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Molecular and Cellular Biology, August 2006, p. 5876-5887, Vol. 26, No. 15
0270-7306/06/$08.00+0     doi:10.1128/MCB.02342-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of the Cldn6 Cytoplasmic Tail Domain in Membrane Targeting and Epidermal Differentiation In Vivo

Azadeh Arabzadeh,^1,2 Tammy-Claire Troy,¹ and Kursad Turksen^1,2,3,4*

Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada,¹ Department of Cellular and Molecular Medicine,² Department of Medicine, Divisions of Dermatology and Endocrinology,³ Department of Obstetrics, Gynaecology, Division of Reproductive Endocrinology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada⁴

Received 7 December 2005/ Returned for modification 4 January 2006/ Accepted 12 May 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (C187) 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-C187 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Claudins (Cldns) comprise a family of integral membrane proteins involved in the formation of tight junction (TJ) fibrils, which are responsible for the formation and maintenance of the epidermal permeability barrier (EPB) (36, 38, 40, 42). Recent studies indicate that Cldn6 overexpression (34, 40) and Cldn1 deletion mutants (15) are associated with EPB defects in vivo and that the level of Cldn expression appears to be crucial for EPB integrity and function (34, 38, 40). Knowledge of the overall structure-function of the Cldn family of proteins is limited; however, they are known to have three distinct and characteristic functional domains: (i) four transmembrane-spanning regions, (ii) two extracellular loops responsible for permeability barrier formation within the paracellular space and specific ion selectivity residing within the first external loop (8, 43, 44), and (iii) a cytoplasmic C terminus that functions to anchor to the cytoskeleton, apparently through scaffolding molecules such as zonula occludens 1 (ZO-1) (12, 22, 36, 38, 42, 43). It has been demonstrated that PDZ-binding sequences at the C terminus of the Cldn cytoplasmic tail are responsible for their association with other PDZ domain proteins (16, 19, 22). However, although predicted to be important, the role of the tail domain in the targeting of Cldn proteins to the membrane as well as the regulation of their function and stability in the stratified epithelium have not been demonstrated.

In this study, we investigated the consequences of expressing a tailless Cldn6, namely, C187, in the suprabasal layer of the mouse epidermis. Expression of C187 resulted in the accumulation of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm and elicited histological and biochemical perturbations in epidermal proliferation and differentiation that were evident after 1 week of age and that persisted throughout life. These results confirm the importance of the cytoplasmic tail domain of Cldn molecules in membrane targeting and hence their function in TJs in vivo.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of transgenic mice. Inv-C187-FLAG transgenic mice were generated by following the same strategy as that used for the generation of our full-length Inv-Cldn6 transgenic mice described previously (40). Briefly, the FLAG epitope tag (Sigma-Aldrich) was fused to mutant tailless mouse Cldn6 by PCR, and C187-FLAG was subcloned into the pCRII vector and sequenced for verification. The lacZ insert of the pInv plasmid (H3700-pL2) (6, 7) was replaced with the C187-FLAG coding sequence to create the Inv-C187-FLAG expression vector. A 585-bp fragment containing C187-FLAG cDNA was introduced into the NotI site of the Inv cassette, and the resultant construct was designated pInv-C187-FLAG. SalI digestion was used to excise the plasmid vector by releasing the Inv promoter as well as the downstream Cldn6/simian virus 40 poly(A) DNA sequence, and purification was done by using the QIAamp tissue kit (QIAGEN) according to the manufacturer's instructions. Ova collected from superovulated CD1 mice were used to generate transgenic mice at the Ottawa Health Research Institute Transgenic Mouse Facility as previously described (40). Genomic DNA extracted from ear trimmings was used to screen for the presence of the transgene by PCR analysis using Cldn6 forward and FLAG reverse primers (Table 1).

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TABLE 1. PCR primers

RNA isolation and RT-PCR. Back skin samples were dissected from the mid-dorsal region of Inv-C187 transgenic mice and their age-matched wild-type counterparts. Samples were immediately frozen in liquid nitrogen and then homogenized in TRIzol (Invitrogen) reagent for total RNA isolation according to the instructions of the manufacturer. The isolated RNA was subjected to DNase (Invitrogen) treatment, and first-strand cDNA was synthesized using random hexamers (Applied Biosystems) and 1 mg of each RNA sample. PCR analysis was then performed as previously described using specific primers (Table 1). Reverse transcription-PCR (RT-PCR) products were visualized on ethidium bromide-containing agarose gels, and images were acquired using Bio-Rad Molecular Analyst software version 1.2 (Bio-Rad Laboratories).

Immunohistochemistry and histology. For immunohistochemical and histological analyses, both Inv-C187 and wild-type back skin samples were collected at the following ages: newborn; 1, 2, and 3 weeks of age; and 1, 3, and 5 months of age.

(i) Sample collection. At the time points described above, back skin samples (1 cm²) were dissected from the mid-dorsal region of Inv-C187 transgenic and wild-type mice. Frozen sections were required for FLAG, Ki67, and occludin immunostaining, whereas all other staining and histology (hematoxylin and eosin) were performed on paraffin sections. For frozen sections, skin samples were orientated in HistoPrep and submerged in isopentane-containing dry ice, and 5-mm sections were cut as previously described (37). Samples were warmed at room temperature for 3 min followed by fixation in methanol at –20°C for 10 min and washing in phosphate-buffered saline (PBS) before immunostaining (37). For paraffin sections, skin samples were fixed in Bouin's fixative (75% saturated picric acid, 20% formaldehyde, and 5% glacial acetic acid) for 12 to 16 h at room temperature and dehydrated through a series of ethanol washes before being embedded and sectioned. Sections (5 mm thick) were dewaxed and rehydrated followed by antigen unmasking and washing steps prior to histological analysis and immunostaining (35).

(ii) Immunohistochemistry. Nonspecific antibody binding was blocked (10% goat serum, 0.8% bovine serum albumin [BSA], and 1% gelatin in PBS) for 30 min at room temperature followed by incubation in wash buffer (0.8% BSA and 1% gelatin in PBS). Primary antibodies were appropriately diluted in incubation buffer (1% goat serum, 0.8% BSA, and 1% gelatin in PBS), and sections were incubated for 1 to 2 h at room temperature followed by incubation in wash buffer. Antibodies against the following proteins were used: FLAG (M2 monoclonal) (1:440; Sigma), K15 (1:100) (UC55), K5 (1:100) (5054), K14 (1:100) (199), K1 (1:100) (UC81), involucrin (1:100; BabCO), filaggrin (1:100; BabCO), loricrin (1:100) (UC84), TGase-3 (1:100; a gift from Len Milstone), K6 (1:100; BabCO), K17 (1:500; a gift from Pierre Coulombe), Ki67 (1:25; Abcam), Cldn1 (6:100; Zymed Laboratories), Cldn6 (1:50), Cldn10 (1:25), Cldn11 (1:50), Cldn18 (1:50), and occludin (1:100; Zymed). Secondary antibodies against rabbit, mouse, rat, and chicken conjugated to fluorescein isothiocyanate or Texas Red (Jackson ImmunoResearch Laboratories) were used at a 1:50 dilution for 1 h at room temperature followed by incubations in wash buffer and PBS. Sections were incubated with Hoechst stain (1:50; Sigma) for 15 min before mounting with Mowiol 4-88 (Calbiochem) containing 2.5% 1,4-diazobicyclo-2,2,2-octane (Sigma).

(iii) Photography. Images were captured with a bright-field/fluorescence-capable Zeiss Axioplan 2 microscope (Carl Zeiss Canada Ltd.) equipped with an AxioCam camera (Carl Zeiss Canada Ltd.) and using Axio Vision 2.05 software (Carl Zeiss Canada Ltd.). Adobe Photoshop version 7.0 (Adobe Systems, Inc.) was used for image processing.

Protein isolation and immunoblotting. Proteins were extracted from freshly dissected back skin samples (0.4g) by homogenization in sodium dodecyl sulfate (SDS) extraction buffer (62.5 mM Tris, pH 6.8, 25% glycerol, 2% SDS, and 2% ß-mercaptoethanol with pepstatin A and a complete mini protease inhibitor cocktail [Roche Diagnostics] tablet) followed by high-speed centrifugation at 4°C. The supernatant containing the proteins was collected and assayed for protein concentration. Proteins were incubated at room temperature for 30 min in sample-reducing buffer (62.5 mM Tris, pH 6.8, 6 M urea, 25% glycerol, 2% SDS, 0.1% bromophenol blue, 2% ß-mercaptoethanol), boiled for 5 min, and centrifuged at high speed for 10 min, and 10-µg samples were then separated on 12% SDS-polyacrylamide gels, transferred onto nitrocellulose, and incubated in blocking buffer (5% skim milk in Tris-buffered saline-0.1% Tween 20 [TBS-T]) for 1 h at room temperature. Primary antibodies were diluted in incubation buffer (1% goat serum, 0.8% BSA, and 1% gelatin in TBS-T), and blots were incubated overnight at 4°C with antibodies against FLAG (polyclonal) (1:500; Sigma) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:20,000; Abcam). After washing in TBS-T, blots were incubated for 1 h at room temperature in horseradish peroxidase-conjugated secondary antibodies against rabbit or mouse (1:20,000; Amersham Biosciences) diluted in 5% skim milk-TBS-T. Following washes in TBS-T, blots were incubated with ECL Western blotting detection reagents (Amersham Biosciences), and proteins were visualized on Kodak BioMax XAR film (Kodak). Films were digitally scanned, and images were processed with Adobe Photoshop version 7.0 (Adobe Systems, Inc.).

Transfection of HaCat cells and immunofluorescence analysis. HaCat cells were plated onto coverslips 24 h prior to transfection at a density of 250,000 cells/35-mm dish in 2 ml Dulbecco's modified Eagle's medium (DMEM) (high glucose without L-glutamine; Invitrogen) supplemented with 10% fetal calf serum (HyClone), 0.1 mM nonessential amino acids (Invitrogen), and 1 mM sodium pyruvate (Invitrogen) without antibiotics. For transfection, 0.5 µg of Inv-c187-FLAG was added to 250 µl unsupplemented DMEM, and in a separate tube, 10 µl Lipofectamine 2000 reagent was mixed into 250 µl unsupplemented DMEM, followed by incubation for 5 min at room temperature. Two hundred fifty microliters of a DMEM-DNA mixture and 250 µl of a DMEM-Lipofectamine 2000 mixture were combined and incubated for 20 min at room temperature. The transfection mixture (500 µl) was added dropwise to each 35-mm dish, and the cells were incubated for 5 h at 37°C under a 5% CO₂ atmosphere. The media were then replaced with DMEM supplemented with 10% fetal calf serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). After 24 h, cells on coverslips were fixed with methanol for 10 min at –20°C and rinsed with PBS. Cells were incubated in a humidified chamber for 30 min with antibodies against FLAG (M2 monoclonal) (1:440; Sigma), and following a wash in PBS, secondary antibodies against mouse conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) were used at a 1:50 dilution for 30 min at room temperature. Following a wash in PBS, cells were incubated with Hoechst stain (1:100; Sigma) for 10 min and mounted onto slides with Mowiol 4-88 containing 2.5% 1,4-diazobicyclo-2,2,2-octane. Observation by epifluorescence and photography was performed as described above.

Animal photography. Animals were euthanized by isofluorane-CO₂, and images were acquired using a Nikon COOL-PIX950 digital camera and processed using Adobe Photoshop version 7.0.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Inv-C187 transgenic mice. We previously demonstrated that Cldn6 cDNA overexpressed in differentiating mouse epidermal cells results in the disruption of the epidermal differentiation program and a dysfunctional EPB, leading to rapid postnatal death from dehydration (34, 40). To begin a structure-function analysis of Cldn6 domains in these activities, we created a "tailless Cldn6 mutant" (C187) by deleting the C-terminal cytoplasmic tail domain after amino acid 187, leaving four residues next to the fourth transmembrane-spanning region (Fig. 1A and B). We used transgenic mouse technology to express FLAG epitope-tagged C187 cDNA under the control of a 3.7-kb 5'-flanking element of the human involucrin gene (Inv), the same promoter used for our full-length Cldn6 model (40), to drive transgene expression to the suprabasal cells of the epidermis, where TJs are localized (Fig. 1C) (6, 7, 40). PCR using Cldn6 forward (from the start codon) and FLAG reverse (from the 3' end) primers (585 bp) revealed that three transgenic founder mice (two females and one male) were generated (Fig. 1D and Table 1), and lines that exhibited indistinguishable phenotypes were established. To examine the localization of C187 in the transgenic epidermis, we used immunohistochemistry with anti-FLAG antibodies (Fig. 1E). As expected, there was no FLAG protein expression in the wild-type epidermis, while expression was restricted to the upper spinous and granular layers of the transgenic epidermis. Notably, however, rather than finding expression primarily localized to cell-cell junctions, FLAG-tagged C187 was abundant in the cytoplasm, presumably due to inefficient membrane targeting (Fig. 1F). In addition, immunoblotting using anti-FLAG antibodies confirmed that there was a 19.5-kDa band in the Inv-C187 back skin samples and not in the wild-type back skin samples (Fig. 1G). To complement our in vivo observations and to verify the cytoplasmic accumulation of C187-Cldn6, we transfected the same construct into cultured basal-like, undifferentiated, exponentially growing monolayers of HaCat cells, where it has been demonstrated that Cldn6 is not expressed (32; K. Turksen and T.-C. Troy, unpublished observations). Again, C187 accumulated in the cytoplasm and not at the cell membrane (Fig. 1H), confirming a defect in the ability of C187 to target to the membrane in vivo and in vitro.

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FIG. 1. Inv-C187 transgenic mice. The tailless Cldn6 (C187) 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 C187 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-C187 (TG) and not the wild-type (WT) back skin samples using anti-GAPDH as a loading control (G). HaCat cells transfected with Inv-C187-FLAG also showed significant cytoplasmic accumulation, confirming a defect in the ability of C187 to target to the membrane both in vivo and in vitro (H). The C187 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.

Next, we explored the consequences of the expression of C187 in the overall phenotype of transgenic mice compared to the phenotype of transgenic mice overexpressing full-length Cldn6 as previously described (34, 40). Phenotypically, Inv-C187 transgenic neonates appeared to be comparable to their wild-type counterparts. In fact, newborn transepidermal water loss measurements (103 dpm) and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) staining (no penetration) confirmed that Inv-C187 transgenic mice did not display the barrier dysfunction at birth, which was seen in their full-length Cldn6 counterparts (data not shown). In addition, cornified envelopes extracted from newborn C187 epidermis were essentially indistinguishable from those from wild-type extracts (data not shown), providing further support that an intact barrier was indeed achieved during development despite the expression of a tailless Cldn6. However, with the emergence of hair fibers, Inv-C187 transgenic mice were easily identifiable by their coat appearance, which was not as sleek as that of their wild-type counterparts (Fig. 1I), a phenotype that persists throughout life. Overall, the hair phenotype of the Inv-C187 transgenic mice is similar to that of the Inv-Cldn6 mice and appears not to be directly related to the other epidermal defects we observed in either case (34); therefore, it has not been investigated further here.

Expression of Inv-C187 elicits morphological changes in the epidermis. A histological analysis of samples of back skin from newborn Inv-C187 mice and Inv-C187 mice at 1 week, 2 weeks, 3 weeks, 1 month, 3 months, and 5 months of age compared to samples from their age-matched wild-type counterparts was done to examine the morphology of the transgenic epidermis. As anticipated from the observations described above, no obvious differences in epidermal morphology in the back skin samples from newborn or 1-week-old Inv-C187 mice versus those of wild-type mice were seen (Fig. 2A and 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 (Fig. 2C to D). After 3 weeks of age and persisting throughout life, a thick epidermis with striking morphological abnormalities in architecture and differentiation was present (see below). The transgenic back skin 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 (Fig. 2E).

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FIG. 2. Histological abnormalities in the Inv-C187 epidermis. A histological analysis of samples of back skin from Inv-C187 mice compared to those of their age-matched wild-type counterparts revealed no obvious differences in the epidermal morphology of the Inv-C187 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).

Inv-C187 transgenic mice exhibit abnormalities in epidermal differentiation. It has been demonstrated that alterations in the normal distribution of keratin markers, as well as scaffold and cornified envelope markers, are excellent means of determining the integrity of the epidermal differentiation program (9, 13, 14, 25, 28, 39). Therefore, immunohistochemistry analysis of early, later, and terminal differentiation markers in back skin samples from Inv-C187 transgenic mice versus those of their age-matched wild-type littermates was done. We report on the analysis of skin samples from newborn (Fig. 3 and 4, left panel) and 1-month-old (Fig. 3 and 4, right panel) mice; samples from animals from 3 weeks to 5 months of age gave similar and consistent results compared to those at 1 month of age. Consistent with the histological results summarized above, expression of differentiation markers was indistinguishable between newborn (Fig. 3 and 4, left panel) and 1-week-old (data not shown) transgenic animals versus wild-type animals. However, there was a progressive alteration in the expression of epidermal differentiation markers from 2 to 4 weeks of age that remained consistent throughout life. For example, K15 expression, while limited to the basal layer, became sporadic in transgenic samples rather than uniform as in the wild type (Fig. 3A, right panel). Expression of K5 and K14 extended beyond the usual/wild-type basal layer through all the suprabasal layers of the transgenic epidermis (Fig. 3B and C, right panel). Expression of K1 also showed a broadened expression pattern, overlapping with the suprabasal expression compartment of K5/K14, in the Inv-C187 back skin (Fig. 4A, right panel). A similarly expanded expression compartment in the transgenic epidermis was observed for various structural proteins including involucrin (Fig. 4B, right panel), filaggrin (Fig. 4C, right panel), loricrin (Fig. 4D, right panel), and TGase-3 (Fig. 4E, right panel).

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FIG. 3. Perturbation of early markers of epidermal differentiation. Early differentiation markers in back skin samples from newborn and 1-month-old Inv-C187 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.

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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-C187 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).

C187 increases basal cell proliferation and induces suprabasal cell proliferation. Histological observations of a thickened epidermis and the apparent perturbations in the epidermal differentiation program were suggestive of an increased proliferation rate in the Inv-C187 transgenic epidermis. Therefore, immunostaining was done for K6 and K17, differentiation markers that are generally not present in the normal adult interfollicular epidermis except under conditions reflective of abnormal cell proliferation and differentiation (20, 23, 24, 41). Results indicated that there was no abnormal proliferation in the samples of back skin from newborn or 1-week-old transgenic mice (data not shown). However, with the thickening of the transgenic epidermis, there was an obvious persistent increase in the expression of these proliferation-associated markers (samples of back skin from 1-month-old animals are shown) (Fig. 5A and B). We also assayed for Ki67, a cell cycle-related nuclear protein expressed by proliferating cells in the G₁, S, G₂, and M phases of the cell cycle. In the wild-type epidermis, Ki67-positive and Hoechst-stained cells were confined to the basal cell layer as expected (2, 29) (Fig. 5C and D, right panels). However, in the Inv-C187 transgenic epidermis, consistent with the other analyses reported above, there were Hoechst-stained nuclei in the upper layers (Fig. 5D, left panel), and not only was the number of Ki67-positive basal cells increased by about fourfold (P < 0.001 using the Student's t test) (Fig. 5E), but a significant proportion of Ki67-positive cells were also found in the suprabasal cell layers (Fig. 5C, left panel). In addition, immunohistochemical staining for macrophages (F4/80 antigen) and T cells (CD3 molecular complex) in sections of Inv-C187 transgenic epidermis did not reveal any accumulation or immune infiltration compared to the wild-type (data not shown), indicating that the observed changes in the proliferation rate in the epidermis of the transgenic mice are likely not due to the expression of immune cell-derived cytokines (17).

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FIG. 5. Proliferation in the Inv-C187 epidermis. To address the potential proliferation defect observed in the Inv-C187 transgenic epidermis, immunostaining for proliferation-associated markers, including K6, K17, and Ki67, was performed. Back skin samples from 1-month-old Inv-C187 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-C187 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).

Perturbations in Cldn expression in Inv-C187 transgenic mice. Different epithelial cells exhibit a complex profile of Cldn TJ molecules, the composition of which has recently been attributed to the formation of a diverse array of selective permeability barriers for different epithelia (1, 10, 18, 34, 38, 42). Concomitantly, it is thought that a precise Cldn expression profile characterizes epidermal differentiation and that in response to injury or disease, modifications in the epidermal differentiation program are reflected in changes in the Cldn profile and vice versa (15, 27, 34, 40). Cldn2, Cldn3, and Cldn5, which are normally not expressed in the epidermis, were not observed in either wild-type or transgenic skin at any age sampled (data not shown). As was observed with the expression of epidermal keratins and other differentiation markers, back skin samples from newborn and 1-week-old transgenic mice also exhibited no differences in the expression or localization of any of the typical epidermal Cldn proteins assayed (data not shown). However, in parallel with the thickening of the epidermis, there was a marked difference in the transgenic expression profile of epidermal Cldn proteins (Fig. 6). Cldn6, Cldn10, Cldn11, and Cldn18, which are normally restricted to the differentiating compartment of the mature epidermis, starting with the upper spinous layers (15, 34, 40), were expressed in a clearly expanded zone (Fig. 6A and B, samples from 1-month-old mice). On the other hand, whereas Cldn1 is expressed in the basal to suprabasal layers of the wild-type epidermis, the basal layer was essentially devoid of Cldn1 expression in the samples from transgenic animals (Fig. 6A and B). Notably, and reminiscent of the expression of the transgene product (Fig. 1F), Cldn6, Cldn10, Cldn11, and Cldn18 localization shifted from the membrane of wild-type mice to the cytoplasm of transgenic mice to various degrees (Fig. 6B). Altered localization of Cldn1 expression to the cytoplasm was less pronounced than that of the other Cldn proteins, and the localization of occludin (another TJ-associated integral membrane protein that is normally associated with the granular layer in epidermal cells) (26, 27, 47) remained membranous (Fig. 6B).

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FIG. 6. Cytoplasmic accumulation of Cldn proteins in the Inv-C187 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-C187 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.

Activation of an unfolded protein signaling pathway in the Inv-C187 epidermis. The cytoplasmic accumulation of Cldn proteins observed in the Inv-C187 transgenic mouse epidermis is reminiscent of certain other proteins where unusual cytoplasmic accumulation has been attributed to signaling activation through an unfolded protein pathway (30, 31). RT-PCR analysis with RNA extracted from the back skin of Inv-C187 transgenic and wild-type mice (Fig. 7) indicated that the unfolded protein signaling pathway components activating transcription factor 4 (Atf4); activating transcription factor 6 (Atf6); eukaryotic translation initiation factor 2 kinase 4 (Eif2ak4); eukaryotic translation initiation factor 2, subunit 1 (Eif2s1); endoplasmic reticulum-to-nucleus signaling 1 (Ern1); and heat shock 70-kDa protein 5 (Hspa5) are activated in our transgenic model.

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FIG. 7. Activation of an unfolded protein response pathway in the C187 epidermis. In an attempt to explain the cytoplasmic accumulation of Cldn proteins observed in the Inv-C187 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-C187 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-C187 epidermis compared to the wild type, including Atf4, Atf6, Eif2ak4, Eif2s1, Ern1, and Hspa5.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we used a structure-function approach to elucidate whether the cytoplasmic tail of Cldn6 plays a crucial role in epidermal differentiation and the formation of a physiologically intact EPB. Using transgenic mouse technology, we expressed a complete tail truncation of Cldn6 at its endogenous site of expression, the TJ compartment of the epidermis, via the Inv promoter (Inv-C187). While Inv-C187 animals displayed apparently normal prenatal epidermal development and a functioning EPB, histological and immunohistochemical characterizations revealed abnormalities in the epidermal differentiation program with progressive thickening of the epidermis and aberrant epidermal marker expression that was evident after 1 week of age and that persisted throughout life. This was accompanied by increased cell proliferation in both the basal and suprabasal compartments of the epidermis. Although some membrane association was evident, a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 mislocalized to the cytoplasm of transgenic epidermal cells, an observation that correlates with the activation of the unfolded protein signaling pathway in the Inv-C187 epidermis. These findings demonstrate the importance of the tail domain in targeting Cldn6 to the membrane for normal proliferation and differentiation in the mouse epidermis.

Loss of the cytoplasmic tail domain of Cldn6 results in epidermal hyperproliferation and differentiation defects. A normal wild-type epidermis starts out as a multilayer structure at birth and undergoes thinning until the mature 2- to 3-cell-layer-thick epidermis is achieved within 2 weeks after birth (46). The abnormal thickening of the Inv-C187 epidermis reflects a disrupted epidermal proliferation and differentiation program, presumably as a result of inefficient membrane targeting and mislocalization of Cldn6 to the cytoplasm (see below). The fact that other Cldn proteins are also both mislocalized and aberrantly expressed points towards the activation of not only coordinate regulatory pathways but also potential compensatory pathways that rescue EPB function but do not rescue the tightly coupled epidermal proliferation-differentiation program. Whether Cldn proteins are linked directly to signaling for epidermal cell proliferation or whether the observed hyperproliferation results from differentiation anomalies is not yet known. However, one potential explanation for the proliferation and differentiation changes observed is the activation of an unfolding protein pathway (30, 31). The cytoplasmic accumulation of C187 is reminiscent of that observed for numerous molecules where a folding defect has inhibited their normal functioning and is consistent with our data showing up-regulation of Atf4, Atf6, Eif2ak4, Eif2s1, Ern1, and Hspa5, all of which are expected if the unfolded protein response signaling pathway is activated. Notably, it is becoming apparent that the prolonged presence of unfolded proteins, as occurs in the Inv-C187 mice, may result in certain disease states, including hyperproliferation situations such as tumorigenesis (for reviews, see references 21 and 48).

Epidermis-specific keratins and epidermal terminal differentiation markers such as involucrin, filaggrin, loricrin, and TGase-3, proteins involved in scaffold function as well as the eventual formation of the cornified envelopes of the stratum corneum (5), are excellent indicators of the orderly progression of epidermal differentiation (4, 9, 14, 25), which is clearly disrupted in the Inv-C187 transgenic mice. In this regard, expression of basal layer-specific K5 and K14 was deregulated and extended throughout the epidermis of the transgenic mice, indicating that the thick epidermis maintains basal layer-like characteristics throughout, supporting the hyperproliferative state. In addition, the observed broad and overlapping expression compartments of the early differentiation marker K1 as well as some of the late differentiation markers (i.e., involucrin, loricrin, filaggrin, and TGase-3) suggest that the terminal differentiation program and processing of late epidermal differentiation markers are also dysregulated. The anomalous expression of K6, which is normally not associated with the interfollicular epidermis except under specific skin conditions such as wound healing and psoriasis as well as during the development of epidermal tumors (11, 41), also supports this view. Similarly, K17 is normally expressed in the basal layer of the epidermis during development and within the first few days after birth before its expression is turned off (24). Finally, among markers with abnormal expression domains, the hyperproliferation state may also be related to the fact that Cldn1 expression is lost in the basal layer of the transgenic epidermis (see also below), which parallels Cldn1 ablation in cancerous cells (33). Taken together, these observations suggest that the disrupted proliferation-differentiation program in the Inv-C187 mice may render them susceptible to epidermal tumors, a possibility being pursued further in these animals.

The cytoplasmic tail domain of Cldn6 is necessary for Cldn homeostasis and membrane targeting in the epidermis. As mentioned above, a precise Cldn expression profile characterizes epidermal differentiation, and modifications to the Cldn expression profile are reflected in changes to the epidermal differentiation program (15, 27, 34, 40). Thus, for example, in our two different transgenic models in which native (Inv-Cldn6) or tail-truncated (Inv-C187) Cldn6 is overexpressed, a common feature is a loss of the usual Cldn expression profile. Our Inv-C187 transgenic mouse model underscores the intimate link between Cldn expression, heterophilic interactions, and normal differentiation in the epidermis. In addition to the accumulation of C187 in the cytoplasm of the transgenic epidermis, there was also increased accumulation of Cldn6, Cldn10, Cldn11, and Cldn18 to various degrees; while the subcellular Cldn1 localization was not as drastically modified, its expression domain was. Since details of potential Cldn-Cldn interactions in the cytoplasm and/or TJ fibrils have not yet been elucidated, our observations support two potential mechanisms whereby C187 acts as a dominant negative to affect other Cldn proteins; dominant-negative mutations encode mutant polypeptides that disrupt the activity of the wild-type gene when they are overexpressed (16a). In the first potential mechanism, heterophilic Cldn-Cldn assembly would occur in the cytoplasm before membrane targeting; in this model, mislocalized mutant Cldn6 would bind to and inhibit (or delay) membrane targeting of other Cldn proteins, causing their accumulation in the cytoplasm. In the second potential mechanism, heterophilic Cldn-Cldn assembly would occur in the membrane; in this model, a small amount of mutant membrane-localized Cldn6 may destabilize not only its own binding but also the binding of other Cldn proteins in TJ strands, leading to fibril dissolution and Cldn accumulation in the cytoplasm. It is also possible that both mechanisms are operative. In any case, the resultant changes in Cldn homeostasis have profound consequences on epidermal proliferation and differentiation.

Although the C-terminal tail domain is diverse among different Cldn proteins, the CXXC (where C is cysteine and X is any amino acid) (see underlined sequences of Fig. 1B) tetrapeptide sequence motif, which is conserved in the cytoplasmic tail domain of several Cldn proteins, signals for palmitoylation, a posttranslational modification in a number of integral membrane molecules essential for their targeting to and stabilization in the membrane (3). The importance of palmitoylation in the posttranslational modification and membrane targeting of Cldn14 has also recently been demonstrated, where its mutation resulted in cytoplasmic accumulation (45). Although the poor membrane localization and cytoplasmic accumulation of C187 in the transgenic mice is reminiscent of Cldn mislocalization when the CXXC is mutated, the tail domain CXXC motif remains intact in our mutant; indeed, there are two additional amino acids in the transgene sequence before the FLAG tag was introduced, suggesting that palmitoylation defects cannot account for mislocalized C187 and its consequences in the epidermis. It also seems unlikely that the second conserved CXXC motif in the Cldn internal loop, well removed from our truncation site, contributes to the mislocalization, although its role in palmitoylation and membrane targeting remains to be investigated. Nevertheless, we cannot rule out the possibility that the tail and/or the internal loop CXXC motif has become dysfunctional due to conformational changes in the C187 mutant or that the introduction of the FLAG epitope tag results in the high turnover of membrane molecules, thereby contributing to excessive cytoplasmic accumulation, issues we are currently exploring. However, our data support the notion that there are other crucial determinants for stable TJ incorporation in the cytoplasmic C-terminal sequence of Cldn proteins that up to now have not been implicated in specific protein-protein interactions.

Most Cldn proteins, including Cldn6, have a conserved PDZ-binding YV sequence at the C-terminal end of the tail domain that serves as an interaction site for Cldn binding with other PDZ domain proteins (16, 19, 22). Such interactions may be responsible for the linking of Cldn proteins to the cytoskeleton and/or to Cldn signaling pathways. However, to date, the only PDZ protein shown to act potentially as a link between the actin cytoskeleton and Cldn proteins is ZO-1 (12). Nevertheless, it seems likely that other TJ-related PDZ domain-containing proteins that interact with Cldn proteins will be identified.

Effect of C187 on the barrier function of epidermal cells. While the disruption of Cldn homeostasis is a common feature of the overexpression of both native and tail-truncated Cldn6, neither the severe defects in developmental EPB formation nor the measurable dehydration across the skin with neonatal lethality that characterizes Inv-Cldn6 mice is seen in the Inv-C187 neonates. Furthermore, and again, in contrast to Inv-Cldn6 mice, the epidermis of Inv-C187 neonates was quite comparable to that of the wild-type mice during development and at birth. Strikingly, even after the postnatal differentiation and skin thickness defects are expressed in the Inv-C187 transgenic epidermis, there is again no measurable barrier defect observed. The reason for this is not obvious but must reflect functionally adequate, even if immunohistochemically abnormal, epidermal TJs. Whether this is due to the fact that there is sufficient residual Cldn6 and other Cldn proteins properly targeted to the membrane to participate in TJs with adequate barrier function, if not normal function for the postnatal epidermal proliferation-differentiation program, is not known. However, it is also important to emphasize that the complete Cldn composition of the epidermis is not yet known, and it is therefore possible that the Inv-C187 epidermis may express other Cldn proteins that can compensate for and rescue barrier function but not the entire proliferation-differentiation sequence in the transgenic mice.

In summary, through the generation of transgenic mice that express tailless Cldn6 in the suprabasal layer of the epidermis, we provide evidence for the importance of the cytoplasmic tail domain of Cldn proteins in membrane targeting and epidermal proliferation-differentiation in vivo. It is likely that the cytoplasmic accumulation of C187 may play an important role in the aberrant proliferation observed in the transgenic mouse epidermis, possibly through "gain-of-function" activity, its precocious interaction with membrane or cytoplasmic molecules, and/or the activation of a protein folding defect pathway. In addition, we speculate that the Cldn6 cytoplasmic tail domain contains additional motifs along with those already known that may play a role in the targeting of C187 to TJ fibrils. The Inv-C187 mice provide an excellent animal model system to further investigate the role of Cldn in all these processes.

. . . . . . .

 
We acknowledge the tremendous support we have received from a number of our colleagues and thank the three anonymous reviewers whose suggestions were key to the improvement of our previous repetitive and long-winded discussion. We are grateful to Mario Tiberi (OHRI) for helpful suggestions regarding integral membrane proteins as well as the statistical analysis and to Pierre Coulonbe (JHU) for his generous gifts of antibodies and his ongoing support of our studies. We thank Fiona Watt (Cancer Research-UK) for providing us with the Inv cassette, and a special thank you goes to AnneMarie Gagnon (OHRI) for her invaluable suggestions. Transgenic mice were generated at the OHRI under the dedicated care of Adriana Gambarotta and Pierre Bradley, and Zaida J. Ticas (MLT) (University of Ottawa) performed histological sectioning. We also acknowledge the tremendous support and encouragement that we continue to receive from Jane Aubin (University of Toronto).

This work was sponsored by a research grant from the Canadian Institutes of Health Research.

 
* Corresponding author. Mailing address: Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario K1Y 4E9, Canada. Phone: (613) 798-5555, ext. 17806. Fax: (613) 761-5365. E-mail: kturksen{at}ohri.ca.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, August 2006, p. 5876-5887, Vol. 26, No. 15
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