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Original Investigation | Association of VA Surgeons

The Correlation Between the Expression of Differentiation Markers in Rat Small Intestinal Mucosa and the Transcript Levels of Schlafen 3

Pavlo L. Kovalenko, DVM, PhD1; Marc D. Basson, MD, PhD, MBA1,2
[+] Author Affiliations
1Department of Surgery, Michigan State University, East Lansing
2Research Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan
JAMA Surg. 2013;148(11):1013-1019. doi:10.1001/jamasurg.2013.3572.
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Importance  The normal absorptive function and structural maintenance of the intestinal mucosa depend on a constant process of proliferation of enterocytic stem cells followed by progressive differentiation toward a mature phenotype. The mechanisms that govern enterocytic differentiation in the mucosa of the small intestine are poorly understood.

Objective  To determine whether schlafen 3 (but not other schlafen proteins) act in vivo and whether its effects are limited to the small intestine. We have previously demonstrated in nonmalignant rat intestinal IEC-6 cells that schlafen 3 levels correlate with the expression of various differentiation markers in vitro in response to differentiation stimuli.

Design  Randomized controlled experiment.

Setting  Animal science laboratory.

Participants  Male Sprague-Dawley rats 8 to 13 weeks old.

Main Outcomes and Measures  Messenger RNA (mRNA) from jejunal and colonic mucosa was isolated, and transcript levels of schlafen proteins 1, 2, 3, 4, 5, 13, and 14; sucrase isomaltase (SI); dipeptidyl peptidase 4 (Dpp4); glucose transporter type 2 (Glut2); and villin were measured by quantitative reverse transcriptase–polymerase chain reaction. We tested parallel variations in protein levels by Western blotting and Dpp4 enzyme activity.

Results  The transcript level of schlafen 3 (Slfn3) correlated with the levels of the differentiation markers SI, Dpp4, Glut2, and villin. However, the expression of schlafen proteins 1, 2, 4, 5, 13, and 14 did not correlate with the expression of the differentiation markers. The mucosal mRNA levels of Slfn3, SI, Glut2, and Dpp4 were all substantially higher in the rat jejunum than in colonic mucosa by a mean (SE) factor of 51.0 (13.2) for 6 rats (P < .05), 599 (99) for 8 rats (P < .01), 12.5 (5.5) for 8 rats (P < .01), and 14.0 (3.9) for 8 rats (P < .01), respectively. In IEC-6 cells, infection with adenovirus-expressing GFP-tagged Slfn3 significantly increased Slfn3 expression and Dpp4-specific activity compared with GFP-expressing virus (in 6 rats; P < .05).

Conclusions and Relevance  Taken together with our previous in vitro observations, the results suggest that small intestinal enterocytic epithelial differentiation in rats may be regulated by Slfn3 in vivo, as in vitro, and that these effects may be specific to the small intestinal enterocytic phenotype as opposed to that of the mature colonocyte. Slfn3 human orthologs may be targeted to stimulate intestinal differentiation in patients with short bowel syndrome.

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Figure 1.
Different Schlafen Proteins Exhibiting Different Patterns of Expression in the Rat Intestinal Mucosa

Transcript levels of Slfn1 (A), Slfn2 (B), Slfn3 (C), Slfn5 (D), Slfn13 (E), and Slfn14 (F) in the jejunum (Jj), upper ileum (IL1), middle ileum (IL2), lower ileum (IL3), and colon (Co) of rats were determined by quantitative reverse transcriptase–polymerase chain reaction. A significant difference is indicated if the letter above one error bar is different from that of another (P< .05). Error bars indicate standard errors.

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Figure 2.
Transcript Levels of Differentiation Markers, Which Vary Along the Length of the Rat intestinal Mucosa

Transcript levels of villin (A), Dpp4 (B), Glut2 (C), and SI (D) were measured by quantitative reverse transcriptase–polymerase chain reaction in the mucosa of the jejunum (Jj), upper ileum (IL1), middle ileum (IL2), lower ileum (IL3), and colon (Co). Ordinate scales were matched to the ordinate scales in Figure 1 to facilitate interpretation. A significant difference is indicated if the letter above one error bar is different from that of another (P < .05). Error bars indicate standard errors.

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Figure 3.
Slfn3 and Differentiation Marker Protein Levels and Dpp4 Activity Mirroring Transcript Level in Rat Intestinal Mucosa

Protein levels of Slfn3 (A), villin (B), Glut2 (C), SI (D), and Dpp4 (E) were measured in the jejunum (Jj), upper ileum (IL1), middle ileum (IL2), lower ileum (IL3), and colon (Co) of rats by Western blotting of mucosal lysates. Dpp4 enzyme activity (F) was measured in intestinal rat mucosal samples. A significant difference is indicated if the letter above one error bar is different from that of another (P < .05). Error bars indicate standard errors. RLU indicates relative luminescence units.

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Figure 4.
Slfn3 Induction Modulating Dpp4 Activity In Vitro

A, 80% confluent IEC-6 monolayers were infected with Ad-Slfn3 for 48 hours (in 6 rats). B, Ad-GFP-Slfn3 infection of IEC-6 cells increased Dpp4-specific activity in lysates from these IEC-6 monolayers (in 3 rats). Error bars indicate standard errors. RLU indicates relative luminescence units.

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