection with siHuR or C-siRNA for 48 h. (C) Changes in Wnt/β-catenin signaling activity and cell growth. Values are the means ± SEM (n = 3). *p < 0.05 compared with C-siRNA. (D) Flow cytometric analysis of cell cycle distribution. Black line, area; blue line, curve fit; FL2-A, DNA content. (E) The relative G1, S, and G2/M compartments calculated from data described in D. Values are the means from three separate experiments. *,+p < 0.05 compared with parent IECs and Wnt3a-IECs transfected with C-siRNA, respectively.
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