Musashi-1 Post-Transcriptionally Enhances Phosphotyrosine-Binding Domain-Containing m-Numb Protein Expression in Regenerating Gastric Mucosa

<div><h3>Objective</h3><p>Upregulation of the RNA-binding protein Musashi-1 (Msi1) has been shown to occur in rat gastric corpus mucosa after ethanol-induced mucosal injury. However, there is no direct evidence linking Msi1 with gastric regeneration. We examined the process of tissue repair after acute gastric mucosal injury with Msi1-knock-out (KO) mice to clarify the role of Msi1 and Msi1-dependent regulation of m-Numb expression in regenerating gastric mucosa.</p> <h3>Methods</h3><p>Acute gastric injury was induced in Msi1-KO and wild-type ICR mice by administering absolute ethanol. Expression of the splicing variants of <em>m-Numb</em> mRNA and protein in the gastric mucosa were analyzed by quantitative RT-PCR and western blotting, respectively.</p> <h3>Results</h3><p>We demonstrated that phosphotyrosine-binding domain-containing m-Numb expression was significantly upregulated at both the mRNA and protein levels in wild-type mice at 3 h after ethanol-induced acute gastric injury. In contrast, in Msi1-KO mice, the m-Numb protein was expressed weakly, and was associated with delayed regeneration of the injured gastric mucosal epithelium. In the Msi1-KO mouse, the ratio of <em>m-Numb</em> mRNA to total <em>m-Numb</em> mRNA in the heavy polysome fractions was lower than that in the wild-type mouse. Further, we showed that m-Numb-enhancement in gastric mucous cells induced the expression of prostate stem cell antigen and metallothionein-2. Under the m-Numb enhancing condition, the gastric cells exhibited enhanced cell proliferation and were significantly more resistant to H₂O₂-induced cell death than control cells.</p> <h3>Conclusions</h3><p>Msi1-dependent post-transcriptional enhancement of m-Numb is crucial in gastric epithelial regeneration.</p> </div

Schematic representation of Msi1-dependent gastric regeneration.

<p>After gastric damage, PTB domain-containing <i>m-numb</i> transcript is induced. Msi1 enhances the <i>m-numb</i> translation. The translated m-Numb protein induces the expression of regeneration-related genes such as <i>PSCA</i> and <i>Mt2</i>, resulting in gastric regeneration.</p

Gastric epithelial cell degeneration after gastric damage.

<p>(A) Hematoxylin and eosin (H&E)-stained sections of the stomachs of wild-type and Msi1-KO mice. Control group (water administration) and ethanol administration group. Bars = 100 µm (B) Gastric epithelial cell degeneration in the fundic area was noted in the H&E-stained sections. *<i>P</i><0.05.</p

Expression of m-Numb protein in the stomach of wild-type and Msi1-KO mice after ethanol-induced gastric damage.

<p>(A) Expression of m-Numb and p21 protein in the stomach of wild-type and Msi1-KO mice. (B) The intensity of each band in the western blot of m-Numb was analyzed and the results were statistically compared. White bars: wild-type; black bars: Msi1-KO. *<i>P</i><0.05, **<i>P</i><0.01.</p

Expression of p21 and m-Numb in the stomach of wild-type and Msi1-KO mice.

<p>(A) Western blots indicating expression of Msi1, p21, and m-Numb protein in the stomach and cerebrum of sham-treated wild-type and Msi1-KO mice. (B) Classification of m-Numb protein by proline-rich region (PRR). (C) m-Numb protein and total RNA expression. Western blotting and quantitative RT-PCR for the expression analysis of m-Numb protein and mRNA in sham-treated wild-type and Msi1-KO mice was performed in triplicate. The density of each m-Numb protein band in western blotting is normalized to actin and represented as the fold-change relative to expression of the protein in wild-type mice. White bars: wild-type mice; black bars: Msi1-KO mice. *<i>P</i><0.01 compared to wild-type mice. (D) Polysome analysis of <i>m-Numb</i> mRNA from mouse stomach. Fractions of a polysome gradient prepared from the stomachs of wild-type (empty circles) and Msi1-KO (filled circles) mice. RNA was extracted from each fraction and used for quantitative RT-PCR. The results are shown as the percentage of the total amount of RNA in each fraction. (E) Knockdown of human <i>Msi1</i> and <i>Msi2</i> in N87 cells by shRNA-containing lentiviral particles. Western blotting was performed using primary antibodies specific for Msi1, Msi2, m-Numb, and β-actin.</p

Primer sequences for quantitative RT-PCR.

<p>Primer sequences for quantitative RT-PCR.</p

Immunohistochemical analysis revealed a defect in cell differentiation in Msi1-KO stomach.

<p>Wild-type (A, C, and E) and Msi1-KO (B, D, and F) mice were administered ethanol. Sections of the gastric mucosa from each mouse at 5 h after ethanol administration were then stained using anti-H⁺, K⁺-ATPase- (A and B), anti-Muc6- (C and D), and anti-pepsinogen- (E and F) antibodies. Bar = 100 µm.</p

m-Numb-induced expression of regeneration-related genes.

<p>(A) Expression of <i>LGR5</i>, <i>DCLK1</i>, <i>PSCA</i>, and <i>Mt2</i> mRNA in the stomachs of sham-treated wild-type (white bars) and Msi1-KO (black bars) mice. **<i>P</i><0.01 compared to wild-type mice. (B) mRNA expression of total <i>m-numb</i>, <i>LGR5</i>, <i>DCLK1</i>, <i>PSCA</i>, and <i>Mt2</i> in LacZ-, Numb1-, and Numb2-overexpressing MGE507 cells. *<i>P</i><0.05, **<i>P</i><0.01 compared to LacZ-overexpressing cells. (C) Cell proliferation assay in LacZ-, Numb1-, and Numb2-overexpressing MGE507 cells. **<i>P</i><0.01 compared to LacZ-overexpressing cells. (D) H₂O₂-induced changes in cell viability in LacZ-, Numb1-, and Numb2-overexpressing MGE507 cells. **<i>P</i><0.01 compared to LacZ-overexpressing cells.</p

Expression levels of the m-Numb splicing variants in the stomach.

<p>(A) Schematic representation of the <i>m-Numb</i> gene and the primer designed for m-Numb <i>mRNA</i> amplification. (B) Expression of the PTBS- and PTBL-types of <i>m-Numb mRNA</i> in the stomach of ethanol-administered wild-type (white bars) and Msi1-KO (black bars) mice. Absolute ethanol was administered to both groups of mice and the mRNA expression of each of the splicing variants of <i>m-Numb</i> was analyzed by real-time PCR using SYBR Green. The expression levels were expressed as fold-change relative to the expression in the wild-type animals at 0 h. GAPDH was used as internal standard. (C) Semi-quantitative PCR was performed to confirm the expression of the complete PTBL-type of <i>m-Numb mRNA</i>, which was induced after gastric damage (at 5 h after ethanol administration). The primers used were the PTBL forward primer, and PRRS and PRRL reverse primers. <i>GAPDH</i> was used as the internal standard. The intensity of each band was analyzed, and the results are shown as fold-change relative to the expression in wild-type mice. *<i>P</i><0.05 compared to Msi1-KO mice at 0 h, **<i>P</i><0.01 compared to wild-type mice at 0 h.</p