Abstract
The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity.
Highlights
Intercellular transfer of proteins is an integral part of communication between cells, involving mechanisms such as tunneling nanotubes bridging neighboring cells or release and binding of protein-containing membrane microparticles and extracellular vesicles[1]
We investigated whether intercellular Pgp transfer as reported for cancer cells is a physiological defense mechanism of brain capillary endothelial cells that form the blood-brain barrier (BBB)
To study whether Pgp-EGFP-transfer occurs in human brain capillary endothelial cells, hCMEC/D3 cells (Pgp-recipient cells) were co-cultured with an equal number of hCMEC/D3-MDR1-EGFP cells (Pgp-donor cells)
Summary
Intercellular transfer of proteins is an integral part of communication between cells, involving mechanisms such as tunneling nanotubes bridging neighboring cells or release and binding of protein-containing membrane microparticles and extracellular vesicles[1]. By using hCMEC/ D3-MDR1-EGFP cells (Pgp-donor cells) co-cultured with hCMEC/D3 wildtype cells (Pgp-recipient cells), we demonstrate intercellular Pgp transfer and its functional relevance for the recipient cells, induction of this process by the major antiepileptic drug (AED) valproate, and possible involvement of inhibition of histone deacetylases (HDACs) in this drug effect. These findings have important implications for BBB functioning and resistance to therapy
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