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Am J Physiol Gastrointest Liver Physiol 288: G1159-G1169, 2005. First published February 3, 2005; doi:10.1152/ajpgi.00407.2004
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MUCOSAL BIOLOGY

Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells

Xin Guo,1,3,* Jaladanki N. Rao,1,3,* Lan Liu,1,3 Tongtong Zou,1,3 Kaspar M. Keledjian,1,3 Dessy Boneva,1,3 Bernard S. Marasa,2,3 and Jian-Ying Wang1,2,3

Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland

Submitted 8 September 2004 ; accepted in final form 31 January 2005

Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by {alpha}-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca2+ concentration ([Ca2+]cyt), because either increased or decreased [Ca2+]cyt did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by ~70% following polyamine depletion, whereas its protein half-life was reduced from ~120 min in control cells to ~75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.

epithelial barrier; paracellular permeability; cdx2 gene; claudin; zonula occludens-1; zonula occludens-2; ornithine decarboxylase; protein stability


Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail:jwang{at}smail.umaryland.edu)




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