Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

Area of Science:

• Cell Biology
• Molecular Biology
• Renal Physiology

Background:

• Na(+), K(+)-ATPase is a known ion pump and also acts as a cell-cell adhesion molecule and digitalis receptor.
• Prolonged blockage of Na(+), K(+)-ATPase by ouabain (OUA) causes cell detachment.
• Understanding the mechanisms of tight junction disassembly is crucial for renal epithelial cell integrity.

Purpose of the Study:

• To investigate the cellular mechanisms of tight junction (TJ) disassembly induced by toxic ouabain (OUA) levels in MDCK cells.
• To identify the signaling pathways involved in OUA-induced TJ disruption.
• To determine the effects of OUA on TJ protein expression and mRNA levels.

Main Methods:

• Exposure of MDCK cells to toxic OUA levels (≥300 nM).
• Measurement of transepithelial electrical resistance (TER) and use of kinase inhibitors (EGFR, cSrc, ERK1/2).
• Analysis of TJ protein (CLDN 2, CLDN-4, OCLN, ZO-1) and mRNA levels.

Main Results:

• Toxic OUA levels progressively decreased TER, a process delayed by EGFR, cSrc, and ERK1/2 inhibitors.
• TER decrease was linked to internalization and degradation of CLDN 2, CLDN-4, OCLN, and ZO-1.
• OUA altered mRNA levels of TJ proteins, with differential sensitivity to ERK1/2 inhibition.
• CLDN-2 protein and mRNA reduction was independent of the EGFR-cSrc-ERK1/2 pathway.

Conclusions:

• Toxic OUA activates the EGFR-cSrc-ERK1/2 pathway, leading to endocytosis and degradation of TJ proteins, causing cell detachment.
• Specific TJ proteins (CLDN-4, OCLN, ZO-1) degradation and mRNA changes are mediated by this pathway.
• CLDN-2 is affected by OUA independently of the EGFR-cSrc-ERK1/2 pathway, highlighting complex regulatory mechanisms.