Proximity Mapping of Desmosomes Reveals a Striking Shift in Their Molecular Neighborhood Associated With Maturation.

Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signaling network to exert their full function is unclear. To investigate this, we carried out protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin, and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterized as desmosome matured from Ca2+ dependence to the mature, Ca2+-independent, hyper-adhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signaling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for the analysis of desmosome function.

Desmosome dualism - most of the junction is stable, but a plakophilin moiety is persistently dynamic.

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with fluorescence recovery after photobleaching (FRAP) and fluorescence loss and localisation after photobleaching (FLAP), we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from Ca2+ dependence to Ca2+-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome downregulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes. This article has an associated First Person interview with the first author of the paper.

Desmocollin 1 is abundantly expressed in atherosclerosis and impairs high-density lipoprotein biogenesis.

Aims: The biogenesis of high-density lipoprotein (HDL) particles by cholesterol-laden foam cells in atherosclerotic lesions is crucial for the removal of excess cholesterol from the lesions. Impairment in the HDL biogenic process contributes to the progression of atherosclerosis. The aim of this study is to identify novel cellular factors regulating HDL biogenesis. Methods and results: HDL biogenesis is a process of apolipoprotein (apo)-mediated solubilization of specific plasma membrane (PM) microdomains generated in cholesterol-accumulated cells. We established a new method to isolate PM microdomains interacting with the major HDL protein constituent, apoA-I. Lipidomic and proteomic analyses of an isolated PM microdomain revealed that apoA-I binds to cholesterol-rich and desmocollin 1 (DSC1)-containing microdomains. In this novel apoA-I binding microdomain, DSC1 binds and prevents apoA-I from interacting with another PM microdomain created by adenosine triphosphate-binding cassette transporter A1 (ABCA1) for the formation of HDL. Inhibition of apoA-I-DSC1 binding by silencing DSC1 expression or using DSC1 blocking antibodies increases apoA-I accessibility to ABCA1-created microdomains and thus enhances HDL biogenesis. Importantly, DSC1 is abundantly expressed in macrophages and human atherosclerotic lesions, suggesting that DSC1 may contribute to cholesterol accumulation in atherosclerotic lesions by sequestering apoA-I and impairing HDL biogenesis. Conclusions: The binding of apoA-I to two functionally opposing PM microdomains, ABCA1 and DSC1 domains, suggests that HDL biogenesis and PM cholesterol levels may be regulated by the relative abundance of the two domains and that novel HDL biogenic therapies may be developed by targeting DSC1.

Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens.

Group 1 allergens of house dust mites (HDM) are globally significant triggers of allergic disease. They are considered as initiator allergens because their protease activity enables the development of allergy to a spectrum of unrelated allergens from various sources. This initiator-perpetuator function identifies Group 1 HDM allergens as attractive drug design targets for the first small-molecule approach directed towards a non-human, root cause trigger of allergic disease. The purpose of this study was to: (i) identify exemplar inhibitors of these allergens using Der p 1 as a design template, and (ii) characterise the pharmacological profiles of these compounds using in vitro and in vivo models relevant to allergy. Potent inhibitors representing four different chemotypes and differentiated by mechanism of action were investigated. These compounds prevented the ab initio development of allergy to the full spectrum of HDM allergens and in established allergy they inhibited the recruitment of inflammatory cells and blunted acute allergic bronchoconstriction following aerosol challenge with the full HDM allergen repertoire. Collectively, the data obtained in these experiments demonstrate that the selective pharmacological targeting of Der p 1 achieves an attractive range of benefits against exposure to all HDM allergens, consistent with the initiator-perpetuator function of this allergen.

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration.

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.

Direct evidence that PKCα positively regulates wound re-epithelialization: correlation with changes in desmosomal adhesiveness.

Non-healing wounds cause considerable patient morbidity and represent a significant economic burden. Central to wound repair is re-epithelialization, a crucial process involving the modulation of cell adhesion to allow keratinocyte migration to cover the exposed underlying tissues. The cellular mechanisms regulating the earliest stages of re-epithelialization are unclear. We present the first direct evidence that protein kinase Cα (PKCα) plays an important role in regulating wound re-epithelialization. In PKCα(-/-) mice re-epithelialization is delayed, while in novel bitransgenic mice over-expressing constitutively active PKCα it is accelerated. These effects are not due to changes in keratinocyte proliferation, apoptosis or intrinsic cell motility. Instead, they correlate with changes in desmosomal adhesiveness, delay being preceded by retained desmosomal hyper-adhesiveness and acceleration with a rapid switch to desmosomal Ca(2+) -dependence. We demonstrate mechanistic conservation in acute human wounds where PKCα localizes to wound edge desmosomes, which become Ca(2+) -dependent. However, in chronic wounds PKCα remains cytoplasmic and desmosomes fail to switch from the hyper-adhesive state. These results throw new mechanistic light on the earliest stages of wound re-epithelialization and suggest activation of PKCα as a new therapeutic strategy for non-healing wounds.

Non-junctional human desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a pathway possibly involved in the pathogenesis of pemphigus vulgaris.

E-cadherin, a classical cadherin, is an adhesion receptor in adherens junctions and has important functions in cell-cell adhesion and cell signalling. Recently we reported that a desmosomal cadherin, desmoglein 3 (Dsg3), an autoantigen in pemphigus vulgaris (PV), associates with E-cadherin and activates Src, which results in tyrosine phosphorylation of adherens junction proteins. However, the nature of such an interaction and its role in cell-cell adhesion remain unclear. In this report, we provide direct evidence that it is the detergent-soluble, non-desmosomal Dsg3 that regulates the activity of Src and its association with E-cadherin in adherens junction formation. Modulation of Dsg3 levels, either by Dsg3 silencing or over-expression, alters Src activity and its association with E-cadherin. Dsg3 silencing caused retardation of calcium-induced E-cadherin junction assembly and a reduction of desmosomal protein expression. Furthermore, we provide evidence that this signalling pathway is involved, at least in part, in the pathophysiology of pemphigus. Along with the reduced expression of Dsg3, loss and disruption of E-cadherin and a concomitant decreased pSrc signalling was identified in the basal keratinocytes surrounding the blisters in PV. These findings suggest a novel function for Dsg3 in the control of E-cadherin-Src signalling and cell-cell adhesion.

Targeting an Initiator Allergen Provides Durable and Expansive Protection against House Dust Mite Allergy.

Whereas treatment of allergic diseases such as asthma relies largely on the targeting of dysregulated effector pathways, the conceptually attractive alternative of preventing them by a pharmaceutical, at-source intervention has been stymied until now by uncertainties about suitable targets and the challenges facing drug design. House dust mites (HDMs) are globally significant triggers of allergy. Group 1 HDM allergens, exemplified by Der p 1, are cysteine proteases. Their degradome has a strong disease linkage that underlies their status as risk and initiator allergens acting directly and through bystander effects on other allergens. Our objective was to test whether target-selective inhibitors of group 1 HDM allergens might provide a viable route to novel therapies. Using structure-directed design to optimize a series of pyruvamides, we undertook the first examination of whether pharmaceutically developable inhibitors of group 1 allergens might offer protection against HDM exposure. Developability criteria included durable inhibition of clinically relevant signals after a single aerosolized dose of the drug. The compounds suppressed acute airway responses of rats and mice when challenged with an HDM extract representing the HDM allergome. Inhibitory effects operated through a miscellany of downstream pathways involving, among others, IL-33, thymic stromal lymphopoietin, chemokines, and dendritic cells. IL-13 and eosinophil recruitment, indices of Th2 pathway activation, were strongly attenuated. The surprisingly expansive benefits arising from a unique at-source intervention suggest a novel approach to multiple allergic diseases in which HDMs play prominent roles and encourage exploration of these pharmaceutically developable molecules in a clinical setting.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Desmosomal cadherins.

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.

Desmosomes: adhesive strength and signalling in health and disease.

Desmosomes are intercellular junctions whose primary function is strong intercellular adhesion, known as hyperadhesion. In the present review, we discuss how their structure appears to support this function as well as how they are assembled and down-regulated. Desmosomal components also have signalling functions that are important in tissue development and remodelling. Their adhesive and signalling functions are both compromised in genetic and autoimmune diseases that affect the heart, skin and mucous membranes. We conclude that much work is required on structure-function relationships within desmosomes in vivo and on how they participate in signalling processes to enhance our knowledge of tissue homoeostasis and human disease.

Desmosomal cadherin misexpression alters beta-catenin stability and epidermal differentiation.

Desmosomal adhesion is important for the integrity and protective barrier function of the epidermis and is disregulated during carcinogenesis. Strong adhesion between keratinocytes is conferred by the desmosomal cadherins, desmocollin (Dsc) and desmoglein. These constitute two gene families, members of which are differentially expressed in epidermal strata. It has been suggested that this stratum-specific expression regulates keratinocyte differentiation. We tested this hypothesis by misdirecting the expression of the basally abundant desmosomal cadherins Dsc3a and Dsc3b to suprabasal differentiating keratinocytes in transgenic mice. No phenotype was apparent until adulthood, when mice developed variable ventral alopecia and had altered keratinocyte differentiation within affected areas. The follicular changes were reminiscent of changes in transgenic mice with an altered beta-catenin stability. Stabilized beta-catenin and increased beta-catenin transcriptional activity were demonstrated in transgenic mice prior to the phenotypic change and in transgenic keratinocytes as a consequence of transgene expression. Hence, a link between desmosomal cadherins and beta-catenin stability and signaling was demonstrated, and it was shown that desmocollin cadherin expression can affect keratinocyte differentiation. Furthermore, the first function for a "b-type" desmocollin cadherin was demonstrated.

Pathways of airway oxidant formation by house dust mite allergens and viral RNA converge through myosin motors, pannexons and Toll-like receptor 4.

INTRODUCTION: Intracellular reactive oxidant species (ROS) are generated in human airway epithelial cells by the prothrombinase action of Group 1 house dust mite (HDM) allergens and by ligation of viral RNA sensor Toll-like receptors (TLRs). We explored signaling convergence between HDM allergens and TLRs in ROS generation because epithelial cells form the primary barrier against inhaled substances and dictate host responses to allergens and viruses. METHODS: ROS formation by Calu-3 human airway cells was studied by measuring dihydrorhodamine 123 oxidation after activation by polyinosinic:polycytidylic acid (to activate TLR3), CL097 (to activate TLR7), a natural mixture of HDM allergens, or BzATP. RESULTS: TLR4 activation was identified as an indispensable response element for all stimuli, operating downstream from myosin motor activation, pannexon gating for ATP release and the endogenous activation of prothrombin. Exogenous prothrombin activation by HDM allergens was prevented by SGUL 1733, a novel inhibitor of the proteolytic activity of Group 1 HDM allergens, which thus prevented TLR4 from being activated at source. CONCLUSIONS: Our data identify for the first time that endogenously-generated prothrombin and TLR4 form a shared effector mechanism essential to intracellular ROS generation activated by a group 1 HDM allergen (itself a prothrombinase) or by ligation of viral RNA-sensing TLRs. These stimuli operate a confluent signaling pathway in which myosin motors, gating of pannexons, and ADAM 10 lead to prothrombin-dependent activation of TLR4 with a recycling activation of pannexons.

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.

Controlled laser texturing of titanium results in reliable osteointegration.

We have developed a laser-textured superhydrophilic Ti-6Al-4V surface with unique surface chemistry and topography that substantially promotes osteoblast adhesion in culture. Here we investigate the osteointegration of laser-textured implants in an ovine model. Our hypothesis was that laser-textured implants, without any surface coating (LT), would encourage comparable amounts of bone-implant contact and interfacial strength when compared with widely accepted hydroxyapatite (HA) coated implants. Additionally, we hypothesized that LT would significantly increase bony integration compared with machine-finished (MF) and grit-blasted (GB) implants. Forty-eight tapered transcortical pins were implanted into six sheep. Four experimental groups (LT, HA, MF, and GB) were investigated (n = 12) and implants remained in vivo for 6 weeks. Bone apposition rates, interfacial shear strength, and bone-implant contact (BIC) were quantified. The interfacial strength of LT and HA implants were found to be significantly greater than GB (p = 0.032 and p = 0.004) and MF (p = 0.004 and p = 0.004, respectively), but no significant difference between LT and HA implants was observed. Significantly increased BIC was measured adjacent to HA implants when compared with both LT and GB implant surfaces (p = 0.022 and p = 0.006, respectively). No significant difference was found when LT and GB implants were compared. However, all surface finishes encouraged significantly increased BIC when compared with the MF surface. Maximizing implant fixation to host bone is vital for its long-term success. The production of an LT surface is a simple and cheap manufacturing process and this study demonstrated that laser-textured implants are a very promising technical development that warrants further research. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:820-828, 2017.

The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma.