Ras mutation impairs epithelial barrier function to a wide range of nonelectrolytes

Area of Science:

• Cell Biology
• Renal Physiology
• Molecular Biology

Background:

• Ras mutations are known to affect epithelial architecture and polarity.
• The specific impact of ras mutations on the integrity and function of tight junctions is not well understood.

Purpose of the Study:

• To investigate how mutated ras affects tight junction permeability in LLC-PK1 renal epithelia.
• To determine the size selectivity of the altered paracellular pathway.
• To explore the molecular mechanisms, including tight junction protein and signaling pathway involvement.

Main Methods:

• Transfection of a valine-12 mutated ras construct into LLC-PK1 cells.
• Measurement of transepithelial permeability to various solutes (D-mannitol, PEG, methylated dextrans) and transepithelial electrical resistance.
• Analysis of tight junction protein (claudins, occludin) and extracellular signal-regulated kinase-2 (ERK-2) phosphorylation levels.

Main Results:

• Mutated ras expression led to increased permeability to D-mannitol and polyethylene glycol, but not large dextrans, indicating size-selective leakiness.
• Transepithelial electrical resistance decreased, suggesting reduced paracellular permeability to ions like NaCl.
• Claudin-2 levels decreased, while occludin and other claudins (1, 4, 7) increased; ERK-2 phosphorylation was elevated.

Conclusions:

• Ras mutations selectively alter tight junction permeability in renal epithelia, creating a barrier leaky to smaller solutes but not large ones.
• These changes in barrier function are associated with specific alterations in tight junction protein expression and MAPK signaling.
• The selective permeability changes induced by ras mutations may play a role in epithelial tumorigenesis.