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

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

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

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

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

Supplemental material for Burden of impaired sleep quality on work productivity in functional dyspepsia

<p>Supplemental Material for Burden of impaired sleep quality on work productivity in functional dyspepsia by Juntaro Matsuzaki, Hidekazu Suzuki, Koji Togawa, Tsuyoshi Yamane, Hideki Mori, Takahiro Komori, Tatsuhiro Masaoka and Takanori Kanai in United European Gastroenterology Journal</p