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

Liu L, Christodoulou-Vafeiadou E, Rao JN, Zou T, Xiao L, Chung HK, Yang H, Gorospe M, Kontoyiannis D, Wang JY - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201 Veterans Affairs Medical Center, Baltimore, MD 21201;

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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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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.

Mentions: To investigate the consequences of HuR-regulated LRP6 expression upon cell proliferation, we used stable Wnt3a-transfected IEC-6 cells (Wnt3a-IECs) that were recently developed in our laboratory (Liu et al., 2012). Stably transfected Wnt3a-IECs expressed high levels of Wnt3a protein (Figure 7Aa) and exhibited a dramatic activation of Wnt/β-catenin signaling pathway, as indicated by an increase in the levels of the Wnt/β-catenin reporter plasmid superTOPFLASH activity (Figure 7Ab). Consistently, ectopic Wnt3a overexpression increased IEC proliferation (Figure 7Ac), although the levels of HuR and LRP6 were unchanged in Wnt3a-IECs (Figure 7B, left). Cell cycle analysis indicated that ∼35% of parent IECs were in the S phase, whereas the population of S-phase cells significantly increased to ∼54% in stable Wnt3a-IECs, along with a decrease in G1-phase cells. Moreover, decreased levels of endogenous LRP6 in stable Wnt3a-IECs by HuR silencing decreased Wnt/β-catenin signaling activity (Figure 7Ca), inhibited cell growth (Figure 7Cb), and returned the cell cycle distribution displayed by parent IECs. When stable Wnt3a-IECs were transfected with siHuR, S-phase cells decreased to ∼27% from ∼53% in cells transfected with C-siRNA (Figure 7, D and E). The inhibitory effects of decreasing endogenous LRP6 in stable Wnt3a-IECs by HuR silencing were not simply due to clonal variation, since two different clonal populations, Wnt3a-IEC-C1 and Wnt3a-IEC-C2, showed similar responses. LRP6 silencing in stable Wnt3a-IECs by transfection with siLRP6 also resulted in inhibition of the Wnt/β-catenin signaling pathway and cell growth, as indicated by decrease in S-phase cells. Conversely, ectopic overexpression of LRP6 in HuR-silenced cells by transfection with the LRP6 expression vector increased cell growth, along with increase in S-phase cells (Supplemental Figure S5). Marginal inhibition of parental IEC-6 cell proliferation by silencing HuR or LRP6 was also observed (Supplemental Figure S6). On the other hand, silencing HuR or LRP6 alone failed to induce apoptosis in parent IEC-6 cells or Wnt3a-IECs, as measured by assessment of cell viability, annexin-V staining, and levels of cleaved caspase-3. These results indicate that HuR-induced LRP6 expression plays an important role in stimulation of IEC proliferation.


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

Liu L, Christodoulou-Vafeiadou E, Rao JN, Zou T, Xiao L, Chung HK, Yang H, Gorospe M, Kontoyiannis D, Wang JY - Mol. Biol. Cell (2014)

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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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.
Mentions: To investigate the consequences of HuR-regulated LRP6 expression upon cell proliferation, we used stable Wnt3a-transfected IEC-6 cells (Wnt3a-IECs) that were recently developed in our laboratory (Liu et al., 2012). Stably transfected Wnt3a-IECs expressed high levels of Wnt3a protein (Figure 7Aa) and exhibited a dramatic activation of Wnt/β-catenin signaling pathway, as indicated by an increase in the levels of the Wnt/β-catenin reporter plasmid superTOPFLASH activity (Figure 7Ab). Consistently, ectopic Wnt3a overexpression increased IEC proliferation (Figure 7Ac), although the levels of HuR and LRP6 were unchanged in Wnt3a-IECs (Figure 7B, left). Cell cycle analysis indicated that ∼35% of parent IECs were in the S phase, whereas the population of S-phase cells significantly increased to ∼54% in stable Wnt3a-IECs, along with a decrease in G1-phase cells. Moreover, decreased levels of endogenous LRP6 in stable Wnt3a-IECs by HuR silencing decreased Wnt/β-catenin signaling activity (Figure 7Ca), inhibited cell growth (Figure 7Cb), and returned the cell cycle distribution displayed by parent IECs. When stable Wnt3a-IECs were transfected with siHuR, S-phase cells decreased to ∼27% from ∼53% in cells transfected with C-siRNA (Figure 7, D and E). The inhibitory effects of decreasing endogenous LRP6 in stable Wnt3a-IECs by HuR silencing were not simply due to clonal variation, since two different clonal populations, Wnt3a-IEC-C1 and Wnt3a-IEC-C2, showed similar responses. LRP6 silencing in stable Wnt3a-IECs by transfection with siLRP6 also resulted in inhibition of the Wnt/β-catenin signaling pathway and cell growth, as indicated by decrease in S-phase cells. Conversely, ectopic overexpression of LRP6 in HuR-silenced cells by transfection with the LRP6 expression vector increased cell growth, along with increase in S-phase cells (Supplemental Figure S5). Marginal inhibition of parental IEC-6 cell proliferation by silencing HuR or LRP6 was also observed (Supplemental Figure S6). On the other hand, silencing HuR or LRP6 alone failed to induce apoptosis in parent IEC-6 cells or Wnt3a-IECs, as measured by assessment of cell viability, annexin-V staining, and levels of cleaved caspase-3. These results indicate that HuR-induced LRP6 expression plays an important role in stimulation of IEC proliferation.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus