Mechanotransduction through hemidesmosomes during aging and longevity.

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.

Bazi Bushen alleviates skin senescence by orchestrating skin homeostasis in SAMP6 mice.

Bazi Bushen, a Chinese-patented drug with the function of relieving fatigue and delaying ageing, has been proven effective for extenuating skin senescence. To investigate the potential mechanism, senescence-accelerated mouse prone 6 (SAMP6) was intragastrically administered with Bazi Bushen for 9 weeks to induce skin homeostasis. Skin homeostasis is important in mitigating skin senescence, and it is related to many factors such as oxidative stress, SASP, apoptosis, autophagy and stem cell. In our study, skin damage in SAMP6 mice was observed using HE, Masson and SA-ß-gal staining. The content of hydroxyproline and the activities of SOD, MDA, GSH-PX and T-AOC in the skin were measured using commercial assay kits. The level of SASP factors (IL-6, IL-1ß, TNF-α, MMP2 and MMP9) in skin were measured using ELISA kits. The protein expressions of p16, p21, p53, Bax, Bcl-2, Cleaved caspase-3, LC3, p62, Beclin1, OCT4, SOX2 and NANOG were measured by western blotting. The expression of ITGA6 and COL17A1 was measured by immunofluorescence staining and western blotting. Our findings demonstrated that Bazi Bushen alleviated skin senescence by orchestrating skin homeostasis, reducing the level of oxidative stress and the expression of SASP, regulating the balance of apoptosis and autophagy and enhancing the protein expressions of ITGA6 and COL17A1 to improve skin structure in SAMP6 mice. This study indicated that Bazi Bushen could serve as a potential therapy for alleviating skin senescence.

Regulation of hemidesmosome dynamics and cell signaling by integrin α6ß4.

Hemidesmosomes (HDs) are specialized multiprotein complexes that connect the keratin cytoskeleton of epithelial cells to the extracellular matrix (ECM). In the skin, these complexes provide stable adhesion of basal keratinocytes to the underlying basement membrane. Integrin α6ß4 is a receptor for laminins and plays a vital role in mediating cell adhesion by initiating the assembly of HDs. In addition, α6ß4 has been implicated in signal transduction events that regulate diverse cellular processes, including proliferation and survival. In this Review, we detail the role of α6ß4 in HD assembly and beyond, and we discuss the molecular mechanisms that regulate its function.

Effects of TP63 mutations on keratinocyte adhesion and migration.

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced AEC iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

Periodic subcellular structures undergo long-range synchronized reorganization during C. elegans epidermal development.

Periodic pattern formation on the cellular and tissue scale is an important process and has been extensively studied. However, periodic pattern formation at the subcellular level still remains poorly understood. The C. elegans epidermis displays a highly ordered parallel stripe pattern as part of its subcellular structure, making it an ideal model to study the formation and reorganization of periodic patterns within cells. Here, we show that the initial formation of periodic striped patterns in the C. elegans epidermis is dependent on actin and spectrin, and requires the apical membrane attachment structures for maintenance. The periodic subcellular structures do not accommodate cell growth by continuously making new stripes. Instead, they increase the number of stripes by going through one round of uniform duplication, which is independent of the increasing epidermal length or the developmental cycles. This long-range synchronized reorganization of subcellular structures is achieved by physical links established by extracellular collagens together with extension forces generated from epidermal cell growth. Our studies uncover a novel strategy employed by evenly spaced and interlinked subcellular structures to maintain their integrity and equidistribution during cell growth and tissue development.

The plakin domain of C. elegans VAB-10/plectin acts as a hub in a mechanotransduction pathway to promote morphogenesis.

Mechanical forces can elicit a mechanotransduction response through junction-associated proteins. In contrast to the wealth of knowledge available for focal adhesions and adherens junctions, much less is known about mechanotransduction at hemidesmosomes. Here, we focus on the C. elegans plectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. In C. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SRs) or a partially hidden Src homology 3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 or SR8 domains in combination with mutations affecting mechanotransduction, or just the part of SR5 shielding the SH3 domain, induced embryonic elongation arrest because hemidesmosomes collapse. Notably, recruitment of GIT-1, the first mechanotransduction player, requires the SR5 domain and the hemidesmosome transmembrane receptor LET-805. Furthermore, molecular dynamics simulations confirmed that forces acting on VAB-10 could make the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly indicate that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.

Tetraspanin CD151 and integrin α3ß1 contribute to the stabilization of integrin α6ß4-containing cell-matrix adhesions.

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3ß1 and α6ß4; however, its precise function in keratinocyte adhesion remains elusive. In this study, we investigated the role of CD151 in the formation and maintenance of laminin-associated adhesions. We show that CD151, through binding to integrin α3ß1, plays a critical role in the stabilization of an adhesion structure with a distinct molecular composition of hemidesmosomes with tetraspanin features. These hybrid cell-matrix adhesions, which are formed early during cell adhesion and spreading and at later stages of cell spreading, are present in the central region of the cells. They contain the CD151-α3ß1/α6ß4 integrin complexes and the cytoskeletal linker protein plectin, but are not anchored to the keratin filaments. In contrast, hemidesmosomes, keratin filament-associated adhesions that contain integrin α6ß4, plectin, BP180 (encoded by COL17A1) and BP230 (encoded by DST), do not require CD151 for their formation or maintenance. These findings provide new insights into the dynamic and complex regulation of adhesion structures in keratinocytes and the pathogenic mechanisms underlying skin blistering diseases caused by mutations in the gene for CD151.

Assembly of the ß4-Integrin Interactome Based on Proximal Biotinylation in the Presence and Absence of Heterodimerization.

Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific ß4-integrin that, as α6ß4-heterodimer, forms the core of HDs. The analysis identified ∼150 proteins that were specifically labeled by BirA-tagged integrin-ß4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-ß4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate ß4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of α6ß4-heterodimers, the assembly of ß4-interactome was not strictly dependent on α6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the ß4-integrin.

BP180 dysfunction triggers spontaneous skin inflammation in mice.

BP180, also known as collagen XVII, is a hemidesmosomal component and plays a key role in maintaining skin dermal/epidermal adhesion. Dysfunction of BP180, either through genetic mutations in junctional epidermolysis bullosa (JEB) or autoantibody insult in bullous pemphigoid (BP), leads to subepidermal blistering accompanied by skin inflammation. However, whether BP180 is involved in skin inflammation remains unknown. To address this question, we generated a BP180-dysfunctional mouse strain and found that mice lacking functional BP180 (termed ΔNC16A) developed spontaneous skin inflammatory disease, characterized by severe itch, defective skin barrier, infiltrating immune cells, elevated serum IgE levels, and increased expression of thymic stromal lymphopoietin (TSLP). Severe itch is independent of adaptive immunity and histamine, but dependent on increased expression of TSLP by keratinocytes. In addition, a high TSLP expression is detected in BP patients. Our data provide direct evidence showing that BP180 regulates skin inflammation independently of adaptive immunity, and BP180 dysfunction leads to a TSLP-mediated itch. The newly developed mouse strain could be a model for elucidation of disease mechanisms and development of novel therapeutic strategies for skin inflammation and BP180-related skin conditions.

BP180/Collagen XVII: A Molecular View.

BP180 is a type II collagenous transmembrane protein and is best known as the major autoantigen in the blistering skin disease bullous pemphigoid (BP). The BP180 trimer is a central component in type I hemidesmosomes (HD), which cause the adhesion between epidermal keratinocytes and the basal lamina, but BP180 is also expressed in several non-HD locations, where its functions are poorly characterized. The immunological roles of intact and proteolytically processed BP180, relevant in BP, have been subject to intensive research, but novel functions in cell proliferation, differentiation, and aging have also recently been described. To better understand the multiple physiological functions of BP180, the focus should return to the protein itself. Here, we comprehensively review the properties of the BP180 molecule, present new data on the biochemical features of its intracellular domain, and discuss their significance with regard to BP180 folding and protein-protein interactions.

Hemidesmosome-Related Keratin Filament Bundling and Nucleation.

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

CCAR-1 affects hemidesmosome biogenesis by regulating unc-52/perlecan alternative splicing in the C. elegans epidermis.

Hemidesmosomes are epithelial-specific attachment structures that maintain tissue integrity and resist tension. Despite their importance, how hemidesmosomes are regulated at the post-transcriptional level is poorly understood. Caenorhabditiselegans hemidesmosomes (CeHDs) have a similar structure and composition to their mammalian counterparts, making C. elegans an ideal model for studying hemidesmosomes. Here, we focus on the transcription regulator CCAR-1, identified in a previous genetic screen searching for enhancers of mutations in the conserved hemidesmosome component VAB-10A (known as plectin in mammals). Loss of CCAR-1 function in a vab-10(e698) background results in CeHD disruption and muscle detachment from the epidermis. CCAR-1 regulates CeHD biogenesis, not by controlling the transcription of CeHD-related genes, but by affecting the alternative splicing of unc-52 (known as perlecan or HSPG2 in mammals), the predicted basement extracellular matrix (ECM) ligand of CeHDs. CCAR-1 physically interacts with HRP-2 (hnRNPR in mammals), a splicing factor known to mediate unc-52 alternative splicing to control the proportions of different UNC-52 isoforms and stabilize CeHDs. Our discovery underlines the importance of post-transcriptional regulation in hemidesmosome reorganization. It also uncovers previously unappreciated roles of CCAR-1 in alternative splicing and hemidesmosome biogenesis, shedding new light on the mechanisms through which mammalian CCAR1 functions in tumorigenesis.

A mutation found in esophageal cancer alters integrin ß4 mRNA splicing.

Integrin ß4 (CD104, mRNA: ITGß4) contributes to anchoring cells to the extracellular matrix and is regulated in many cancer types where it contributes to tumor progression. One splice variant, integrin ß4E, is poorly characterized. We extracted several mutations from tumor samples within ITGB4 near the splice site that controls ITGß4E production, and computational analysis predicted six of these would alter splicing to alter ITGß4E abundance. One of these mutations, from an esophageal squamous cell carcinoma sample, was predicted to increase splicing toward ITGß4E. We verified this effect using a minigene, and observed that integrin ß4E slows esophageal squamous cell migration while other variants enhance migration, demonstrating that integrin ß4E regulation through mutations may contribute to esophageal squamous cell tumorigenesis.

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment.

The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal-epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein's triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain's folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly.

The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy.

Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.

Structural proteins of the dermal-epidermal junction targeted by autoantibodies in pemphigoid diseases.

The dermal-epidermal junction consists of a network of several interacting structural proteins that strengthen adhesion and mediate signalling events. This structural network consists of hemidesmosomal-anchoring filament complexes connecting the basal keratinocytes to the basement membrane. The anchoring filaments in turn interact with the anchoring fibrils to attach the basement membrane to the underlying dermis. Several of these structural proteins are recognized by autoantibodies in pemphigoid diseases, a heterogeneous group of clinically and immunopathologically diverse entities. Targeted proteins include the two intracellular plakins, plectin isoform 1a and BP230 (also called bullous pemphigoid antigen (BPAG) 1 isoform e (BPAG1e)). Plectin 1a and BP230 are connected to the intermediate filaments and to the cell surface receptor α6ß4 integrin, which in turn is connected to laminin 332, a component of the anchoring filaments. Further essential adhesion proteins are BP180, a transmembrane protein, laminin γ1 and type VII collagen. Latter protein is the major constituent of the anchoring fibrils. Mutations in the corresponding genes of these adhesion molecules lead to inherited epidermolysis bullosa emphasizing the importance of these proteins for the integrity of the dermal-epidermal junction. This review will provide an overview on the structure and function of the proteins situated in the dermal-epidermal junction targeted by autoantibodies.

UV exposure modulates hemidesmosome plasticity, contributing to long-term pigmentation in human skin.

Human skin colour, ie pigmentation, differs widely among individuals, as do their responses to various types of ultraviolet radiation (UV) and their risks of skin cancer. In some individuals, UV-induced pigmentation persists for months to years in a phenomenon termed long-lasting pigmentation (LLP). It is unclear whether LLP is an indicator of potential risk for skin cancer. LLP seems to have similar features to other forms of hyperpigmentation, eg solar lentigines or age spots, which are clinical markers of photodamage and risk factors for precancerous lesions. To investigate what UV-induced molecular changes may persist in individuals with LLP, clinical specimens from non-sunburn-inducing repeated UV exposures (UVA, UVB or UVA + UVB) at 4 months post-exposure (short-term LLP) were evaluated by microarray analysis and dataset mining. Validated targets were further evaluated in clinical specimens from six healthy individuals (three LLP+ and three LLP-) followed for more than 9 months (long-term LLP) who initially received a single sunburn-inducing UVA + UVB exposure. The results support a UV-induced hyperpigmentation model in which basal keratinocytes have an impaired ability to remove melanin that leads to a compensatory mechanism by neighbouring keratinocytes with increased proliferative capacity to maintain skin homeostasis. The attenuated expression of SOX7 and other hemidesmosomal components (integrin α6ß4 and plectin) leads to increased melanosome uptake by keratinocytes and points to a spatial regulation within the epidermis. The reduced density of hemidesmosomes provides supporting evidence for plasticity at the epidermal-dermal junction. Altered hemidesmosome plasticity, and the sustained nature of LLP, may be mediated by the role of SOX7 in basal keratinocytes. The long-term sustained subtle changes detected are modest, but sufficient to create dramatic visual differences in skin colour. These results suggest that the hyperpigmentation phenomenon leading to increased interdigitation develops in order to maintain normal skin homeostasis in individuals with LLP.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and ß4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.

Implant surface physicochemistry affects keratinocyte hemidesmosome formation.

Previous studies have shown hydrophilic/hydrophobic implant surfaces stimulate/hinder osseointegration. An analogous concept was applied here using common biological functional groups on a model surface to promote oral keratinocytes (OKs) proliferation and hemidesmosomes (HD) to extend implant lifespans through increased soft tissue attachment. However, it is unclear what physicochemistry stimulates HDs. Thus, common biological functional groups (NH2 , OH, and CH3 ) were functionalized on glass using silanization. Non-functionalized plasma-cleaned glass and H silanization were controls. Surface modifications were confirmed with X-ray photoelectron spectroscopy and water contact angle. The amount of bovine serum albumin (BSA) and fibrinogen, and BSA thickness, were assessed to understand how adsorbed protein properties were influenced by physicochemistry and may influence HDs. OKs proliferation was measured, and HDs were quantified with immunofluorescence for collagen XVII and integrin ß4. Plasma-cleaned surfaces were the most hydrophilic group overall, while CH3 was the most hydrophobic and OH was the most hydrophilic among functionalized groups. Modification with the OH chemical group showed the highest OKs proliferation and HD expression. The OKs response on OH surfaces appeared to not correlate to the amount or thickness of adsorbed model proteins. These results reveal relevant surface physicochemical features to favor HDs and improve implant soft tissue attachment.