Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

Abstract

Background In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells. Methods MDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison. Results All cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport. Conclusions The present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.

A Microfluidic In Vitro Three-Dimensional Dynamic Model of the Blood–Brain Barrier to Study the Transmigration of Immune Cells

“…Hence, it is mandatory that a high-throughput and low-cost alternative for excessive animal testing demonstrates Pg-p expression. However, it was previously demonstrated that wild-type hCMEC/D3 cells exhibit a low expression of Pg-p and low junctional tightness under routine culture conditions [ 56 ]. For this, we studied the percentage of positive cells expressing P-glycoprotein (CD243), PECAM-1 (CD31), ICAM-1 (CD54), transferrin receptor (CD71) and CD45 in the static BBB model as well as the dynamic TripleB model using flow cytometry.…”

Characterization of P‐glycoprotein orthologs from human, sheep, pig, dog, and cat

“…A potential drawback of tagging a protein is that the tag may affect the structure of the protein, and thereby, alter its function. However, to reduce the potential effects of the tag on protein behavior, the tag was attached to the C‐terminus of the protein as has been done in previous reports studying P‐gp (Chen et al, 2001; Chen & Simon, 2000; Gericke et al, 2021). Additionally, we included a linker to tag the P‐gp orthologs to permit some flexibility in the structure and minimize any steric interference on protein function.…”

Genetic disorders and genetic manipulation at the blood-brain barriers

“…There can be concerns when inducing expression of proteins that abnormal cellular distributions may occur. Gericke et al [21] have examined this for P-glycoprotein in brain endothelial cell lines in vitro.…”