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Differential regulation of junctional complex assembly in renal epithelial cell lines
Shobha Gopalakrishnan1, Mark A Hallett, Simon J Atkinson
1Department of Medicine, Indiana University Medical Center, Indianapolis, IN 46202-5181, USA.
Abstract:
Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and beta-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.
Insights
Calpeptin differentially affects epithelial cell junctions. This compound disrupts stress fibers and cell-cell adhesion in MDCK cells but promotes them in LLC-PK cells, impacting cell line-specific signaling.
Area of Science:
- Cell biology
- Molecular signaling
- Epithelial cell biology
Background:
- Tight junctions and adherens junctions are crucial for epithelial cell integrity.
- Signaling pathways regulating these junctions are complex and under investigation.
- Calpeptin is known to influence cytoskeletal dynamics and Rho-GTPase signaling in fibroblasts.
Purpose of the Study:
- To investigate the effects of calpeptin on stress fiber and junctional complex assembly in two distinct epithelial cell lines (MDCK and LLC-PK).
- To elucidate the cell line-specific responses to calpeptin treatment regarding cytoskeletal organization and cell-cell adhesion.
Main Methods:
- Treatment of Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells with calpeptin.
- Analysis of stress fiber formation and Rho GTPase activity.
- Immunofluorescence microscopy to assess the localization of tight junction proteins (zonula occludens-1, occludin) and adherens junction proteins (E-cadherin, beta-catenin, p120ctn).
- Measurement of tight junction permeability and paracellular permeability.
Main Results:
- Calpeptin induced stress fiber disassembly and inhibited Rho GTPase activity in MDCK cells.
- Conversely, calpeptin augmented stress fiber formation in LLC-PK cells.
- In MDCK cells, calpeptin caused displacement of ZO-1 and occludin from cell-cell junctions and reduced phosphotyrosine on ZO-1/ZO-2, without affecting tight junction permeability.
- Calpeptin increased paracellular permeability in LLC-PK cells despite no effect on tight junction assembly.
- Adherens junction assembly was modulated in MDCK cells (E-cadherin, beta-catenin redistribution; reduced p120ctn association) but not in LLC-PK cells.
Conclusions:
- Calpeptin exhibits differential effects on stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells.
- The regulatory pathways governing cytoskeletal organization and cell-cell adhesion in epithelial cells are likely cell line-specific.
- Calpeptin serves as a valuable tool for dissecting these cell-specific signaling mechanisms.