Characterization of inter-Sertoli cell tight and gap junctions in the testis of turtle: Protect the developing germ cells from an immune response.

It is conceivable that early developing germ cells must across the basal to the luminal region of seminiferous tubules (STs) during spermatogenesis is associated with extensive restructuring of junctional complex. However, very limited information is documented about these junctional complexes in reptiles. In the present study we have determined the localization of inter-Sertoli cell tight junctions (TJ's), protein CLDN11 and gap junction protein Cx43 during spermatogenesis in the testis. In early spermatogenesis, weak immunoreactivity of CLDN11and focal localization of Cx43 was observed around the Sertoli cell in the luminal region, but completely delaminated from the basal compartment of STs. In late spermatogenesis, strong focal to linear localization of CLDN11and Cx43 was detected at the points of contact between two Sertoli cells and around the early stages of primary spermatocytes in the basal compartment of STs. In late spermatogenesis, localization of CLDN11and Cx43 was drastically reduced and seen only around Sertoli cells and spermatogonia near the basal lamina. However, transmission electron microscopy revealed that inter-Sertoli cell tight junctions were present within the basal compartment of STs, leaving the spermatogonia and early primary spermatocytes in the basal region during mid spermatogenesis. Gap junctions were observed between Sertoli cells, and Sertoli cells with spermatogonia and primary spermatocytes throughout spermatogenesis. Moreover, adherens and hemidesmosomes junctions were observed during spermatogenesis. The above findings collectively suggest that the intensity and localization of TJ's and gap junctions vary according to the spermatogenetic stages that might be protected the developing germ cells from own immune response.

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

Hemidesmosome integrity protects the colon against colitis and colorectal cancer.

OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6ß4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6ΔIEC) or in adults (α6ΔIEC-TAM). RESULTS: Strikingly, all α6ΔIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6ΔIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1ß secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.

A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.-Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes.

Leucine-enkephalin promotes wound repair through the regulation of hemidesmosome dynamics and matrix metalloprotease.

The skin responds to environmental stressors by coordinated actions of neuropeptides and their receptors. An endogenous peptide for δ-opioid receptor (DOPr), Leu-enkephalin (L-ENK), is expressed in the skin and its expression is altered in pathological conditions. Although the importance of DOPr is rapidly gaining recognition, the molecular mechanisms underlying its effects on wound healing are largely undefined. We show here that L-ENK induced activation of Erk, P90(RSK), and Elk-1 and promoted the disruption of hemidesmosomes and the expression of matrix metalloprotease (MMP)-2 and MMP-9, important processes for wound healing. Treatment with Erk inhibitor blocked activation of P90(RSK) and Elk-1 and significantly blunted wound repair. Therefore, our results suggest that activation of Erk and its downstream effectors, P90(RSK) and Elk-1, are critical for DOPr-mediated skin homeostasis.

The role of hemidesmosomes and focal contacts in the skin visualized by dual-color live cell imaging.

Live cell imaging is a powerful tool to elucidate dynamics of protein(s). Our group has concentrated on dynamics of two major cell-matrix adhesion devices, hemidesmosome and focal contact in the keratinocytes. Firstly, we observed the fate of hemidesmosome protein or focal contact protein by single-color live cell imaging in the physiological setting of keratinocytes. Both hemidesmosome proteins and focal contact proteins were highly dynamic. Next, in order to observe the interaction between hemidesmosome protein and focal contact protein, we observed the fate of these proteins at the same time by dual-color live cell imaging in physiological setting and in wound setting of keratinocytes. These hemidesmosome proteins and focal contact proteins showed individual dynamics with minimal overlap expressions in physiological settings. In sharp contrast, both proteins showed highly regulated interaction in wound setting of keratinocytes. Finally, we observed the fate of BP180 protein, which is a major target of autoimmune bullous disease, bullous pemphigoid, and component of hemidesmosome, under the existence of anti-BP180 autoantibody. In results, under such a circumstance, BP180 molecules were internalized and thus keratinocyte showed weakened adhesion to the cell matrix. Our work has elucidated dynamic aspects of cell-matrix adhesion devices under both physiological and pathological conditions.

Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium.

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through ß4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6ß4 integrin clustering into hemidesmosomes.

Penfigoide ampolloso: guía de manejo práctico / Bullous Pemphigoid: Clinical Practice Guidelines

El penfigoide ampolloso (PA) es una enfermedad ampollosa autoinmune causada por anticuerpos dirigidos contra componentes de la membrana basal. La mayoría de estos anticuerpos son de clase IgG y se unen principalmente a 2 proteínas hemidesmosómicas, los antígenos BP180 y BP230.Se trata de la enfermedad ampollosa más frecuente en los países desarrollados en la población adulta, con una incidencia estimada en nuestro medio de 0,2 a 3 casos nuevos por cada 100.000 habitantes. Afecta principalmente a pacientes ancianos, aunque puede presentarse en jóvenes e incluso niños. En los últimos años hemos incorporado a la práctica clínica nuevas técnicas diagnósticas (ELISA para BP180) y nuevos fármacos con distintas dianas terapéuticas en el tratamiento del PA. Esto ha permitido un avance en el conocimiento y el manejo de esta entidad. A pesar de ello, no existen a día de hoy unas guías consensuadas internacionalmente sobre el manejo del PA. Este artículo es una revisión actualizada de la literatura científica sobre el tratamiento de esta enfermedad, destacando las recomendaciones basadas en la evidencia y desde un punto de vista práctico basado en la experiencia del manejo diario de estos pacientes

Bullous pemphigoid (BP) is an autoimmune subepidermal bullous disease in which autoantibodies are directed against components of the basement membrane. Most of these antibodies belong to the immunoglobulin G class and bind principally to 2 hemidesmosomal proteins: the 180-kD antigen (BP180) and the 230-kD antigen (BP230). It is the most common blistering disease in the adult population in developed countries, with an estimated incidence in Spain of 0.2 to 3 cases per 100,000 inhabitants per year. The disease primarily affects older people, although it can also occur in young people and even in children. In recent years, advances in clinical practice have led to a better understanding and improved management of this disorder. These advances include new diagnostic techniques, such as enzyme-linked immunosorbent assay for BP180 and new drugs for the treatment of BP, with diverse therapeutic targets. There is, however, still no international consensus on guidelines for the management of BP. This article is an updated review of the scientific literature on the treatment of BP. It focuses primarily on evidence-based recommendations and is written from a practical standpoint based on experience in the routine management of this disease

Expression of odontogenic ameloblast-associated and amelotin proteins in the junctional epithelium.

Two novel proteins - odontogenic ameloblast-associated protein and amelotin - have recently been identified in maturation-stage ameloblasts and in the junctional epithelium. This article reviews the structure and function of the junctional epithelium, the pattern of expression of odontogenic ameloblast-associated and amelotin proteins and the potential involvement of these proteins in the formation and regeneration of the junctional epithelium.

Keratins mediate localization of hemidesmosomes and repress cell motility.

The keratin (K)-hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells.

Actinin-4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions.

During wound repair, epidermal cells at the edge of an injury establish front-rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin-4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin-4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin-4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin-332 matrix. In actinin-4-knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6ß4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin-4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.

New insights in wound response and repair of epithelium.

Epithelial wounds usually heal relatively quickly, but repair may be impaired by environmental stressors, such as hypoxic or diabetic states, rendering patients vulnerable to a number of corneal pathologies. Though this response appears simple, at first, years of research have uncovered the complicated biochemical pathways coordinating the wound healing response. Here, we investigate signaling cascades and individual proteins involved in the corneal epithelium's self-repair. We will explore how an epithelial cell migrates across the wound bed and attaches itself to its new post-injury surroundings, including its neighboring cells and the basement membrane, through focal adhesions and hemidesmosomes. We will also discuss how the cell coordinates this motion physiologically, through calcium signaling and protein phosphorylation, focusing on the communication through purinergic, glutamatergic, and growth factor receptors. Many of these aspects reflect and can be extended to similar epithelial surfaces, and can be used to facilitate wound healing in patients with various underlying pathologies. The collective library of laboratory and clinical research done around the world has demonstrated how important precise regulation of these processes is in order for the injured corneal epithelium to properly heal.

Hemidesmosomes and focal contact proteins: functions and cross-talk in keratinocytes, bullous diseases and wound healing.

The outer most layer of the skin, the epidermis, is attached to the dermis via a sheet of extracellular matrix proteins termed the basement membrane zone (BMZ). In the intact skin, adhesion of the keratinocytes in the basal layer of the epidermis to the BMZ is facilitated primarily by hemidesmosomes which associate with the keratin cytoskeleton. Cultured keratinocytes do not assemble bona fide hemidesmosomes although hemidesmosome protein clusters (stable anchoring contacts) are found along the substrate-attached surface of the cells and towards the leading edge of keratinocytes repopulating scratch wounds. Actin cytoskeleton-associated matrix adhesion devices termed focal contacts are not thought to play an important role in the adhesion of keratinocytes to the BMZ in intact skin but are prominent in cultured keratinocytes where they are believed to regulate cell migration. We review the molecular components, functions, dynamics and cross-talk of hemidesmosomes and focal contacts in keratinocytes. In addition, we briefly describe what is known about their role in autoimmune and genetic blistering diseases of the skin. We also discuss recent publications which indicate, contrary to expectation, that certain focal contact proteins retard keratinocyte migration while hemidesmosomal proteins regulate directed keratinocyte motility during wound healing.

PAT-12, a potential anti-nematode target, is a new spectraplakin partner essential for Caenorhabditis elegans hemidesmosome integrity and embryonic morphogenesis.

Caenorhabditis elegans embryonic elongation depends on both epidermal and muscle cells. The hemidesmosome-like junctions, commonly called fibrous organelles (FOs), that attach the epidermis to the extracellular matrix ensure muscle anchoring to the cuticular exoskeleton and play an essential role during elongation. To further define how hemidesmosomes might control elongation, we searched for factors interacting with the core hemidesmosome component, the spectraplakin homolog VAB-10. Using the VAB-10 plakin domain as bait in a yeast two-hybrid screen, we identified the novel protein T17H7.4. We also identified T17H7.4 in an independent bioinformatic search for essential nematode-specific proteins that could define novel anti-nematode drug or vaccine targets. Interestingly, T17H7.4 corresponds to the C. elegans equivalent of the parasitic OvB20 antigen, and has a characteristic hemidesmosome distribution. We identified two mutations in T17H7.4, one of which defines the uncharacterized gene pat-12, previously identified in screens for genes required for muscle assembly. Using isoform-specific GFP constructs, we showed that one pat-12 isoform with a hemidesmosome distribution can rescue a pat-12 null allele. We further found that lack of pat-12 affects hemidesmosome integrity, with marked defects at the apical membrane. PAT-12 defines a novel component of C. elegans hemidesmosomes, which is required for maintaining their integrity. We suggest that PAT-12 helps maintaining VAB-10 attachment with matrix receptors.

Cross-talk between hemidesmosomes and focal contacts: understanding subepidermal blistering diseases.

Because hemidesmosomes and focal contacts (FCs) play major roles in epidermal wound healing and in the pathogenesis of subepidermal blistering diseases, it is of particular importance to understand their cross-talk in the regulation of their assembly and disassembly. In this issue, Ozawa et al. demonstrate that hemidesmosome-enriched protein complex (HPC) and FC dynamics are tightly coregulated in keratinocytes undergoing migration by employing HaCat cells that express fluorescent protein-tagged beta4 integrin and alpha-actinin as markers of HPCs and FCs.

The making of hemidesmosome structures in vivo.

Hemidesmosomes are evolutionarily conserved attachment complexes linked to intermediate filaments that connect epithelial cells to the extracellular matrix. They provide tissue integrity and resistance to mechanical forces. Alterations in hemidesmosome structures are responsible for skin blistering, carcinoma invasion, and wound-healing defects. Valuable information about hemidesmosome assembly and disassembly has been obtained from in vitro cell culture studies. However, how these processes take place in vivo still remains elusive. Here, we discuss recent data about the formation and reorganization of hemidesmosomes in several in vivo model systems, particularly zebrafish and Caenorhabditis elegans, focusing on various factors affecting their dynamics. Mechanisms found in different organisms reveal that hemidesmosome formation and maintenance in vivo are carefully controlled by ECM protein folding, ECM-receptor expression and trafficking, and by post-translational modification of hemidesmosome components. These findings validate and extend the in vitro studies, and shed light on our understanding about hemidesmosomes across species.

Dynamic relationship of focal contacts and hemidesmosome protein complexes in live cells.

Epidermal cells adhere to the basement membrane zone through cell-matrix junctions termed hemidesmosomes. During wound healing, hemidesmosomes are disassembled to allow keratinocytes to move over wound sites. Such movement is mediated by both hemidesmosome protein complexes (HPCs) and focal contacts (FCs). In this study, we analyzed the interaction between HPCs and FCs in live HaCat cells expressing yellow fluorescent protein (YFP)-tagged beta4 integrin and cyan fluorescent protein (CFP)-tagged alpha-actinin as markers of HPCs and FCs, respectively. In HaCat cells migrating to repopulate wounds, FC proteins cluster rapidly in the direction of the wound. HPC assembly then follows and the newly formed HPCs occupy sites vacated by the disassembled FCs. HPC dynamics are dramatically reduced, and HaCat cells cease migration upon treatment with reagents that affect FC integrity/function. Upon treatment with reagents that destabilize HPCs, the dynamics of FCs in HaCat cells at the edges of wounds are enhanced, although FC assembly is irregular and the migration of the cells is aberrant. We also show that the complex interaction between hemidesmosomes and FCs in keratinocytes is myosin dependent and requires energy. In summary, we suggest that HPCs and FCs dynamics are tightly co-regulated in keratinocytes undergoing migration during wound healing.

Flii control: balancing migration and adhesion.

Wound healing in the skin requires a compromise between adhesion and migration. Both processes include modulation of the cytoskeleton, cell-surface receptors, and receptor ligands., In this issue, Kopecki et al. demonstrate that overexpression of Flii, an actin-remodeling protein, impedes wound healing but inhibits hemidesmosome formation. In contrast, Flii deficiency results in enhanced wound healing while promoting hemidesmosome assembly. We discuss potential mechanisms that could explain how this unique gelsolin family member might regulate both stable keratinocyte adhesion and motility.

Flightless I regulates hemidesmosome formation and integrin-mediated cellular adhesion and migration during wound repair.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.