Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells

Abstract

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

Figure 1

Specificity of pAbs. (A) Amino acid sequences of the cytoplasmic domains of claudin-5/TMVCF and claudin-6. The COOH-terminal KNYV is shared by these two claudin species. Two polypeptides corresponding to these sequences were used as antigens to produce pAbs in rabbits. (B) GST fusion proteins with the cytoplasmic domains of claudin-1 to -8 as well as the cytoplasmic domain of claudin-6 lacking its COOH-terminal KNYV (see claudin-6Δ in A) were produced in E. coli. The lysates of E. coli were separated by SDS-PAGE (C.B.B. staining), followed by immunoblotting with two distinct pAbs, which were raised against the cytoplasmic domain of claudin-5/TMVCF or claudin-6, respectively. The former recognized both GST–claudin-5/TMVCF and GST–claudin-6, but not GST–claudin-6Δ, indicating that this pAb (referred to as anti–claudin-5/6 pAb) specifically bound to the COOH-terminal KNYV sequence. The latter recognized GST–claudin-6 and GST–claudin-6Δ but not GST–claudin-5/TMVCF, indicating that this pAb (referred to as anti–claudin-6 pAb) was specific for the YSTSVPHSRGPSEYPT sequence of claudin-6 (see A). (C) Anti–claudin-5/6 pAb immunofluorescently stained L transfectants expressing claudin-5/TMVCF (C5L cells) and claudin-6 (C6L cells), whereas anti–claudin-6 pAb stained only C6L cells. Bar, 10 μm.

Figure 2

Exclusive expression of claudin-5/TMVCF in endothelial cells in the brain. (a–d) Whole-mount immunostaining. Mouse 12.5-d embryos were labeled with anti–claudin-5/6 pAb (a and b), anti–claudin-6 pAb (c), or anti-PECAM pAb (d). Both anti–claudin-5/6 pAb and anti-PECAM pAb showed similar tree-like staining of blood vessels in the head portion. In contrast, anti–claudin-6 pAb yielded no signals in the head portion. (e–h) Immunofluorescence staining. Frozen sections of adult mouse brain were double labeled with anti–claudin-5/6 pAb (e) and anti–VE-cadherin mAb (f) or anti–claudin-6-pAb (g) and anti–VE-cadherin mAb (h). Since anti–claudin-6 pAb yielded no signal at all, anti–claudin-5/6 pAb staining can be considered to represent the distribution of claudin–5/TMVCF. All blood vessels in the brain were VE-cadherin–positive, and claudin-5/TMVCF was precisely colocalized with VE-cadherin. Bars: 1 mm for a, c, and d (d); 0.2 mm (b); 40 μm for e–h (h). Cln, claudin; Cad, cadherin.

Figure 3

The precise colocalization of claudin-5/TMVCF and VE-cadherin at the cell–cell borders of endothelial cells in the brain. Blood vessels in frozen sections of adult mouse brain were double stained with anti–claudin-5/6 pAb (a, green) and anti–VE-cadherin mAb (b, red). Merged image (c) revealed that both staining patterns precisely overlapped. Anti–claudin-6 pAb yielded no signals (data not shown). Bar, 10 μm. Cln, claudin; Cad, cadherin.

Figure 4

Localization of claudin-5/TMVCF in the lung. (a–f) Frozen sections of the lung were double stained with anti–claudin-5/6 pAb (a) and anti–VE-cadherin mAb (b), or anti–claudin-5/6 pAb (d) and antioccludin mAb (e). Since anti–claudin-6 pAb yielded no signals (data not shown), the anti–claudin-5/6 pAb staining can be considered to represent the distribution of claudin-5/TMVCF. Merged images (c and f) then revealed that claudin-5/TMVCF was precisely colocalized with VE-cadherin and that the claudin-5/TMVCF signals were complementary to the occludin signals. Considering that VE-cadherin and occludin were expressed exclusively in endothelial and epithelial cells, respectively, in the lungs, these findings indicated that the expression of claudin-5/TMVCF was restricted to endothelial cells. (g) Freeze–fracture replicas of the lung were labeled with anti–claudin-5/6 pAb. Note the specific labeling on the TJ strands of endothelial cells, which delineated the capillary lumen (indicated by asterisks). RC, erythrocytes; Cln, claudin; cad, cadherin. Bars: 20 μm in a–f (f); 100 nm (g).

Figure 5

Localization of claudin-5/TMVCF in the kidney and the intestine. Frozen sections of the kidney (a–h) and the intestine (i and j) were double labeled with anti–claudin-5/6 pAb (a) and antioccludin mAb (b), anti–claudin-5/6 pAb (c) and anti–VE-cadherin mAb (d), anti–claudin-6 pAb (e) and anti–VE-cadherin mAb (f), or anti–claudin-5/6 pAb (i) and antioccludin mAb (j). Since anti–claudin-6 pAb yielded no signals in the kidney (e) and the intestine (data not shown), the anti–claudin-5/6 pAb staining can be considered to represent the distribution of claudin-5/TMVCF. Claudin-5/TMVCF appeared to be expressed in a subset of blood vessels (arrows in a and i) that were occludin-negative. Occludin was concentrated at TJs in distal tubules in the kidney (arrowheads in b) and intestinal epithelial cells (arrowheads in j). In the kidney, VE-cadherin–positive intertubular capillaries (arrowheads in d and f) and glomerular capillaries (asterisk in d and f) did not express claudin-5/TMVCF, but afferent and efferent arterioles (arrows in c and d) expressed both claudin-5/TMVCF and VE-cadherin. When transverse frozen sections of the thicker artery and vein (arrows and arrowheads, respectively, in g and h) of the kidney were stained with anti–claudin-5/6 pAb, only the artery was intensely stained (g). (h) Phase–contrast image. Bars: 40 μm for a–f (f); 70 μm in g and h (h); 40 μm in i and j (j). Cln, claudin; Cad, cadherin.

Figure 6

L transfectants expressing claudin-5/TMVCF. (a and b) Immunofluorescence (a) and corresponding phase–contrast images (b) of stable L transfectants expressing claudin-5/TMVCF. Cells were stained with anti–claudin-5/6 pAb. Expressed claudin-5/TMVCF was concentrated at cell–cell borders as planes (arrows). (c) Freeze–fracture images of cell–cell contact planes of stable L transfectants expressing claudin-5/TMVCF. Well-developed TJ strand/groove networks were reconstituted. In these reconstituted TJs, most TJ particles were recovered on the E-face; in the E-face (E), the grooves were completely occupied by chains of particles (arrow in upper inset), whereas only particle-free ridges were observed on the P-face (P) (arrow in lower inset). Bars: 15 μm in a and b (b); 200 nm (c); 50 nm (insets).