Gastroenterology

Gastroenterology

Volume 120, Issue 6, May 2001, Pages 1381-1392
Gastroenterology

Alimentary Tract
Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation☆,☆☆

https://doi.org/10.1053/gast.2001.24044Get rights and content

Abstract

Background & Aims: Phosphatidylinositol 3-kinase (PI3K), an important mediator of intracellular signal transduction, has been shown to affect proliferation, differentiation, and apoptosis in a number of cells; the role of PI3K in intestinal cell differentiation is not known. Methods: The effect of PI3K inhibition on enterocyte-like differentiation of the human colon cancer cells, HT29 and Caco-2, was assessed using complementary approaches (i.e., chemical inhibition with wortmannin, transfection with a dominant negative p85 mutant, or overexpression of the tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 [PTEN]). Brush-border enzyme (intestinal alkaline phosphatase [IAP] and sucrase) activities, IAP messenger RNA levels, and IAP promoter induction were measured. Results: The PI3K inhibitor, wortmannin, in combination with sodium butyrate, synergistically induced IAP and sucrase enzyme activities and IAP messenger RNA levels in a time- and dose-dependent fashion. Consistent with these results, cotransfection using the dominant negative mutant of p85 (Δp85) induced IAP promoter activity. Moreover, overexpression of PTEN, which antagonizes PI3K, significantly augmented the induction of IAP enzyme activity in HT29 and Caco-2 cells treated with sodium butyrate and in spontaneously differentiated Caco-2 cells. Conclusions: Our results show that inhibition of PI3K significantly enhances enterocyte-like differentiation of HT29 and Caco-2 cells. Taken together, our findings suggest a contributory role for the PI3K/PTEN pathway in intestinal cell differentiation.

GASTROENTEROLOGY 2001;120:1381-1392

Section snippets

Materials

Wortmannin, NaBT, phosphatidylinositol, and phosphatidylserine were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-PI3K p85α (06-195) and a monoclonal antibody to phosphotyrosine (05-321) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against PTEN (sc 6818) and actin (sc 1616) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Thin layer chromatography plates of Silica Gel were from Whatman Ltd. (Hillsboro, OR). The pSVhiap-2-Luc, encoding the

Wortmannin induces IAP and sucrase enzyme activities in HT29 cells

The human colon cancer cell line HT29 has been extensively used as a model of intestinal cell differentiation.30, 41, 42, 43, 44 Treatment of HT29 cells with the short chain fatty acid NaBT results in enterocyte-like differentiation by 24 to 48 hours of treatment, as noted by induction of the brush-border enzymes IAP and sucrase.30 To determine whether the PI3K pathway plays a role in the differentiation of intestinal cells, we first treated HT29 cells with the PI3K inhibitor wortmannin (1

Discussion

The cellular signaling pathways that are required for intestinal cell differentiation are largely undefined. Delineating these mechanisms will provide a better understanding of normal intestinal homeostasis and the processes contributing to intestinal cell differentiation. In our present study, we identify a role for the PI3K/PTEN pathway in the modulation of enterocyte-like differentiation using well-characterized in vitro models of intestinal cell differentiation. Treatment of the human colon

Acknowledgements

The authors thank Drs. Aubrey Thompson and Mark Hellmich for their helpful suggestions throughout the course of their studies. In addition, the authors thank Mr. Tatsuo Uchida for statistical analyses and Eileen Figueroa and Karen Martin for manuscript preparation.

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    ☆

    Address requests for reprints to: B. Mark Evers, M.D., Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536. e-mail: [email protected]; fax: (409) 747-4819.

    ☆☆

    Supported by grants RO1 DK48498, R01 AG10885, P01 DK35608, and T32 DK07639 from the National Institutes of Health.

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    Copyright © 2001 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.