Role of the Cldn6 cytoplasmic tail domain in membrane targeting and epidermal differentiation in vivo

Abstract

It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CDelta187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CDelta187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.

FIG. 1.

Inv-CΔ187 transgenic mice. The tailless Cldn6 (CΔ187) mutant was created by deleting the C-terminal cytoplasmic tail domain after amino acid 187, leaving four residues next to the fourth transmembrane-spanning region (A). The protein sequence of Cldn6 is shown with the transmembrane-spanning regions enclosed within a box, the CXXC motifs underlined, and the truncation at amino acid 187 indicated (B). Transgenic mice were created using the Inv promoter to drive the expression of CΔ187 to the suprabasal cells of the epidermis, where TJs are localized (C), and transgenic mice were identified using Cldn6 forward primers (FP) and FLAG reverse primers (RP) (585 bp) (D) (positions are marked with red arrows in C). Transgene localization was restricted to the upper spinous and granular layers of the transgenic epidermis as visualized by immunohistochemistry using anti-FLAG antibodies (E); however, there was a prominent cytoplasmic accumulation (F), presumably due to inefficient membrane targeting. Immunoblot analysis with anti-FLAG antibodies confirmed a ∼19.5-kDa band in the Inv-CΔ187 (TG) and not the wild-type (WT) back skin samples using anti-GAPDH as a loading control (G). HaCat cells transfected with Inv-CΔ187-FLAG also showed significant cytoplasmic accumulation, confirming a defect in the ability of CΔ187 to target to the membrane both in vivo and in vitro (H). The CΔ187 transgenic mice are easily identifiable by their coat appearance, which is not sleek, compared to that of the wild-type, a phenotype that persists throughout life (I). SV40, simian virus 40.

FIG. 2.

Histological abnormalities in the Inv-CΔ187 epidermis. A histological analysis of samples of back skin from Inv-CΔ187 mice compared to those of their age-matched wild-type counterparts revealed no obvious differences in the epidermal morphology of the Inv-CΔ187 mice (left panel) compared to that of wild-type mice (right panel) as newborns (A) or at 1 week of age (B). However, after 1 week of age, a progressive thickening of the transgenic epidermis, rather than the normal thinning pattern of the wild-type epidermis, was seen (C and D). After 3 weeks of age and persisting throughout life, a thick epidermis was present in the transgenic samples, which was characterized by an increase in the number of spinous layers, wherein cells exhibited some degree of disorganization, abnormalities in the upper differentiating layers including parakeratosis with the prevalent appearance of nuclei as well as an obvious but improperly packed granular layer, and a thicker stratum corneum (E).

FIG. 3.

Perturbation of early markers of epidermal differentiation. Early differentiation markers in back skin samples from newborn and 1-month-old Inv-CΔ187 transgenic mice were evaluated by immunofluorescence and compared to those of their age-matched wild-type littermates. Consistent with our histological observations, in the newborn transgenic mouse epidermis (left panel), there was no deviation from wild-type expression of K15 (A), K5 (B), or K14 (C), where expression was restricted to the basal layer. However, in the samples from 1-month-old animals (right panel), K15 expression, while limited to the basal layer, became sporadic in transgenic samples rather than uniform, as seen in the wild type (A). Expression of K5 (B) and K14 (C) extended beyond the usual/wild-type basal layer through all the suprabasal layers of the transgenic epidermis.

FIG. 4.

Aberrance of later and terminal differentiation. Markers of later and terminal epidermal differentiation in samples of back skin from age-matched wild-type and Inv-CΔ187 mice were also evaluated by immunohistochemical analysis. Concurrent with the expression of early epidermal differentiation markers, there was no apparent modification in the expression of K1 (A), involucrin (B), filaggrin (C), loricrin (D), or TGase-3 (E) in the newborn transgenic epidermis compared to the wild-type epidermis (left panel). However, in the samples from 1-month-old animals (right panel), K1 showed a broadened expression pattern (A), and a similarly expanded expression compartment in the transgenic epidermis was observed for various structural proteins including involucrin (B), filaggrin (C), loricrin (D), and TGase-3 (E).

FIG. 5.

Proliferation in the Inv-CΔ187 epidermis. To address the potential proliferation defect observed in the Inv-CΔ187 transgenic epidermis, immunostaining for proliferation-associated markers, including K6, K17, and Ki67, was performed. Back skin samples from 1-month-old Inv-CΔ187 transgenic mice revealed that K6 (A) and K17 (B) expression was precociously localized to the basal to suprabasal layers of the transgenic epidermis (left panel), compared to that of age-matched wild-type animals, while there was no interfollicular expression in the wild-type epidermis (right panel). In addition, the Inv-CΔ187 transgenic epidermis was characterized with Hoechst-stained nuclei in the upper layers (D), and not only was the number of Ki67-positive basal cells increased by about fourfold (P < 0.001 using the Student's t test) (E), but a significant proportion of Ki67-positive cells were also found in the suprabasal cell layers (C).

FIG. 6.

Cytoplasmic accumulation of Cldn proteins in the Inv-CΔ187 epidermis. Given the complex profile of Cldn TJ molecules in epithelial cells, we systematically analyzed the expression of Cldn1, Cldn6, Cldn10, Cldn11, and Cldn18 in back skin samples from 1-month-old Inv-CΔ187 transgenic mice and compared it to that of their age-matched wild-type counterparts. In the transgenic epidermis, Cldn6, Cldn10, Cldn11, and Cldn18 were expressed in a clearly expanded zone (A and B [higher magnification]). Cldn1 is expressed in the basal to suprabasal layers of the wild-type epidermis, while the basal layer of the transgenic epidermis was essentially devoid of Cldn1 expression. Reminiscent of the expression of the transgene product (Fig. 1F), Cldn6, Cldn10, Cldn11, and Cldn18 localization shifted to various degrees from the membrane of wild-type mice to the cytoplasm of transgenic mice (B). Altered localization of Cldn1 expression to the cytoplasm was less pronounced than that of the other Cldn proteins, and the localization of occludin remained membranous.

FIG. 7.

Activation of an unfolded protein response pathway in the CΔ187 epidermis. In an attempt to explain the cytoplasmic accumulation of Cldn proteins observed in the Inv-CΔ187 transgenic epidermis, the potential activation of a pathway involving protein folding was investigated by RT-PCR of RNA extracted from the back skin of Inv-CΔ187 transgenic (TG) mice and compared to that of the wild type (WT). There was up-regulated expression of various genes involved in protein folding in the Inv-CΔ187 epidermis compared to the wild type, including Atf4, Atf6, Eif2ak4, Eif2s1, Ern1, and Hspa5.