Glucocorticoid-mediated Regulation of Tight Junction Organization in Human Endometrial Cancer Cells

Communication between cells is important to multi-cellular organisms for processes such as cell survival, development, proliferation, differentiation, adhesion, and migration. Junctional complexes that are comprised of intracellular and intercellular protein components govern these cell-cell interactions. The loss of junctional integrity is implicated in the development and progression of cancer. Although intracellular signaling molecules such as phosphatases and kinases and extracellular signaling molecules such as steroids hormones and growth factors have been shown to regulate cellular junctions, the exact mechanism by which these junctional complexes are assembled, disassembled, and maintained is largely unknown. Glucocorticoids have been shown to regulate junctional complexes in a variety of tissue types. However, their effect on the human endometrium, a tissue that is primarily regulated by steroid hormones, has not been evaluated. Using human endometrial cancer cells, the present work details the effects of glucocorticoids on junctional organization and delineates glucocorticoid-mediated signaling cascades that regulate protein complexes within these cellular junctions. We show that treatment of Ishikawa cells, a well-established human endometrial cancer cell line, with dexamethasone (DEX), a synthetic glucocorticoid, results in organization of tight junctional proteins ZO-1 and occludin to the cell periphery. This effect does not involve changes in cell cycle progression and is attenuated upon treatment with RU-486, a glucocorticoid receptor (GR) antagonist. Furthermore, DEX treatment stimulates the interaction between ZO-1 and occludin that occurs before the localization to the cellular membrane. Total protein expression levels of the GR, ZO-1, occludin and other junctional proteins such as E-cadherin and beta-catenin do not change. The DEX-dependent increase in interaction between ZO-1 and occludin and the subsequent localization of the proteins at the cell periphery is a co-dependent process that requires the expression of both ZO-1 and occludin. An evaluation of various kinases known to regulate interactions between ZO-1 and occludin reveals that activation of AMP-activated protein kinase (AMPK), a serine/threonine kinase, is required for the DEX-induced organization of the ZO-1 and occludin to the cell periphery, as siRNA mediated knockdown of AMPK reverses the effect. Inhibition of c-Src, a non-receptor protein tyrosine kinase, increases the localization of ZO-1 and occludin to the cell membrane where as the inhibition of protein kinase C (PKC), a serine/threonine kinase, disrupts the membranous organization of the two proteins. Activation of AMPK and inhibition of c-Src and PCK also results in changes in interaction between ZO-1 and occludin. Gene expression profile of Ishikawa cells reveals that Kruppel-like factor (KLF9), a transcription factor that is associated with a differentiated endometrium, is significantly up-regulated upon treatment with DEX. Clinical data suggests that loss-of-expression of KLF9 leads to endometrial carcinogenesis. We show that knockdown of KLF9 disrupts the DEX-induced organization of ZO-1 and occludin to the cell periphery and attenuates the interactions between the two proteins. Similarly, gene expression level of GRIM-19, a tumor-suppressor and a known inhibitor of c-Src kinase is also increased upon treatment with DEX. si-RNA mediated knockdown of GRIM-19 disrupts DEX-induced organization of ZO-1 and occludin to the cell periphery and attenuates the interactions between the two proteins. The effect is somewhat reversed upon addition of SRC-inhibitor1 in cells where GRIM-19 is knocked-down. Taken together, our work shows that DEX confers a differentiated phenotype with a lower tumorigenic potential in human endometrial cancer cells by increasing gene expression levels of KLF9 and GRIM-19 that leads to alterations in the dynamics of ZO-1 and occludin through a mechanism that involves kinases associated with regulating interactions between the two tight junctional proteins.

Advisor: Firestone, Gary