Junctions and Inflammation in the Skin

Abstract

The skin forms a life-sustaining barrier between the organism and physical environment. The physical barrier of skin is mainly localized in the stratum corneum (SC); however, nucleated epidermis also contributes to the barrier through tight, gap, and adherens junctions (AJs), as well as through desmosomes and cytoskeletal elements. Many inflammatory diseases, such as atopic dermatitis (AD) and psoriasis, are associated with barrier dysfunction. It is becoming increasingly clear that the skin barrier function is not only affected by inflammatory signals but that defects in structural components of the barrier may be the initiating event for inflammatory diseases. This view is supported by findings that mutations in filaggrin, a key structural epidermal barrier protein, cause the inflammatory skin disease AD, and that a loss of AJ components, namely epidermal p120 catenin or α-catenin results in skin inflammation.

Keywords::

• adhesion
• inflammation
• skin
• catenin
• junctions

INTRODUCTION

The skin represents the largest organ of the body and provides a vital interface between the body and the environment. It is challenged to control the extent of an immune response during normal homeostasis or wound healing in order to maintain its integrity. If an immune response is inadequate then the overwhelming infections or tumors may arise, but if an immune response is excessive then chronic inflammation and autoimmunity may develop.

The skin consists of the epidermis and the dermis, which are separated by the basement membrane (Fuchs, 2009). The epidermis is composed mainly of keratinocytes, which undergo a tightly regulated program of proliferation and differentiation to form a stratified epithelium that ensures the renewal of epithelial tissues without compromising their functional properties. This is a particularly difficult task, considering that epithelial tissues are constantly challenged by threats coming from the outside world and should to be able to adjust their proliferative and differentiation programs to maintain and restore basal homeostasis after injury. Specialized immune cell populations such as Langerhans cells and the γδT-cell receptor-bearing dendritic epidermal T cells, the latter being present in mice but not in humans (Romani et al., 2010; Jameson et al., 2004; Pasparakis, 2012), are also found in the epidermis. The dermis contains primarily fibroblasts and immune cells and also structures essential for supporting skin function such as blood vessels, nerves, muscles, hair follicles, and glands. Resident skin macrophages and monocytes constitute the major immune cell population found in the dermis, which also contains tissue dendritic cells, mast cells, and small numbers of T cells. In vivo, epidermal keratinocytes produce basal levels of IL-1α sufficient to initiate cutaneous inflammation (Groves et al., 1995). Accumulating evidence indicates that in both the basal and diseased states, keratinocytes produce many other cytokines/chemokines, including IL-6, IL-10, and TNF-α, which recruit immune cells to the epidermis/dermis and initiate inflammation. In line with their ability to secrete cytokines/chemokines, epidermal keratinocytes express several Toll-like receptors (Nestle et al., 2009), arguing for an autonomous role for keratinocytes in sensing danger signals to initiate the inflammatory responses. In humans, keratinocytes in allergic skin appear to be activated before the immune cells (e.g., T cells) enter the skin (Griffiths & Nickoloff, 1989), again supporting the notion that skin plays an active role in controlling immune responses. An intense cross-talk between epithelial, stromal, and immune cells is critical for the regulation of skin homeostasis. Deregulation of these interactions causes the pathogenesis of inflammatory skin diseases such as psoriasis, atopic dermatitis (AD), and contact dermatitis (Nestle et al., 2009).

Cell–cell connectivity is crucial for adult tissues to perform their unique functions, preserve their architectural integrity, and coordinate the precision with which cells are able to remodel their tissues during normal homeostasis and repair them in response to injury. At the level of the individual cell, direct cell–cell adherence is mediated by the intercellular junction complexes: adherens junctions (AJs), tight junctions (TJs), and desmosomes (Perez-Moreno et al., 2003; Matter et al., 2005; Yin & Green, 2004).

Cadherin-mediated AJs are particularly important in controlling the specificity, formation, and maintenance of intercellular adhesion (Gumbiner et al., 1988; Lewis et al., 1994; Takeichi, 1990). In addition, they direct coordinated cellular organization and movements within epithelia and transmit information from the environment to the interior of cells (Brembeck et al., 2006; Gumbiner, 2000; Nelson & Nusse, 2004). E-cadherin is the prototypic member of the subfamily of classical cadherins and is expressed in most epithelial tissues. It physically links neighboring cells in a calcium-dependent manner through homotypic interactions (Patel et al., 2003). Classical cadherins preferentially associate with p120-catenin and β-catenin and are connected to actin microfilaments (Perez-Moreno & Fuchs, 2006). Desmosomes are composed of two kinds of desmosomal cadherins: desmoglein (DSG) and desmocollin (DSC). Desmosomal cadherins bind to the cytoplasmic plaque proteins plakoglobin and desmoplakin (DSP), and are linked to keratin intermediate filaments (Yin & Green, 2004). Despite their sequence similarities, p120-catenin and β-catenin bind to distinct sites on the cadherin tail. p120-catenin interacts with the membrane-proximal or juxtamembrane domain involved in regulating the lateral clustering and stabilization of cadherins at the membrane (Perez-Moreno & Fuchs, 2006). By contrast, β-catenin interacts with the distal part of the cytoplasmic tail and with α-catenin, an unrelated catenin that can bind directly to filamentous actin (Perez-Moreno & Fuchs, 2006) as well as to several actin-binding proteins (Kobielak et al., 2004). In this review, we discuss how epithelial cells within skin can sense signals through cell–cell adhesion components and control skin homeostasis and bring forth the tightly regulated anti-microbial and wound-healing responses.

SKIN BARRIER FUNCTION AND INFLAMMATORY SKIN DISEASES

Cutaneous barrier plays an important role in the pathophysiology of skin disorders. In some cases barrier abnormality represents a primary or intrinsic process like in irritant contact dermatitis, allergic contact dermatitis, burns, ulcers, bullous disorders, premature infant's skin, and ichthyosis. In other cases immunological abnormality can trigger barrier abnormality. This is found in T-cell lymphoma and autoimmune bullous diseases (Proksch et al., 2008). Barrier disruption and immunological mechanisms may reciprocally play enhancing roles in initiating and sustaining skin lesions. This might be the case in AD and psoriasis. In AD and in psoriasis, it is debatable whether permeability barrier disruption is followed by inflammation or whether inflammation leads to epidermal changes including barrier dysfunction. Many reports on the pathogenesis of AD and psoriasis focused on the primary role of abnormalities in the immune system (reviewed in [Leung, 2013] and [Ong & Leung, 2006]). However, others have proposed an “outside-inside” pathogenesis for AD and other inflammatory dermatoses with barrier abnormalities (Elias & Feingold, 2001; Jensen et al., 2004). The theory of outside–inside pathogenesis is also supported by mouse models which exhibit a psoriasis-like phenotype because of alterations in keratinocytes. Inducible downregulation of JunB and c-Jun in keratinocytes of adult mice results in a psoriasis-like skin phenotye and arthritic lesions. The skin phenotype was also found but to a weaker extent in mice without functional T cells (Zenz et al., 2005). Also mice with a knockdown of IKK2 in epidermal keratinocytes showed a psoriasis-like skin phenotype which was independent of T cells (Pasparakis et al., 2002). The existence of defective permeability barrier function in AD is now widely accepted. Genetically impaired skin barrier function is already present in nonlesional skin and is more pronounced in lesional skin of AD. Increased epidermal proliferation and disturbed differentiation including changes in keratins and cornified envelope proteins, like involucrin, loricrin, and filaggrin, and in lipid content and composition cause impaired barrier function (Proksch et al., 2006; Ruether et al., 2006). Genome-wide linkage scans have identified a locus linked to the epidermal differentiation complex on chromosomes 1q21 in AD (Bowcock & Cookson, 2004). Recently, loss-of-function genetic variants in the gene encoding filaggrin with very high significance have been shown to be strong predisposing factors for AD and have been found in about 20% of all AD patients and in about 50% of patients with severe disease (Palmer et al., 2006; Irvine & McLean, 2006; Ruether et al., 2006). As in AD, a genetic linkage of psoriasis to the epidermal differentiation complex 1q21 has been found alongside other markers (Bowcock & Cookson, 2004). However, mutations with a very high significance comparable with the filaggrin mutations in AD have not been found yet (Bowcock & Cookson, 2004).

α-CATENIN AND NF-κB ACTIVATION

α-E-catenin is the prototypic member of the α-catenin family and was initially characterized as a component of the cadherin–catenin complex. α-catenins bind indirectly to cadherins via interaction with β-catenin. In mammals, there are three α-catenins: α-E-catenin is most prevalent in epithelial tissues; α-N-catenin is restricted to neural tissues; α-T-catenin is expressed primarily in the heart. Additionally, the distant member α-catulin, an α-catenin-like protein, is ubiquitously expressed (Kobielak & Fuchs, 2004). α-catenins share an overall similarity with vinculin, an actin-binding protein, and differ considerably in sequence from the other catenins (β-catenin, p120-catenin, plakoglobin, and plakophilins), which unlike α-catenin belong to the armadillo family of proteins.

Genetic studies in mice have shown that α-E-catenin is required to sustain adhesion between cells during mammalian morphogenetic events (Torres et al., 1997; Vasioukhin et al., 2001). In Drosophila, cell adhesion is disrupted when the single α-catenin gene harbors a mutation in its encoded binding site for armadillo, the fly homolog of β-catenin (Orsulic & Peifer, 1996), emphasizing the importance of α-catenin in sustaining cell adhesion. Surprisingly, studies using conditional loss of function of α-catenin have uncovered unexpected cell-autonomous behaviors that could not be readily explained by defects in cell–cell adhesion alone. Conditional targeting of the α-catenin gene in the skin epidermis of E14.5 mouse embryos (Vasioukhin et al., 2001) followed by skin grafting resulted in formation of papilloma-like undulations, followed by breakdown of the underlying basement membrane and finally epithelial-mesenchymal transitions resembling invasive squamous cell carcinoma (SCC) (Kobielak & Fuchs, 2006).

In vivo and in culture, α-catenin null keratinocytes display enhanced activation of NFkB and immune infiltration, which are also features of human SCC (Kobielak & Fuchs, 2006). The correlation between cancer and inflammation has been recognized for decades, but only in recent years has evidence begun to suggest that the inflammation might be a prerequisite rather than a consequence of tumorigenesis (Balkwill & Coussens, 2004; Greten et al., 2004; Mantovani, 2005; Colotta et al., 2009). Conditional loss of epithelial α-catenin suggests that loss of intercellular adhesion and α-catenin is an unanticipated instigator of a proinflammatory and cell survival response. Moreover, because α-catenin null epidermal cells not only intrinsically activate NF-κB, but also have the surface receptors to respond to its downstream targets, this activation not only protects the cells against apoptosis but also triggers a vicious proinflammatory signaling circuit through infiltrating immune cells. When accompanied by defects in intercellular adhesion and polarity, the α-catenin null epidermal barrier is also exposed to infection, further fueling this cascade.

How loss of α-catenin leads to the initial difference in NF-κB activity is an intriguing question. Interestingly, it was recently reported that α-catenin functions as a tumor suppressor in E-cadherin-negative basal-like breast cancer cells by inhibiting NF-κB signaling. Mechanistically, α-catenin interacts with the IκBα protein, and stabilizes IκBα by inhibiting its ubiquitination and its association with the proteasome. This stabilization in turn prevents nuclear localization of RelA and p50, leading to decreased expression of TNF-α, IL-8 and RelB (Piao et al., 2014).

ROLE FOR IKK/NF-κB SIGNALING IN EPITHELIAL TISSUE HOMEOSTASIS

Reduced α-catenin at intercellular borders and activation of NF-κB also occur during wound healing, where normal epidermal cells must transiently downregulate cell–cell contact, increase migration, activate an inflammatory response, and remodel extracellular matrix. Multiple studies support an important role for IKK/NF-κB signaling in epithelial cells in the maintenance of epithelial tissue homeostasis (reviewed in [Pasparakis, 2012]). Due to its dual cytoprotective and immunoregulatory functions, the IKK/NF-κB pathway is ideally suited for the regulation of the physical and immunological properties of epithelial barriers at the interface with the environment. Under steady-state conditions, NF-κB signaling supports the survival of epithelial cells in response to cytokines such as TNF that are locally induced at low levels in epithelial tissues by receptors sensing environmental or commensal microbes. When epithelial tissues are challenged by pathogens, chemical, or immunological insults, activation of IKK/NF-κB signaling on the one hand performs cytoprotective functions supporting the epithelial cell survival and on the other hand induces the expression and release of cytokines and growth factors that activate the essential host defense immunological responses. At the same time, NF-κB coordinates proliferative wound healing responses to restore the epithelial barrier.

In order to perform its important role in epithelial cells, NF-κB activity needs to be tightly regulated. The response to microbial and environmental stresses must be finely tuned to ensure that the cytoprotective functions of NF-κB are preserved while potent proinflammatory responses are transiently induced to facilitate efficient host defense and wound healing but are rapidly downregulated to prevent excessive tissue damage and chronic inflammation. Deregulation of NF-κB activity could therefore have an important role in the pathogenesis of chronic inflammatory diseases of epithelial tissues. If a temporary modification, downregulation, or sequestering of α-catenin is a natural response to injury, the cross-talk between cell–cell adhesion and NF-κB pathway could explain why chronic wound conditions ranging from ulcers to leprosy render patients susceptible to cancer.

p120 CATENIN AND INFLAMMATION

p120-catenin belongs to the family of armadillo repeat domain proteins. Like its structural homologues, β- and γ-catenin, p120-catenin is an essential component of AJs. p120-catenin binds directly to the cytoplasmic domain of cadherin and contributes to the regulation of cell–cell junctional integrity. p120-catenin can influence cell adhesion by dynamic regulation of the actin cytoskeleton (Anastasiadis & Reynolds, 2001), transport of cadherins to the membrane, and stability of cadherins at the membrane (Reynolds & Carnahan, 2004). Studies have demonstrated that p120-catenin plays important roles in cell–cell adhesion, embryonic development, cell proliferation and polarity, tumor cell migration, and cancer progression. However, recent insights have generated an entirely new perspective, suggesting that p120-catenin is implicated in the anti-inflammatory responses in the absence and presence of infection (Perez-Moreno et al., 2006).

The activation of RhoA and NF-κB were cell autonomous and independent of p120's role in cell–cell junction formation, as they occurred in cultured p120-catenin null keratinocytes and under conditions in which junctions do not form. In vivo, NF-κB activation in the p120-catenin null epidermis results in the production and release of cytokines and chemokines, which in turn triggers a robust immune response and chronic inflammation. NF-κB activation occurred in p120 null epidermal cells through stimulation of RhoA in the absence of inflammatory stimuli. In addition the p120-catenin null epidermis differs from the α-catenin null state in that the epidermis is only mildly hyperproliferative, and this appears to be an indirect consequence of the infiltration of immune cells. In skin, overall adhesion is maintained, and the epidermis retains its impermeable barrier detected by inside-out and outside-in dye penetration assays. Despite significant progress in understanding the role of p120-catenin in the development of inflammation, our knowledge is far from complete with regard to the cellular and molecular mechanisms of p120-catenin in the regulation of innate immunity. Some additional data are coming from the use of a genetic approach (Hu, 2012) which revealed that p120-catenin significantly inhibited IκB-α degradation and subsequent activation of NF-κB induced by LPS in lungs and pulmonary microvascular endothelial cells. However, p120 knockdown alone in mouse pulmonary endothelial cells did not affect NF-κB activation but instead significantly enhanced LPS-induced NF-κB activation, suggesting the important role of endothelial p120 in the modulation of LPS/TLR4-dependent NF-κB signaling (Hu, 2012).

E-CADHERIN SIGNALING

The lack of proliferative defects in cadherin-deficient skin contrasted with α-catenin and/or p120-catenin cKO skins (Perez-Moreno et al., 2006; Kobielak & Fuchs, 2006). An additional perturbation arising from α-catenin and p-120 catenin loss was a striking inflammatory cell infiltrate and epidermal NF-κB activation. Surprisingly, however, neither inflammatory cell recruitment nor NF-κB activation were features of cadherin-deficient epidermis (Tinkle et al., 2008). In the work published by Tinkle et al, the loss of both Ecad and Pcad unveiled defects in intercellular adhesion, survival, and epidermal integrity that were not present in skin lacking only one of these cadherins (Tinkle et al., 2008). To some extent, these defects resembled those of α-catenin deficient skin (Vasioukhin et al., 2001). The Pcad/Ecad and α-catenin mutant mice differed in whether cadherin-β-catenin complexes were localized at cell borders, and this difference may account for other distinctions in the observed phenotypes. Although epidermal hyperthickening was observed in both mutants, the hyperthickening arising from cadherin inhibition was not accompanied by significant changes in proliferation or MAPK activation like it was observed in α-catenin deficient skin (Vasioukhin et al., 2001). In addition, while intercellular adhesion was compromised in both cases, only the α-catenin cKO mice displayed an inflammatory cell infiltrate and enhanced epidermal NF-κB activation (Kobielak & Fuchs, 2006). Together, those in vivo studies suggest that proliferative and proinflammatory defects arising from loss of α-catenin within epidermis are independent of cadherin-mediated adhesion (Kobielak & Fuchs, 2006; Vasioukhin et al., 2001) .

Further studies will be necessary to fully appreciate the functional parallels between cadherins and α-catenin; however, the data so far suggest that these AJ components function coordinately in mediating keratinocyte adhesion, yet differ in their ability to influence proliferative and inflammatory responses in skin. Although mice deficient in Pcad and Ecad did not display an inflammatory cell infiltrate and enhanced epidermal NF-κB activation, even though the TJs and barrier are affected it would be very interesting to test whether they are more susceptible to epicutaneous allergen sensitization.

DESMOSOMES AND KERATIN NETWORK IN SKIN INFLAMMATION

Skin barrier formation is also critically dependent on desmosomes. Those transmembrane structures that connect the cell surface to the intermediate filament cytoskeleton consist of heterodimers of desmosomal cadherins, desmogleins (DSG1–DSG4) and desmocollins (DSC1–DSC3). The cytoplasmic part of the desmosomal plaque contains a number of associated proteins, such as plakoglobin and plakophilins that associate with DSP and thereby link to the keratin cytoskeleton (Desai et al., 2009). Desmosomal cadherins and their associated proteins play a role in instructing the development and differentiation of complex tissues in vertebrates. In addition, they are frequently mutated in inherited diseases of the skin and heart and are targeted by autoimmune antibodies and bacterial toxins, the latter of which can result in blistering of complex epithelia (Desai et al., 2009). Recently a new syndrome featuring severe dermatitis, multiple allergies, and metabolic wasting (SAM syndrome) caused by homozygous mutations in DSG1 supports the hypothesis that impaired epidermal barrier function may also contribute to the development of inflammatory skin diseases (Samuelov et al., 2013). Data from mice deficient in plakophilin-3 (PKP3), a member of the Armadillo-repeat family, and a component of desmosomes also strongly supports this hypothesis (Sklyarova et al., 2008). In the basal layer of PKP3-null epidermis, densities of desmosomes, and AJs were remarkably altered. PKP3-null mice were prone to dermatitis providing in vivo evidence that PKP3 plays a critical role in limiting inflammatory responses in the skin (Sklyarova et al., 2008).

Functional link between keratin 1 (KRT1) and human inflammatory skin diseases was also recently suggested (Roth et al., 2012). Keratin 1 and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. Roth et al., demonstrated that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and the S100A8 and S100A9 proteins, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1(-/-) mice. IL-18 release was keratinocyte-autonomous, KRT1 and caspase-1 dependent, supporting an upstream role of KRT1 in the pathology (Roth et al., 2012). Above data strongly suggest the role of desmosomal cadherins and their associated proteins and keratin filament in the development of inflammatory skin diseases.

TIGHT JUNCTIONS AND ATOPIC DERMATITIS

Gene expression profiling of non-lesional epithelium from patients with extrinsic AD, non-atopic subjects, and patients with psoriasis recently revealed a strikingly lower level of the TJ proteins, claudin-1, and claudin-23, in patients with AD (De Benedetto et al., 2011). TJs are cell–cell junctions that connect neighboring cells, control the paracellular pathway of molecules (barrier function), and separate the apical from the basolateral part of a cell membrane. They are made up of a complex of adhesive proteins that control the passage of fluids and solutes through the paracellular pathway. In human epidermis, various TJ proteins have been identified, including occludin, claudins 1, 4, and 7, junctional adhesion molecule-1, zonula occludens protein 1, and MUPP-1 (multi-PDZ protein-1) (Proksch et al., 2008). In human skin, TJ proteins are found in the interfollicular epidermis as well as in the skin appendages. In various diseases with perturbed SC barrier function, including psoriasis vulgaris, lichen planus, and ichthyosis vulgaris, TJ proteins that were formerly restricted to the stratum granulosum and upper stratum spinosum such as occludin and claudin 4 were also found in deeper layers of the epidermis (Proksch et al., 2008). The composition of TJ, especially concerning claudins, seems to be very important for their barrier function in the epidermis. Downregulation as well as overexpression of certain proteins perturbs this barrier (Brandner et al., 2002). Werner's group using different combinations of FGF receptor (FGFR)-deficient mice unraveled their functions in the skin by influencing the expression of TJs components (Yang et al., 2010). Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. They identified loss of FGF-induced expression of TJ components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier caused activation of keratinocytes and epidermal γδ T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiated an inflammatory response and induced a double paracrine loop through production of keratinocyte mitogens by dermal cells. Those results identified essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlighted the importance of stromal–epithelial interactions in skin homeostasis and disease (Yang et al., 2010).

CONCLUSION

Our skin acts as a sentinel, determining how and when to respond to a broad array of environmental insults during both homeostatic and pathologic states. Epidermal keratinocytes are constantly challenged by numerous stresses such as ultraviolet (UV) radiation, chemical, mechanical, and microbial insults. In order to perform their functions, keratinocytes need to integrate environmental stimuli into the network of cellular interactions that control skin homeostasis and elicit tightly regulated anti-microbial and wound-healing responses. Epithelial barrier dysfunction and inflammation are major contributors to the pathogenesis of skin disease; however, much remains unknown about how these two processes overlap and how they contribute independently to disease initiation.

Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.