Rab25 regulates integrin expression in polarized colonic epithelial cells

Moorthy Krishnan^a, Lynne A. Lapierre^a,b, Byron C. Knowles^a,c, and James R. Goldenring^a,b,c,d,1

^aSection of Surgical Sciences and the Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN 37232 ^cDepartment of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232 ^dVanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232 ^bNashville Veterans Affairs Medical Center, Nashville, TN 37212

Monitoring Editor: Benjamin Margolis

Abstract: Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.

M.K. performed experiments, prepared figures, and cowrote the manuscript; L.A.L. performed experiments and reviewed the manuscript; B.C.K. performed experiments and cowrote the manuscript; J.R.G. designed experiments, prepared figures, and cowrote the manuscript.

None of the authors has any conflict of interest.

¹Address correspondence to: James R. Goldenring (jim.goldenring{at}vanderbilt.edu).

Abbreviations: EGF, epidermal growth factor • ETV4, translocation variant 4 • KD, knockdown • RT-PCR, reverse transcription-PCR • shRNA, short hairpin RNA • SI, sucrase Isomaltase • TER, transepithelial resistance