QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Turksen, K. Articles by Troy, T.-C. Search for Related Content PubMed PubMed Citation Articles by Turksen, K. Articles by Troy, T.-C.

Permeability barrier dysfunction in transgenic mice overexpressing claudin 6

¹ Ottawa Health Research Institute,
² Divisions of Dermatology and Endocrinology, Department of Medicine, Ottawa Hospital and
³ Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

View larger version (37K): [in a new window]   Fig. 1. Claudin 6 structure, distribution and transgenic phenotype. (A) Molecular structure analysis of claudin 6 predicts it to be a 4 trans-membrane domain integral membrane protein that has varying tissue distribution in newborn mice. (B) Expression of claudin 6 was detected strongly in liver and kidney while there were varying degrees of expression in heart, thymus, spleen, calveria, brain, lung, tongue, stomach, tail and skin. There was no detectable expression in intestine. (C) RT-PCR results were supported by immunohistochemical staining of wild-type sections of intestine, lung and kidney tissues. (D) Mouse claudin 6 was cloned into the NotI site of an expression vector containing the human involucrin promoter/enhancer and 17 transgenic animals were generated. (E) PCR analysis was used to detect expression of the claudin 6 transgene using primers spanning the involucrin promoter to the claudin 6 coding sequence (Table 1). A 590 bp band was detected in lane 2 (plasmid DNA control) as well as in lanes 6-10 (positive transgenic mice). There was no band detectable in lane 1 (PCR control) and lanes 3-5 (non-transgenic mice). (F) RT-PCR was also performed using primers spanning the exon sequences present in the involucrin promoter to the claudin 6 coding sequence in order to identify only the transgene expression (692 bp). (G) claudin 6 forward and reverse primers also identified the endogenous expression (660 bp), which is approximately 8-fold less than that of the transgenic expression. Western blotting of proteins extracted from the epidermis of transgenic and wild-type mice with claudin 6 antibodies revealed a 2.5-fold increase in the transgenic samples over the wild-type expression (H). The increased transgene expression was also demonstrated through indirect immunofluorescence with a marked increase of claudin 6 protein detected in the upper spinous and granular layers of transgenic backskin samples as compared to their wild-type counterparts (I).

View larger version (64K): [in a new window]   Fig. 2. claudin 6 overexpression disturbs the overall claudin distribution. (A,B) RT-PCR analysis of various claudins using RNA extracted from newborn epidermis revealed that the overexpression of claudin 6 resulted in modification of the expression profiles of other claudins in transgenic epidermis. Semi-quantitative RT-PCR analysis at 25, 30 and 35 cycles shows the range of signal detection between the epidermis of wild-type and Inv-Cldn6 transgenic mice. claudin 2 and 9 were undetectable in both samples. (C) Immunofluorescence analysis of transgenic and wild-type backskin samples supports the RT-PCR results, showing virtually no difference in claudin 1 staining and no claudin 2 detection. Labelling in the SC is due to non-specific binding of the secondary antibody.

View larger version (50K): [in a new window]   Fig. 3. Inv-Cldn6 skin. (A) As a consequence of the transgene, the skin of Inv-Cldn6 neonates is red and shiny in appearance and becomes dehydrated and cracked within a few hours of life. (B,C) Newborn backskin samples from transgenic and wild-type mice were fixed with Bouin’s solution, processed and embedded in paraffin and sections were stained with Haematoxylin/Phloxine/Safranin. The transgenic epidermis is highly disorganised with gaps apparent throughout in addition to non-uniform basal cells. (D,E) It is also apparent that there is a dramatic decrease in keratohyalin granular cells and that the stratum corneum is thicker and often fragmented in the transgenic samples. (F,G) There is also an obvious reduction in the occurrence of subcutaneous fat pads resulting in the overall reduction in the thickness of transgenic skin.

View larger version (46K): [in a new window]   Fig. 4. Inv-Cldn6 transgenic mice have a defective EPB. (A) ß-gal assays were performed to assess the integrity of the EPB of transgenic animals, as compared to the wild type, at different stages of development. Under normal conditions, an intact EPB exists at E17.5 days thereby preventing the penetration of substances such as X-gal. When X-gal enters the epidermis a striking blue staining results. At E16.5 neither the wild-type nor the transgenic embryos possessed an intact EPB, yet at E18.5 there was no penetration of X-gal through the EPB of the wild-type embryos while transgenic embryos remained blue. Even after birth, transgenic neonates did not possess an EPB. (B) In addition to the ß-gal assays, DPM measurements were performed to assess the trans-epidermal water loss of transgenic and wild-type neonates. In representative experiments, there was greater than a 3-fold increase in the dehydration of transgenic neonates as compared to the wild type further supporting the notion that the defective EPB causes the massive dehydration and death of the transgenic animals. (C-F) Isolated CE preparations of newborn transgenic and wild-type epidermis showed that the rigid, polygonal structure of the wild-type CEs (D and F) disappeared in the transgenic samples. Transgenic CEs were mostly rounded in shape (E) and overall less abundant (C) leading to the fragile, less compact nature of the stratum corneum that is observed.

View larger version (53K): [in a new window]   Fig. 5. Transgenic epidermal differentiation markers are perturbed. Newborn transgenic and wild-type epidermis was processed for immunofluorescence to evaluate epidermal differentiation markers. K5 expression was not altered in the transgenic epidermis, however, K1 expression was increased indicating a perturbation in the differentiation program of the transgenic epidermis. In addition, K6, which is not normally expressed in the epidermis, shows some patchy expression in the transgenic epidermis; while normal hair follicle (HF) distribution was not altered. In addition, filaggrin, loricrin and transglutaminase-3 were also assessed. There was aberrance in the expression of these markers in the transgenic epidermis in that they were much more diffuse and expressed in a much broader zone, especially in the case of filaggrin.

View larger version (23K): [in a new window]   Fig. 6. The overexpression of claudin 6 causes an overall disturbance in the expression of other markers. (A) Western blot analysis revealed the enhanced processing of profilaggrin. The transgenic samples revealed a ‘bottom heavy’ expression of the smaller sized processed filaggrin while wild-type samples were ‘top heavy’ indicating a greater abundance of unprocessed profilaggrin in the wild-type newborn epidermis. In fact, processed filaggrin was increased 13-fold. In addition, the expression of loricrin, transglutaminase-3 and involucrin was increased 1.5 fold in the transgenic samples. (B) RT-PCR analysis indicated no significant differences with filaggrin, loricrin, transglutaminase-3 and involucrin in the transgenic epidermis versus wild-type epidermis. (C) RT-PCR analysis further indicated that repetin, members of the SPRR family and Klf4 (genes involved in CE formation) were altered in transgenic epidermis. Repetin, SPRR1A, 2A and Klf4 were downregulated in the transgenic samples while the expression of SPRR2D and 2G was increased. SPRR1B, 2B, 2C and 3 remained unchanged.