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 determine the functional consequences of [HuR/Lrp6 mRNA] associations, we reduced HuR levels by small interfering RNA (siRNA) targeting the HuR mRNA (siHuR), as reported previously (Liu et al., 2009). IEC-6 cells transfected with siHuR showed <10% of HuR levels as compared with the levels seen in cells transfected with control siRNA (C-siRNA; Figure 6A, top). Of importance, HuR silencing reduced LRP6 protein by ∼85% (Figure 6A, middle), but it decreased Lrp6 mRNA levels by only ∼40% (Figure 6B). The reduction in Lrp6 mRNA by HuR silencing was due to the destabilization of Lrp6 mRNA, since silencing HuR selectively lowered the Lrp6 mRNA half-life (Figure 6C). To examine whether HuR silencing also alters the translation of Lrp6 mRNA, we examined changes in the level of new LRP6 protein synthesis after transfection with siHuR and demonstrated that newly synthesized LRP6 protein decreased significantly in HuR-silent cells compared with cells transfected with C-siRNA (Figure 6D). Inhibition of LRP6 protein synthesis by HuR silencing was specific, since there was no change in nascent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesis after transfection with siHuR. To further define the role of HuR in the regulation of LRP6 translation, we examined the relative distribution of Lrp6 mRNA in individual fractions from polyribosome gradients. Although decreasing the levels of HuR did not affect global polysomal profiles (unpublished data), the abundance of Lrp6 mRNA associated with actively translating fractions (fractions 9–11) decreased in HuR-silent cells with a shift of Lrp6 mRNAs to low-translating fractions (fractions 6–8; Figure 6E, top); it is important to note that even small shifts in polysome size can reflect large changes in protein synthesis. In contrast, Gapdh mRNA, encoding the housekeeping protein GAPDH, distributed similarly in both groups (Figure 6E, bottom). HuR regulates the stability and translation of Lrp6 mRNA by interacting with Lrp6 3′-UTR, since HuR silencing decreased the levels of Lrp6-3′UTR-F2 luciferase reporter activity (Figure 6F) but did not affect the activities of Luc-5′-UTR and Luc-CR-F1 reporter genes. On the other hand, ectopic overexpression of HuR by infection with the adenoviral vector containing the corresponding HuR cDNA (AdHuR) increased LRP6 expression by increasing Lrp6 mRNA stability and translation via interaction with its 3′-UTR (Supplemental Figure S4). These results indicate that HuR increases LRP6 expression by stabilizing the Lrp6 mRNA and enhancing its translation.
Affiliation: Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201 Veterans Affairs Medical Center, Baltimore, MD 21201;