Overexpression of claudin-3 in a breast cancer cell line

The endogenous overexpression of claudin-3 in MCF-7 cells changes the cellular physiology of this breast cancer cell line. Suppression of claudin-3 expression with siRNA transfection of MCF-7 cells, which endogenously overexpress claudin-3, causes reduced growth rate and reduced transepithelial resistance. Claudin-3 immunoreactivity is found in membranous, cytosolic, nuclear, and cytoskeletal fractions of MCF-7 cells, which overexpress claudin-3.
Claudin proteins have a major role in tight junction function of regulating the diffusion of specific ions between epithelial cells. Claudins also interact with a tight junction cytoplasmic scaffold, a supramolecular complex that contains links to cytoskeletal proteins, signaling proteins with PDZ binding domains, regulators of membrane traffic, transcription factors, kinases, phosphatases, and trimeric G-proteins. Although cancer researchers have previously reported the delocalization of some claudins away from the plasma membrane (and tight junctions) of some cancer cells, previous publications have not shown a possible truncated claudin in the cytosol and previous publications have not offered explanations as to how an integral membrane protein, with four hydrophobic domains, can dissolve in the cytosol and translocate to the nucleus.
We assessed the levels of claudin-3 protein in a panel of breast and ovarian cancer cell lines. Two breast cancer cell lines, MCF-7 and MDA-MB-415, overexpressed claudin-3. We used siRNA to suppress claudin-3 to near normal levels in MCF-7 cells and observed a decrease in the cellular growth rate and transepithelial resistance. Because the incomplete suppression of claudin-3 levels caused relatively dramatic changes in measures of cellular physiology, we investigated the subcellular location of claudin-3 in untransfected MCF-7 cells (elevated levels of claudin-3) to determine if claudin-3 was exclusively located in cell membranes. Claudin-3 was apparent in the membranous, cytosolic, cytoskeletal, and nuclear MCF-7 fractions isolated by detergent extractions.

See Figure 4 in the Poster. Claudin-3 immunoreactivity in MCF-7 cells (overexpressing claudin-3) was apparent in Membranous (M), cytosolic ©, cytoskeletal (Csk), and nuclear fractions (N; n=6). The membrane fraction contained the most intense signal at the molecular weight of claudin-3, 23 kDa. These data suggested that most of the claudin-3 was functional in the expected location and corresponded to the relatively high TER values we observed for MCF-7 monolayers (e.g. >400 ohm x cm2). The cytoplasmic fraction contained much less immunoreactivity at 23kDa, but instead contained a lower molecular weight signal, possibly representing a truncated form of claudin-3. The cytoskeletal and nuclear fractions contained relatively intense immunoreactivity only at 23 kDa.
The delocalization of claudins away from the plasma membrane, the site of tight junctions, may indicate a role for claudin proteins in novel cell signaling pathways involving proteins of the TJ cytoplasmic scaffold.

We would like to thank Southwestern University, the Andrew W. Mellon Foundation, and the Joe and Jessie Crump Fund at JPMorgan Chase Bank for funding this research.