Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.     

The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice

The importance of the epidermal permeability barrier (EPB) in protecting the mammalian species against harmful UV irradiation, microorganism invasion and water loss is well recognized, as is the role of calcium (Ca(2+)) in keratinocyte differentiation, cell-cell contact and the EPB. In a previous study, we reported that the overexpression of the Ca(2+)-sensing receptor (CaSR) in the undifferentiated basal cells of the epidermis induced a modified epidermal differentiation program including an accelerated EPB formation in transgenic mice, suggesting a role for CaSR signaling in the differentiation of embryonic epidermal cells during development. We now describe the expression profile of claudins (Cldns) and keratin markers in the accelerated EPB formation of K14-CaSR transgenic mice during development as compared to the wild type from E12.5 to newborn stages. Our data show that the transgenic epidermis undergoes an advanced epidermal differentiation program as compared to the wild type as evidenced morphologically as well as by the expression of K14, K1, loricrin, Cldn6, Cldn18 and Cldn11. In addition, we report for the first time the sequential expression of Cldns in epidermal development and describe that the localization of some Cldns change within the epidermis as it matures. Furthermore, we demonstrate that Cldn6 is expressed very early in epidermal morphogenesis, followed by Cldn18, Cldn11 and Cldn1.     

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.     

Claudin immunolocalization in neonatal mouse epithelial tissues

Emerging evidence supports the notion that claudins (Cldns) are dynamically regulated under normal conditions to respond to the selective permeability requirements of various tissues, and that their expression is developmentally controlled. We describe the localization of those Cldns that we have previously demonstrated to be functionally important in epidermal differentiation and the formation of the epidermal permeability barrier, e.g., Cldn1, Cldn6, Cldn11, and Cldn18, and the presence of Cldn3 and Cldn5 in various neonatal mouse epithelia including the epidermis, nail, oral mucosa, tongue, and stomach. Cldn1 is localized in the differentiated and/or undifferentiated compartments of the epidermis and nail and in the dorsal surface of the tongue and glandular compartment of the stomach but is absent from the oral mucosa and the keratinized compartment of the stomach. Cldn3 is present in the basal cells of the nail matrix and both compartments of the murine stomach but not in the epidermis, oral mucosa, or tongue. Cldn5 is found in the glandular compartment of the stomach but not in the epidermis, nail unit, oral mucosa, forestomach, and tongue. Cldn6, Cldn11, and Cldn18 occur in the differentiating suprabasal compartment of the epidermis, nail, and oral mucosa and in the dorsal and ventral surfaces of the tongue and the keratinized squamous epithelium of the stomach. The simple columnar epithelium of the glandular stomach stains for Cldn18 and reveals a non-membranous pattern for Cldn6 and Cldn11 expression. Our results demonstrate differential Cldn protein profiles in various epithelial tissues and their differentiation stages. Although the molecular mechanisms regulating Cldn expression are unknown, elucidation of their differential localization patterns in tissues with diverse permeability requirements should provide a better understanding of the role of tight junctions in tissue function. This work was supported by a research grant from the Canadian Institutes of Health Research (MOP-69087).     

Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo

The calcium sensing receptor (CaSR) has emerged as an important mediator of a wide range of Ca(2+)-dependent physiological responses (Ca(2+) signaling) in various tissues. To explore the role of CaSR in the epidermis, we utilised the keratin 14 promoter to express CaSR cDNA constitutively in the basal cells of the stratified squamous epithelium of transgenic mice. Analysis of the transgenic mice revealed that a sensitized response to CaSR signaling accelerates the epidermal differentiation program with the precocious formation of the epidermal permeability barrier (EPB) during development and an accelerated hair growth at birth. Our observations indicate that overexpression of CaSR in the undifferentiated basal cells leads to changes in the differentiation program of the transgenic epidermis, including the stimulation of keratins 1 and 6 as well as the overexpression of several markers of terminal differentiation such as filaggrin, loricrin and involucrin. Our data suggest that the observed modifications in the differentiation pathway are a consequence of a CaSR-induced enhancement of Ca(2+) signaling involving cross-talk with other signaling pathways (e.g. EGF and Wnt/Ca(2+)). These studies provide new insights into the role of CaSR in epidermal differentiation including EPB development and hair follicle morphogenesis.     

Immunolocalization of a p53/p63‐like protein in the regenerating tail of the wall lizard (Podarcis muralis) suggests it is involved in the differentiation of the epidermis

During tail regeneration in lizards, the epidermis forms new scales comprising a hard beta‐layer and a softer alpha‐layer. Regenerated scales derive from a controlled folding process of the wound epidermis that gives rise to epidermal pegs where keratinocytes do not invade the dermis. Basal keratinocytes of pegs give rise to suprabasal cells that initially differentiate into a corneous wound epidermis and later in corneous layers of the regenerated scales. The immunodetection of a putative p53/63 protein in the regenerating tail of lizards shows that immunoreactivity is present in the nuclei of basal cells of the epidermis but becomes mainly cytoplasmic in suprabasal and in differentiating keratinocytes. Sparse labelled cells are present in the regenerating blastema, muscles, cartilage, ependyma and nerves of the growing tail. Ultrastructural observations on basal and suprabasal keratinocytes show that the labelling is mainly present in the euchromatin and nucleolus while labelling is more diffuse in the cytoplasm. These observations indicate that the nuclear protein in basal keratinocytes might control their proliferation avoiding an uncontrolled spreading into other tissues of the regenerating tail but that in suprabasal keratinocytes the protein moves from the nucleus to the cytoplasm, a process that might be associated to keratinocyte differentiation.     

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.     

Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.

In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.     

The class II phosphoinositide 3-kinase C2beta is not essential for epidermal differentiation

Phosphoinositide 3-kinases (PI3Ks) regulate an array of cellular processes and are comprised of three classes. Class I PI3Ks include the well-studied agonist-sensitive p110 isoforms; however, the functions of class II and III PI3Ks are less well characterized. Of the three class II PI3Ks, C2alpha and C2beta are widely expressed in many tissues, including the epidermis, while C2gamma is confined to the liver. In contrast to the class I PI3K p110alpha, which is expressed throughout the epidermis, C2beta was found to be localized in suprabasal cells, suggesting a potential role for C2beta in epidermal differentiation. Overexpressing C2beta in epidermal cells in vitro induced differentiation markers. To study a role for C2beta in tissue, we generated transgenic mice overexpressing C2beta in both suprabasal and basal epidermal layers. These mice lacked epidermal abnormalities. Mice deficient in C2beta were then generated by targeted gene deletion. C2beta knockout mice were viable and fertile and displayed normal epidermal growth, differentiation, barrier function, and wound healing. To exclude compensation by C2alpha, RNA interference was then used to knock down both C2alpha and C2beta in epidermal cells simultaneously. Induction of differentiation markers was unaffected in the absence of C2alpha and C2beta. These findings indicate that class II PI3Ks are not essential for epidermal differentiation.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

Tight junction regulates epidermal calcium ion gradient and differentiation

It is well known that calcium ions (Ca²⁺) induce keratinocyte differentiation. Ca²⁺ distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca²⁺ gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca²⁺ gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca²⁺ flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca²⁺ gradient.     

Spatial and temporal expression of POF1B, a gene expressed in epithelia

Mammalian epithelia possess specialized cellular components that provide an impermeable barrier between two different environments. In particular, in the skin, mitotically dividing cells undergo a programmed set of morphological and biochemical changes leading to the establishment of the epidermal permeability barrier (EPB) to prevent escape of moisture and entrance of toxic molecules. Many different skin proteins are involved in the process but not all have been identified. We report here the results of the expression studies of a novel gene, highly and specifically expressed in the granular layer of the epidermis and in the epithelia of the oro-pharyngeal and gastro-intestinal tracts. Our data show that during mouse development Pof1b expression is activated in the external layers of the epidermis just prior to formation of the EPB.     

Thematic review series: skin lipids. Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology

The epidermis is a very active site of lipid metabolism, and all peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) isoforms are expressed in the epidermis. Activation of PPARalpha, -beta/delta, or -gamma or LXRs stimulates keratinocyte differentiation. Additionally, activation of these receptors also improves permeability barrier homeostasis by a number of mechanisms, including stimulating epidermal lipid synthesis, increasing lamellar body formation and secretion, and increasing the activity of enzymes required for the extracellular processing of lipids in the stratum corneum, leading to the formation of lamellar membranes that mediate permeability barrier function. The stimulation of keratinocyte differentiation and permeability barrier formation also occurs during fetal development, resulting in accelerated epidermal development. PPAR and LXR activation regulates keratinocyte proliferation and apoptosis, and studies have shown that these receptors play a role in cutaneous carcinogenesis. Lastly, PPAR and LXR activation is anti-inflammatory, reducing inflammation in animal models of allergic and irritant contact dermatitis. Because of their broad profile of beneficial effects on skin homeostasis, PPAR and LXR have great potential to serve as drug targets for common skin diseases such as psoriasis, atopic dermatitis, and skin cancer.     

Expression of the helix-loop-helix protein ID1 in keratinocytes is upregulated by loss of cell-matrix contact

To analyze the inhibitor of DNA-binding type 1 (ID1) in the human epidermis and in cultured keratinocytes we generated and characterized ID1-specific monoclonal antibodies. Immunohistological studies on human skin biopsies revealed that ID1 is not detectable in normal human epidermis but in lesional epidermis of bullous pemphigoid. In the latter case we found ID1 in the cytoplasm of basal and proximal suprabasal keratinocytes. Cultured normal human epidermal keratinocytes displayed ID1 in the cytoplasm; upon differentiation into a multilayered keratinocyte sheet, ID1 was no longer detectable. It was reexpressed after dispase-mediated detachment of the keratinocyte cultures from the growth substratum. In this case ID1 was localized to the cytoplasm and the nucleus. Our data indicate that after epidermal injury-in our case loss of cell-matrix contact-ID1 is upregulated in affected keratinocytes. In view of the ID1 function in other cell types, we speculate that ID1 facilitates the transition from the resting to the migrating and proliferating keratinocyte required for efficient repair of epidermal lesions by reepithelialization. Taken together we suggest that ID1 is an important player in epidermal (patho-)physiology.     

The chromatin remodeler Mi-2beta is required for establishment of the basal epidermis and normal differentiation of its progeny

Using conditional gene targeting in mice, we show that the chromatin remodeler Mi-2beta is crucial for different aspects of skin development. Early (E10.5) depletion of Mi-2beta in the developing ventral epidermis results in the delayed reduction of its suprabasal layers in late embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5) loss of Mi-2beta in the dorsal epidermis does not interfere with suprabasal layer differentiation or maintenance of the basal layer, but induction of hair follicles is blocked. After initiation of the follicle, some subsequent morphogenesis of the hair peg may proceed in the absence of Mi-2beta, but production of the progenitors that give rise to the inner layers of the hair follicle and hair shaft is impaired. These results suggest that the extended self-renewal capacity of epidermal precursors arises early during embryogenesis by a process that is critically dependent on Mi-2beta. Once this process is complete, Mi-2beta is apparently dispensable for the maintenance of established repopulating epidermal stem cells and for the differentiation of their progeny into interfollicular epidermis for the remainder of gestation. Mi-2beta is however essential for the reprogramming of basal cells to the follicular and, subsequently, hair matrix fates.     

Cellular responses to disruption of the permeability barrier in a three-dimensional organotypic epidermal model

Repeated injury to the stratum corneum of mammalian skin (caused by friction, soaps, or organic solvents) elicits hyperkeratosis and epidermal thickening. Functionally, these changes serve to restore the cutaneous barrier and protect the organism. To better understand the molecular and cellular basis of this response, we have engineered an in vitro model of acetone-induced injury using organotypic epidermal cultures. Rat epidermal keratinocytes (REKs), grown on a collagen raft in the absence of any feeder fibroblasts, developed all the hallmarks of a true epidermis including a well-formed cornified layer. To induce barrier injury, REK cultures were treated with intermittent 30-s exposures to acetone then were fixed and paraffin-sectioned. After two exposures, increased proliferation (Ki67 and BrdU staining) was observed in basal and suprabasal layers. After three exposures, proliferation became confined to localized buds in the basal layer and increased terminal differentiation was observed (compact hyperkeratosis of the stratum corneum, elevated levels of K10 and filaggrin, and heightened transglutaminase activity). Thus, barrier disruption causes epidermal hyperplasia and/or enhances differentiation, depending upon the extent and duration of injury. Given that no fibroblasts are present in the model, the ability to mount a hyperplastic response to barrier injury is an inherent property of keratinocytes.