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Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

Kovalenko PL, Yuan L, Sun K, Kunovska L, Seregin S, Amalfitano A, Basson MD - PLoS ONE (2013)

Bottom Line: This Slfn3 overexpression was associated with increases in all four differentiation markers.Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity.Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.

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Slfn3 modulates expression of enterocyte differentiation markers in vivo.(A) 1011 vector particles in 200 µL of Ad-GFP or Ad-GFP-Slfn3 were injected intraluminally into temporarily obstructed jejunal segments of anesthetized 8-12 weeks old male rats. 72 hours after exposure to the Ad-GFP-Slfn3 virus there was a significant increase of Slfn3 transcript (A), villin (B), Glut2 (C), and SI (D) expression compared to respective mRNA from control animals inoculated with intraluminal Ad-GFP by real-time qPCR of mRNA from jejunal mucosal scrapings (n = 6–7, *p<0.01). Induced gene expression was confirmed by grading of slides stained with appropriate antibody. Overexpression of Slfn3 significantly increase expression of Glut2 (C) on brush border membrane (BMM, arrows) and compared to Ad-GFP treated mucosa. (F) Intestinal mucosa villous length was significantly increased after Ad-GFP-Slfn3 infection (n = 3, *p<0.05, #p<0.1). Representative images of control (Ad-GFP) and Ad-GFP-Slfn3 virus injected intestines. Scale bar in (A–E, 400× magnification) is 20 µm and in (F, 100× magnification) 100 µm.
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pone-0079745-g002: Slfn3 modulates expression of enterocyte differentiation markers in vivo.(A) 1011 vector particles in 200 µL of Ad-GFP or Ad-GFP-Slfn3 were injected intraluminally into temporarily obstructed jejunal segments of anesthetized 8-12 weeks old male rats. 72 hours after exposure to the Ad-GFP-Slfn3 virus there was a significant increase of Slfn3 transcript (A), villin (B), Glut2 (C), and SI (D) expression compared to respective mRNA from control animals inoculated with intraluminal Ad-GFP by real-time qPCR of mRNA from jejunal mucosal scrapings (n = 6–7, *p<0.01). Induced gene expression was confirmed by grading of slides stained with appropriate antibody. Overexpression of Slfn3 significantly increase expression of Glut2 (C) on brush border membrane (BMM, arrows) and compared to Ad-GFP treated mucosa. (F) Intestinal mucosa villous length was significantly increased after Ad-GFP-Slfn3 infection (n = 3, *p<0.05, #p<0.1). Representative images of control (Ad-GFP) and Ad-GFP-Slfn3 virus injected intestines. Scale bar in (A–E, 400× magnification) is 20 µm and in (F, 100× magnification) 100 µm.

Mentions: Three days after exposure of the jejunal mucosa to the Ad-GFP-Slfn3 virus, we observed a 2.3±0.4 fold increase in Slfn3 transcript levels (Fig. 2A, n = 6–7, p<0.01). Ad-GFP-Slfn3 virus also induced a 1.8±0.4 fold increase in villin expression, a 3.8±0.3 fold increase in SI expression, a 2.9±0.6 fold increase in Glut2 expression compared to respective mRNA from control animals (Fig. 2B-D, n = 6–7, p<0.01 for each). We further investigated the effect of injecting the Slfn3-overexpressing virus on the protein level of these differentiation markers by IHC. Ad-GFP-Slfn3 injection significantly increased jejunal mucosal immunoreactivity for Slfn3, villin, Glut2, SI (p<0.05, n = 6–10 each). While in normal control rats GLUT2 was primarily located at the basement membrane (BM) overexpression of Slfn3 significantly increased availability of to the brush border membrane (BBM) (Figure 2C).


Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

Kovalenko PL, Yuan L, Sun K, Kunovska L, Seregin S, Amalfitano A, Basson MD - PLoS ONE (2013)

Slfn3 modulates expression of enterocyte differentiation markers in vivo.(A) 1011 vector particles in 200 µL of Ad-GFP or Ad-GFP-Slfn3 were injected intraluminally into temporarily obstructed jejunal segments of anesthetized 8-12 weeks old male rats. 72 hours after exposure to the Ad-GFP-Slfn3 virus there was a significant increase of Slfn3 transcript (A), villin (B), Glut2 (C), and SI (D) expression compared to respective mRNA from control animals inoculated with intraluminal Ad-GFP by real-time qPCR of mRNA from jejunal mucosal scrapings (n = 6–7, *p<0.01). Induced gene expression was confirmed by grading of slides stained with appropriate antibody. Overexpression of Slfn3 significantly increase expression of Glut2 (C) on brush border membrane (BMM, arrows) and compared to Ad-GFP treated mucosa. (F) Intestinal mucosa villous length was significantly increased after Ad-GFP-Slfn3 infection (n = 3, *p<0.05, #p<0.1). Representative images of control (Ad-GFP) and Ad-GFP-Slfn3 virus injected intestines. Scale bar in (A–E, 400× magnification) is 20 µm and in (F, 100× magnification) 100 µm.
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Related In: Results  -  Collection

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pone-0079745-g002: Slfn3 modulates expression of enterocyte differentiation markers in vivo.(A) 1011 vector particles in 200 µL of Ad-GFP or Ad-GFP-Slfn3 were injected intraluminally into temporarily obstructed jejunal segments of anesthetized 8-12 weeks old male rats. 72 hours after exposure to the Ad-GFP-Slfn3 virus there was a significant increase of Slfn3 transcript (A), villin (B), Glut2 (C), and SI (D) expression compared to respective mRNA from control animals inoculated with intraluminal Ad-GFP by real-time qPCR of mRNA from jejunal mucosal scrapings (n = 6–7, *p<0.01). Induced gene expression was confirmed by grading of slides stained with appropriate antibody. Overexpression of Slfn3 significantly increase expression of Glut2 (C) on brush border membrane (BMM, arrows) and compared to Ad-GFP treated mucosa. (F) Intestinal mucosa villous length was significantly increased after Ad-GFP-Slfn3 infection (n = 3, *p<0.05, #p<0.1). Representative images of control (Ad-GFP) and Ad-GFP-Slfn3 virus injected intestines. Scale bar in (A–E, 400× magnification) is 20 µm and in (F, 100× magnification) 100 µm.
Mentions: Three days after exposure of the jejunal mucosa to the Ad-GFP-Slfn3 virus, we observed a 2.3±0.4 fold increase in Slfn3 transcript levels (Fig. 2A, n = 6–7, p<0.01). Ad-GFP-Slfn3 virus also induced a 1.8±0.4 fold increase in villin expression, a 3.8±0.3 fold increase in SI expression, a 2.9±0.6 fold increase in Glut2 expression compared to respective mRNA from control animals (Fig. 2B-D, n = 6–7, p<0.01 for each). We further investigated the effect of injecting the Slfn3-overexpressing virus on the protein level of these differentiation markers by IHC. Ad-GFP-Slfn3 injection significantly increased jejunal mucosal immunoreactivity for Slfn3, villin, Glut2, SI (p<0.05, n = 6–10 each). While in normal control rats GLUT2 was primarily located at the basement membrane (BM) overexpression of Slfn3 significantly increased availability of to the brush border membrane (BBM) (Figure 2C).

Bottom Line: This Slfn3 overexpression was associated with increases in all four differentiation markers.Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity.Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.

Show MeSH
Related in: MedlinePlus