RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway.
Bottom Line: 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.
Affiliation: Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201 Veterans Affairs Medical Center, Baltimore, MD 21201;Show MeSH
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Mentions: To investigate the mediators of the HuR-elicited effects, we found that the HuR-deficient intestinal epithelium was associated with decreased levels of Lrp6 mRNA and LDL-receptor-related protein 6 (LRP6; Figure 4, A–C). In contrast, HuR deletion increased expression of p65 and Smad7 in the intestinal mucosa, although it failed to alter the levels of Frizzled-7 (Fzd7) or E-cadherin (Figure 4D). Because there are several potential hits of HuR motif in the Lrp6 mRNA, we further examined whether HuR directly interacted with the Lrp6 mRNA in cultured IEC-6 cells by performing ribonucleoprotein immunoprecipitation (RIP) assays using anti-HuR antibody under conditions that preserved RNP integrity (Lal et al., 2004). The interaction of Lrp6 mRNA with HuR was examined by isolating RNA from the immunoprecipitated material and subjecting it to reverse transcription (RT), followed by either conventional PCR or real-time quantitative PCR (qPCR) analyses. As shown in Figure 5A, the Lrp6 PCR products were highly enriched in HuR samples compared with control immunoglobulin G (IgG) samples. HuR was also found to bind the p65 mRNA, although it did not preferentially associate with Fzd7, E-cad, and Smad7 mRNAs (Supplemental Figure S3). To determine whether HuR binds to specific regions of the Lrp6 5′-UTR, CR, and 3′-UTR, we further tested [HuR/Lrp6 mRNA] associations by using biotinylated transcripts that spanned the Lrp6 mRNA regions shown (Figure 5B, schematic). After incubation with cytoplasmic lysates, the interaction between the biotinylated Lrp6 transcripts and HuR was examined by biotin pull-down, followed by Western blot analysis (Abdelmohsen et al., 2007; Chen et al., 2008). As shown, HuR readily associated with the Lrp6 3′-UTR, particularly the fragment 3′UTR-F2 (spanning positions 5881–6441), but not with Lrp6 5′-UTR and fragments of CR transcripts. Moreover, the abundance of [HuR/Lrp6 mRNA] complexes was enriched in the intestinal mucosa isolated from littermate control mice but not in the mucosa from IE-HuR−/− mice, as measured by RIP/qPCR analysis (Figure 5C). These results indicate that the Lrp6 mRNA is a novel target of HuR in the intestinal epithelium.
Affiliation: Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201 Veterans Affairs Medical Center, Baltimore, MD 21201;