Ryan F.L. O'Shaughnessy

Desmoglein 4 in Hair Follicle Differentiation and Epidermal Adhesion: Evidence from Inherited Hypotrichosis and Acquired Pemphigus Vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.

AKT-dependent HspB1 (Hsp27) Activity in Epidermal Differentiation

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation

Human epidermal stem cells express higher levels of β1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of β1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2‐fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA‐FABP, also known as E‐FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA‐FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA‐FABP is a novel marker of epidermal transit amplifying cells.

Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous beta HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.

Interleukin-1 alpha blockade prevents hyperkeratosis in an in vitro model of lamellar ichthyosis

The autosomal recessive congenital ichthyoses are a family of related diseases, causing a severe defect in the barrier function of the epidermis. Neonates are usually born as collodion babies, but later form scales characteristic of the disease, due to a combination of thickening of the cornified layer and an increase in the production of non-polar lipids. Current treatments of choice are exfoliative creams and moisturizing agents and the use of oral retinoids. The skin condition and treatment impact significantly on quality of life and, with oral retinoids, there are potential complications associated with long-term use. A greater understanding of the mechanisms that result in scaling should lead to better directed therapies, not only for the inherited ichthyoses, but also other hyperkeratotic disorders. Using siRNA knockdown of the principle gene mutated in lamellar ichthyosis (LI), transglutaminase-1, in rat keratinocytes, we created an in vitro organotypic culture model that closely mimics the disease. Interleukin-1 alpha (IL1A) expression was increased and there was a lack of loricrin cross-linking. All LI patients tested had an increased IL1A and treatment of wild-type organotypic cultures with IL1A was sufficient to induce hyperkeratosis. Treatment of disease mimic organotypic cultures with IL-1 receptor antagonist led to a dose-dependent decrease in hyperkeratosis without a reduction in non-polar lipids in the cornified layer, which has the potential to reduce scaling without the requirement to constantly apply emollients.

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development.

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55α or B55α), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development.

The mTOR inhibitor rapamycin opposes carcinogenic changes to epidermal Akt1/PKBα isoform signaling

Epidermal squamous cell carcinoma (SCC) is the most aggressive non-melanoma skin cancer and is dramatically increased in patients undergoing immunosuppression following solid organ transplantation, contributing substantially to morbidity and mortality. Recent clinical studies show that use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin as a post-transplantation immunosuppressive significantly reduces SCC occurrence compared with other immunosuppressives, though the mechanism is not fully understood. We show that rapamycin selectively upregulates epidermal Akt1, while failing to upregulate epidermal Akt2. Rapamycin increases epidermal Akt1 phosphorylation via inhibition of the mTOR complex 1-dependent regulation of insulin receptor substrate-1. Epidermal Akt1 is commonly downregulated in SCC while Akt2 is upregulated. We now demonstrate similar Akt1 downregulation and Akt2 upregulation by ultraviolet (UV) radiation, the most important skin carcinogen. Hence, rapamycin’s upregulation of Akt1 signaling could potentially oppose the effects of UV radiation and/or tumor-associated changes on Akt1 signaling. We show in skin culture that rapamycin does enhance restoration of Akt1 phosphorylation in skin recovering from UV radiation, suggesting a mechanism for rapamycin’s antitumor activity in epidermis in spite of its efficient immunosuppressive properties.

The role of BMP signalling in the control of ID3 expression in the hair follicle

Abstract:  Both the production of the hair shaft in anagen and the initiation of a new hair cycle at telogen are the result of reciprocal interactions between the dermal papilla and the overlying epithelial cells. Secreted factors, such as those of the bone morphogenetic protein (BMP) family, play a crucial role in moderating these interactions. Analysis of hair follicles in different stages of the hair cycle showed that BMP signalling was only active during anagen and again during telogen. During catagen, no BMP signalling occurred in the dermal papilla. ID3, a gene expressed in the dermal papilla of both vibrissa and pelage follicles, is a BMP target, and as such, we found that ID3 was expressed from the earliest stages of morphogenesis. During the hair cycle, ID3 was only expressed in the dermal papilla at middle anagen and telogen. To test the significance of ID3 expression in the dermal papilla, we cultured dermal papilla cells and found that ID3 expression fell significantly after a single passage. ID3 expression was returned to in vivo levels in low‐ and high‐passage cells by culturing to high confluence or by the addition of BMP4. These studies reinforce the requirement for active BMP signalling and cell–cell contacts in the dermal papilla during specific stages in the hair cycle.

Stem Cells in the Epidermis

The epidermis consists of three actively proliferating units, the interfollicular epidermis, the hair follicle, and the sebaceous gland. Stem cells in the epidermis have the capacity to produce all three of these units. The fate of the epidermal stem cells and some of their progeny can be altered, dependent on the environment in which they reside and the genes they express. In this review, we describe the major experiments that have contributed to the understanding of the epidermal stem cells and the control of their fate.