Posttranscriptional regulation of 14-3-3ζ by RNA-binding protein HuR modulating intestinal epithelial restitution after wounding

Abstract

The 14-3-3ζ is a member of the family of 14-3-3 proteins and participates in many aspects of cellular processes, but its regulation and involvement in gut mucosal homeostasis remain unknown. Here, we report that 14-3-3ζ expression is tightly regulated at the posttranscription level by RNA-binding protein HuR and plays an important role in early intestinal epithelial restitution after wounding. The 14-3-3ζ was highly expressed in the mucosa of gastrointestinal tract and in cultured intestinal epithelial cells (IECs). The 3' untranslated region (UTR) of the 14-3-3ζ mRNA was bound to HuR, and this association enhanced 14-3-3ζ translation without effect on its mRNA content. Conditional target deletion of HuR in IECs decreased the level of 14-3-3ζ protein in the intestinal mucosa. Silencing 14-3-3ζ by transfection with specific siRNA targeting the 14-3-3ζ mRNA suppressed intestinal epithelial restitution as indicated by a decrease in IEC migration after wounding, whereas ectopic overexpression of the wild-type 14-3-3ζ promoted cell migration. These results indicate that HuR induces 14-3-3ζ translation via interaction with its 3' UTR and that 14-3-3ζ is necessary for stimulation of IEC migration after wounding.

Keywords: Epithelial cell migration; mucosal injury; posttranscriptional regulation; rapid epithelial repair.

Figure 1

Levels of 14‐3‐3ζ and HuR in gut mucosa in mice and different lines of cultured IECs. (A) Representative immunoblots of 14‐3‐3ζ and HuR in the mucosa isolated from stomach, duodenum, jejunum, ileum, and colon as examined by western blot analysis. GAPDH immunoblotting was performed as an internal control for equal loading. (B) Representative immunoblots of 14‐3‐3ζ and HuR in IEC‐6, differentiated IEC‐Cdx2L1, and Caco‐2 cells. Three separate experiments were performed that showed similar results. IECs, intestinal epithelial cells.

Figure 2

HuR binds the 14‐3‐3ζ mRNA. (A) Schematic representation of the 14‐3‐3ζ mRNA and the predicted hits of the HuR signature motif in its 3′ UTR. (B) Association of endogenous HuR with endogenous 14‐3‐3ζ mRNA. After IP of RNA–protein complexes from cell lysates using either anti‐HuR antibody (Ab) or control IgG, RNA was isolated and used in reverse transcription reactions. Values were means ± SEM from triplicate samples. HuR binding to STIM1 was shown as a positive control; its binding to GAPDH served as a negative control in this study. (C) HuR binding to different fractions of 14‐3‐3ζ 5′ UTR, CR, and 3′ UTR. Top panel, schematic representation of the 14‐3‐3ζ 3′ UTR biotinylated transcripts. After incubation of cytoplasmic lysates with the full‐length (FL) or various fractions (F) of the 14‐3‐3ζ 3′ UTR, the resulting RNP complexes were pulled down, and the abundances of HuR and actin proteins in the pull‐down material were examined.

Figure 3

HuR silencing represses 14‐3‐3ζ translation in IECs. (Aa) Representative immunoblots of HuR and 14‐3‐3ζ. Cells were transfected with control siRNA (C‐siRNA) or siHuR, and whole‐cell lysates were harvested 48 h thereafter. Levels of HuR and 14‐3‐3ζ proteins were measured by western immunoblot analysis. (Ab) Levels of 14‐3‐3ζ mRNA as measured by Q‐PCR analysis in cells treated as described in Aa. Values are the mean ± SEM (n = 3). (B) Levels of reporter activities as measured by analysis of 14‐3‐3ζ 5′ UTR, CR, and 3′ UTR luciferase reporters in cells described in (Aa) Top panel, schematic of plasmids of different chimeric firefly luciferase 14‐3‐3ζ reporters. *P < 0.05 compared with C‐siRNA. IECs, intestinal epithelial cells.

Figure 4

Conditional HuR deletion in IECs inhibits 14‐3‐3ζ expression. (Aa) Representative immunoblots of HuR and 14‐3‐3ζ in the small intestinal mucosa in IE‐HuR^−/− mice and control littermates. (Ab) Quantitative analysis of the immunoblotting signals as measured by densitometry. Values are the means ± SEM (n = 5). *P < 0.05 compared with IE‐control littermate mice. (B) Levels of 14‐3‐3ζ mRNA in IE‐HuR^−/− mice as described in (A). IECs, intestinal epithelial cells.

Figure 5

14‐3‐3ζ silencing inhibits cell migration after wounding in different lines of IECs. (A) Representative immunoblots of 14‐3‐3ζ and HuR. IEC‐6 cells were transfected with control siRNA (C‐siRNA) or si14‐3‐3ζ, and whole‐cell lysates were harvested 48 h thereafter. (B) Images of cell migration after wounding in IEC‐6 cells: (a) 0 h after wounding, (b) 6 h after wounding, (c) 6 h after wounding in cells transfected with C‐siRNA, and (d) 6 h after wounding in cells transfected with si14‐3‐3ζ. (C) Summarized data of cell migration 6 h after removal of part of the monolayer in (a) IEC‐6 cells, (b) IEC‐Cdx2L1 cells, and (c) Caco‐2 cells transfected with C‐siRNA or si14‐3‐3ζ for 48 h followed by cell migration assays. Data are means ± SEM from six dishes. *P < 0.05 compared with cells transfected with C‐siRNA. IECs, intestinal epithelial cells.

Figure 6

Ectopic overexpression of 14‐3‐3ζ enhances cell migration after wounding. (A) Representative immunoblots of 14‐3‐3ζ and HuR protein. Caco‐2 cells were transiently transfected with the 14‐3‐3ζ expression vector or empty vector (Null), and the levels of 14‐3‐3ζ and HuR were examined by western immunoblotting analysis 48 h after the transfection. (B) Summarized data showing cell migration 6 h after removal of part of the monolayer in cells described in A. Values are means ± SEM from six dishes. *P < 0.05 compared with the cells transfected with empty vector.