Role of zonula occludens in reduced epithelial cell proliferation by hypertonic stress

Abstract

In addition to their role as epithelial barriers, tight junctions (TJs) function as dynamic signaling reservoirs involved in regulating the proliferative potential of epithelial cells. Hypertonic stress reduces cell proliferation and alters cell morphology. Here, we examined whether TJ zonula occludens (ZO) modulate cell proliferation under conditions of chronic hypertonic stress. As revealed by microscopy analysis and FACS, proliferation of spontaneously immortalized renal principal collecting duct (CD) mCCDcl1 cells is strongly dependent on cell confluency when grown under isotonic conditions (300 mOsmol/kg). Low levels of cell proliferation were accompanied by a reduced S phase and increased G0/G1 phase. Real‐Time PCR and Western blot analysis showed that ZO‐3 abundance increased with cell confluency while ZO‐1 and ZO‐2 abundance was unaffected. Confocal microscopy analysis revealed that expression of all three ZO isoforms at the cell surface increased with cell confluency. All observed changes were enhanced in slowly proliferating cells adapted to NaCl‐hypertonic medium (500 mOsmol/kg). Under isotonic conditions, knockdown of ZO‐1 and ZO‐2 by siRNA increased G1 phase, while knockdown of ZO‐3 promoted cell cycle progress. Similar results were obtained in cells habituated to hypertonic medium. These data reveal differential effects between ZO isoforms on renal CD cell proliferation. In addition, they indicate that ZO submembrane recruitment may influence slow cell proliferation under hypertonic conditions.