Mesenchymal Stromal Cells Accelerate Epithelial Tight Junction Assembly via the AMP-Activated Protein Kinase Pathway, Independently of Liver Kinase B1

الملخص EN

Background.

Mesenchymal stromal cells (MSC) are fibroblast-like multipotent cells capable of tissue-repair properties.

Given the essentiality of tight junctions (TJ) in epithelial integrity, we hypothesized that MSC modulate TJ formation, via the AMP-activated kinase (AMPK) pathway.

Liver kinase-β1 (LKB1) and Ca2+-calmodulin-dependent protein kinase kinase (CaMKK) represent the main kinases that activate AMPK.

Methods.

The in vitro Ca2+ switch from 5 μM to 1.8 mM was performed using epithelial Madin-Darby canine kidney (MDCK) cells cultured alone or cocultured with rat bone marrow-derived MSC or preexposed to MSC-conditioned medium.

TJ assembly was measured by assessing ZO-1 relocation to cell-cell contacts.

Experiments were conducted using MDCK stably expressing short-hairpin-RNA (shRNA) against LKB1 or luciferase (LUC, as controls).

Compound STO-609 (50 μM) was used as CaMKK inhibitor.

Results.

Following Ca2+ switch, ZO-1 relocation and phosphorylation/activation of AMPK were significantly higher in MDCK/MSC compared to MDCK.

No difference in AMPK phosphorylation was observed between LKB1-shRNA and Luc-shRNA MDCK following Ca2+ switch.

Conversely, incubation with STO-609 prior to Ca2+ switch prevented AMPK phosphorylation and ZO-1 relocation.

MSC-conditioned medium slightly but significantly increased AMPK activation and accelerated TJ-associated distribution of ZO-1 post Ca2+ switch in comparison to regular medium.

Conclusions.

MSC modulate the assembly of epithelial TJ, via the CaMKK/AMPK pathway independently of LKB1.