Expression of junctional proteins in choroid plexus epithelial cell lines: a comparative study

Abstract

Background: There is an increasing interest in using choroid plexus (CP) epithelial cell lines to study the properties of the blood-cerebrospinal fluid barrier (BCSFB). Currently, there are three major CP-derived cell lines available. Z310 and TR-CSFB3, two immortalized cell lines carrying the simian virus 40 large T-antigen gene, were derived from rat CP epithelium, whereas the CPC-2 cell line was derived from human CP carcinoma. Although these cell lines have previously been used in various functional studies, the expression of adherens junction (AJ) and tight junction (TJ) proteins in these epithelial cells has not been systematically studied. Accordingly, in the present study, we sought to characterize the expression of these junctional proteins in these three cell lines.

Methods: The cells were grown in six-well cell culture plates. Reverse-transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry were used to characterize the expression of AJ and TJ proteins in the CP cell lines.

Results: Z310 and TR-CSFB3 cells expressed a TJ protein, occludin, and its cytosolic binding partner, zonula occludens 1, as well as an AJ protein, E-cadherin, and beta-catenin, a cytoplasmic protein that interacts with E-cadherin. However, the expression of occludin and E-cadherin in TR-CSFB3 cells at both the mRNA and protein level was weaker than that found in Z301 cells. The immunocytochemical analysis also demonstrated that the staining pattern for these junctional proteins in TR-CSFB3 cells was discontinuous and the staining intensity was weaker than that observed in Z310 cells. The message for claudin 1 and claudin 2 was expressed at low levels in TR-CSFB3 cells and these cells were weakly immunopositive for claudin 1. In comparison, the message for these TJ proteins could not be detected in Z310 cells. CPC-2 cells expressed occludin, which was localized to areas of cell-cell contact, but the staining pattern for this TJ protein was found to be variable and irregular. Although CPC-2 cells expressed mRNA for claudin 1, claudin 2, and claudin 11, only claudin 1 was expressed at the protein level and it was localized to the nuclei rather than to areas of cell-cell contact. An AJ protein, E-cadherin, was also found to be mislocalized in CPC-2 cells, even though its cytosolic binding partner, beta-catenin, was restricted to areas of cell-cell contact, as in normal CP.

Conclusion: The three CP cell lines analyzed in this study vary considerably with regard to the expression of AJ and TJ proteins, which is likely reflected by different barrier properties of these in vitro models of BCSFB.

Figure 1

Schematic representation of AJ and TJ complexes in choroidal epithelial cells. AJs are formed by E-cadherin, a single pass transmembrane protein that exhibits Ca²⁺-dependent homophilic interactions. The intracellular domain of E-cadherin binds to β-catenin, which binds to α-catenin, which in turn interacts with actin, and this catenin-mediated anchorage of E-cadherin to the actin cytoskeleton is required for strong cell-cell adhesion. Occludin and the members of the family of claudin proteins are the major constituents of TJs. Occludin and claudins are connected to the actin filaments through cytoplasmic adaptor protein, ZO-1.

Figure 2

RT-PCR analysis of expression of junctional proteins in CP cell lines. A. Expression of TJ and AJ proteins in Z310 and TR-CSFB3 cells, two immortalized cell lines derived from rat CP epithelium. B. Expression of TJ and AJ proteins in the CPC-2 cell line derived from human CP carcinoma. The number of cycles of amplification is shown for each lane. Cldn 1, Cldn 2, Cldn 11, Ocln, and E-cdh are claudin 1, claudin 2, claudin 11, occludin, and E-cadherin, respectively. rCP and hCP are rat and human CP tissues used as a positive control.

Figure 3

Western blot analysis of expression of junctional proteins in CP cell lines. A, B, and C. Expression of TJ proteins, claudin 1, claudin 2, and occludin. D. Expression of AJ protein, E-cadherin. Z310 and TR-CSFB3 cell lines were derived from rat CP epithelium, whereas the CPC-2 cell line was derived from human CP carcinoma. The amount of protein loaded is shown for each lane. rCP and hCP are rat and human CP tissues used as a positive control.

Figure 4

Immunocytochemical analysis of expression of junctional proteins in CP cell lines. A and B. Z310 and TR-CSFB3 cell lines derived from rat CP epithelium. C. CPC-2 cell line derived from human CP carcinoma. Scale bars = 10 μm.