Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

Molecular mechanism of intermediate filament recognition by plakin proteins.

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.

Binding of the periplakin linker requires vimentin acidic residues D176 and E187.

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.

Bioinformatics Analysis of Stromal Molecular Signatures Associated with Breast and Prostate Cancer.

This study aimed to identify stromal molecular signatures associated with breast and prostate cancer. The microarray data GSE26910 was downloaded from Gene Expression Omnibus database, including six invasive breast tumor stroma, six matched normal controls, six invasive prostate tumor stroma, and six matched controls. The differentially expressed genes (DEGs) in invasive breast and prostate tumors stroma were, respectively, identified. Then common stromal genes (B_P.DEGs) were further screened. Protein-protein interaction (PPI) network was constructed and Gene Ontology analysis was performed. Besides, gene-chemical interactions were mapped in Comparative Toxicogenomics Database to screen the chemicals related to feature genes. The results showed that, in total, 16 B_P.DEGs were identified. Thereinto, only seven B_P.DEGs were mapped into PPI, and only four functional modules (adenylate cyclase activating polypeptide 1 (pituitary) receptor type I (ADCYAP1R1) module, aspartoacylase (ASPA) module, glutathione S-transferase mu 5 (GSTM5) module, and periplakin (PPL) module) were involved in important biological processes associated with cancer progression. In addition, the chemicals, such as dihydrotestosterone, apocarotenal, testosterone, and progesterone, were screened for the roles of feature genes in the progression of breast and prostate cancer. In conclusion, ADCYAP1R1, GSTM5, and PPL were stromal molecular signatures and might play a key role in the progression of breast and prostate cancer.

The 4717C > G polymorphism in periplakin modulates sensitivity to EGFR inhibitors.

The use of EGFR inhibitors on oral squamous cell carcinoma (OSCC) as monotherapy yielded modest clinical outcomes and therefore would benefit from biomarkers that could predict which patient subsets are likely to respond. Here, we determined the efficacy of erlotinib in OSCC cell lines, and by comparing sensitive and resistant lines to identify potential biomarkers. We focused on the 4717C > G polymorphism in periplakin (PPL) where the CC genotype was associated with erlotinib resistance. To validate this, erlotinib-resistant cell lines harbouring CC genotype were engineered to overexpress the GG genotype and vice versa. Isogenic cell lines were then studied for their response to erlotinib treatment. We demonstrated that overexpression of the GG genotype in erlotinib-resistant lines sensitized them to erlotinib and inhibition of AKT phosphorylation. Similarly, the expression of the CC genotype conferred resistance to erlotinib with a concomitant increase in AKT phosphorylation. We also demonstrated that cell lines with the CC genotype generally are more resistant to other EGFR inhibitors than those with the GG genotype. Overall, we showed that a specific polymorphism in the PPL gene could confer resistance to erlotinib and other EGFR inhibitors and further work to evaluate these as biomarkers of response is warranted.

Identification of periplakin as a major regulator of lung injury and repair in mice.

Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-ß1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung.

Mammalian Plakins, Giant Cytolinkers: Versatile Biological Functions and Roles in Cancer.

Cancer is a highly lethal disease that is characterized by aberrant cell proliferation, migration, and adhesion, which are closely related to the dynamic changes of cytoskeletons and cytoskeletal-adhesion. These will further result in cell invasion and metastasis. Plakins are a family of giant cytolinkers that connect cytoskeletal elements with each other and to junctional complexes. With various isoforms composed of different domain structures, mammalian plakins are broadly expressed in numerous tissues. They play critical roles in many cellular processes, including cell proliferation, migration, adhesion, and signaling transduction. As these cellular processes are key steps in cancer development, mammalian plakins have in recent years attracted more and more attention for their potential roles in cancer. Current evidence shows the importance of mammalian plakins in various human cancers and demonstrates mammalian plakins as potential biomarkers for cancer. Here, we introduce the basic characteristics of mammalian plakins, review the recent advances in understanding their biological functions, and highlight their roles in human cancers, based on studies performed by us and others. This will provide researchers with a comprehensive understanding of mammalian plakins, new insights into the development of cancer, and novel targets for cancer diagnosis and therapy.

Microtubule-actin crosslinking factor 1 (Macf1) domain function in Balbiani body dissociation and nuclear positioning.

Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead our results suggest that Macf1a functions by linking one cytoskeletal system, cortical actin, to another structure, the Bb, where Macf1a is localized. Through this novel linking process, it dissociates the Bb at the oocyte cortex, thus specifying the AV axis of the oocyte and future egg. To our knowledge, this is also the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker.

Position matters: multiple functions of LINC-dependent chromosome positioning during meiosis.

Chromosome positioning is crucial for multiple chromosomal events, including DNA replication, repair, and recombination. The linker of nucleoskeleton and cytoskeleton (LINC) complexes, which consist of conserved nuclear membrane proteins, were shown to control chromosome positioning and facilitate various biological processes by interacting with the cytoskeleton. However, the precise functions and regulation of LINC-dependent chromosome positioning are not fully understood. During meiosis, the LINC complexes induce clustering of telomeres, forming the bouquet chromosome arrangement, which promotes homologous chromosome pairing. In fission yeast, the bouquet forms through LINC-dependent clustering of telomeres at the spindle pole body (SPB, the centrosome equivalent in fungi) and detachment of centromeres from the SPB-localized LINC. It was recently found that, in fission yeast, the bouquet contributes to formation of the spindle and meiotic centromeres, in addition to homologous chromosome pairing, and that centromere detachment is linked to telomere clustering, which is crucial for proper spindle formation. Here, we summarize these findings and show that the bouquet chromosome arrangement also contributes to nuclear fusion during karyogamy. The available evidence suggests that these functions are universal among eukaryotes. The findings demonstrate that LINC-dependent chromosome positioning performs multiple functions and controls non-chromosomal as well as chromosomal events, and that the chromosome positioning is stringently regulated for its functions. Thus, chromosome positioning plays a much broader role and is more strictly regulated than previously thought.

Loss of periplakin expression is associated with the tumorigenesis of colorectal carcinoma.

Periplakin (PPL), a member of the plakin protein family, has been reported to be down-expressed in urothelial carcinoma. The role of PPL in human colorectal cancer, however, remains largely unknown. Also little is known about the contribution of PPL to the malignant property of colorectal cancer and the intracellular function of PPL. In this study, we demonstrated that PPL was apparently down-expressed in colon carcinomas compared with normal and para-carcinoma tissues, which was correlated with the tumor size. Enforced expression of PPL in HT29 cells inhibited its proliferation evidenced by decreased expression of phosphorylated ERK and PCNA. Furthermore, PPL overexpression could reduce metastasis and epithelial-mesenchymal transition (EMT) of HT29 cells, with decreased expression of N-cadherin, Snail, Slug and α-SMA while increased expression of E-cadherin. On the contrary, the PPL knockdown could promote the cell proliferation, migratory, invasive and EMT ability of HT29 cells. Moreover, enforced expression of PPL induced G1/G0 cell cycle arrest, with decreased cyclin D1, p-Rb and increased expression of p27kib, which could be reversed by PPL knockdown. In addition, PPL overexpression inhibited the growth of colon cancer allograft in vivo. Taken together, acted as a tumor suppressor in colon cancer progression, PPL could be a new biomarker or potential therapeutic target in colon cancer.

[Current approaches to the morphologic diagnosis of different types of congenital epidermolysis bullosa]. / Sovremennye podkhody k morfologicheskoi diagnostike razlichnykh tipov vrozhdennogo bulleznogo epidermoliza.

Congenital epidermolysis bullosa (CEB) is an extensive group of hereditary skin diseases, the differential diagnosis of which is a challenge due to the rarity of this pathology and the diversity of its clinical manifestations. The determination of the type of CEB makes it possible to estimate its prognosis and to facilitate a prenatal diagnosis. AIM: to optimize the morphological diagnosis of different types of CEB. MATERIAL AND METHODS: 28 skin biopsies from 14 patients with different types of CEB were investigated. The investigators performed routine histological examination of skin fragments taken from a bullous area and immunofluorescence antigen mapping using the indirect immunofluorescence test (IIFT) with antibodies against structural proteins of the dermal-epidermal junction (laminin α3, ß3, and γ2 chains, keratins 5 and 14, types VII and XVII collagen, α6 and ß4 integrin subunits, desmoplakin, plectin, kindlin-1, and plakophillin) of the apparently unaffected skin. The intact skin of healthy individuals, which had been obtained during cosmetic operations, was used as controls in IIFT. RESULTS: Immunofluorescence antigen mapping could determine the type of CEB in all cases and in 86% of cases identify the protein, the impaired production of which was responsible for the development of the disease. CONCLUSION: Immunofluorescence antigen mapping is an integral part of the comprehensive morphological diagnosis of CEB, acting as an intermediate between the morphological verification of CEB diagnosis and the targeted search for mutations by a molecular genetic method.

Activation of human γδ T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 µM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 µM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Aberrant DNA hypermethylation reduces the expression of the desmosome-related molecule periplakin in esophageal squamous cell carcinoma.

Periplakin (PPL), a member of the plakin family of proteins that localizes to desmosomes and intermediate filaments, is downregulated in human esophageal squamous cell carcinoma (ESCC). Little is known, however, about the molecular mechanism underlying the regulation of PPL expression and the contribution of PPL loss to the malignant property of the cancer is unclear. We demonstrated that PPL mRNA expression was significantly reduced in ESCC tissues compared with that in normal tissues. Therefore, we hypothesized that CpG hypermethylation is the cause of the downregulation of PPL. Bisulfite-pyrosequencing of 17 cases demonstrated that the frequency of PPL methylation was higher in ESCC tissues than in normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, PPL transcription was induced. Human KYSE270 ESCC cells do not stratify under ordinary culture conditions and rarely produce desmosomes; however, the forced expression of PPL promoted cell stratification. PPL induction also promoted adhesion to extracellular matrix but delayed cell migration. The abundance of desmosome-like structures was greatly increased in PPL transfectant as determined by transmission electron microscopy. Very low expression of another desmosome protein EVPL in ESCC, even in PPL transfectant, also supported the significant role of PPL in desmosome formation and cell stratification. Our results first indicate that the downregulation of PPL mediated by DNA hypermethylation, which may play an important role in the loss of ESCC stratification and likely in metastatic phenotype.

Loss of periplakin expression is associated with pathological stage and cancer-specific survival in patients with urothelial carcinoma of the urinary bladder.

The objective of this study was to determine periplakin expression in normal urothelium and bladder cancer tissues and the relationship to clinicopathological findings. Immunohistochemical staining for periplakin was carried out in 92 archival radical cystectomy specimens, with immunoreactivity being stratified on a 0-6 scale. Immunohistochemical staining for periplakin was shown to be significantly lower in bladder cancer tissues compared to non-cancerous tissues including inflammation,hyperplasia and normal urothelium. Loss of periplakin expression was associated with pathological stage (P=0.04). In multivariate Cox regression analysis, loss of periplakin expression and positive lymph node status were independent prognostic factors for cancer-specific survival (P=0.03 and 0.015; odds ratio=2.29 and 2.66; 95% confidence interval=1.085-4.814 and 1.214-5.845, respectively). This new molecular marker may aid in identifying and selecting bladder cancer patients undergoing radical cystectomy who may potentially benefit from neoadjuvant or adjuvant therapy.

Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility.

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults.DOI: http://dx.doi.org/10.7554/eLife.01888.001.

Plakins, a versatile family of cytolinkers: roles in skin integrity and in human diseases.

The plakin family consists of giant proteins involved in the cross-linking and organization of the cytoskeleton and adhesion complexes. They further modulate several fundamental biological processes, such as cell adhesion, migration, and polarization or signaling pathways. Inherited and acquired defects of plakins in humans and in animal models potentially lead to dramatic manifestations in the skin, striated muscles, and/or nervous system. These observations unequivocally demonstrate the key role of plakins in the maintenance of tissue integrity. Here we review the characteristics of the mammalian plakin members BPAG1 (bullous pemphigoid antigen 1), desmoplakin, plectin, envoplakin, epiplakin, MACF1 (microtubule-actin cross-linking factor 1), and periplakin, highlighting their role in skin homeostasis and diseases.

Brain-metastatic triple-negative breast cancer cells regain growth ability by altering gene expression patterns.

UNLABELLED: BACKGROUD/AIM: Triple-negative breast cancer (TNBC) frequently metastasizes to the brain (BrM). However, genes responsible for BrM of TNBC are yet to be identified. MATERIALS AND METHODS: Gene expression profiling of TNBC and BrM was conducted, and studies with cultured cells in vitro were performed to verify functions of genes identified in these analyses. RESULTS: According to gene expression analyses of TNBC and BrM, periplakin (PPL) and mitogen-activated protein kinase 13 (MAPK13) were chosen for further investigations. PPL and MAPK13 were highly expressed in TNBC compared to BrM. While silencing of either PPL or MAPK13 in TNBC cells increased cell growth and reduced cell motility, overexpression of either PPL or MAPK13 in BrM cells, retarded growth rates and facilitated cell motility. CONCLUSION: Gene expression patterns in TNBC and BrM reflect cancer cell growth in regions of metastasis.

Hinged plakin domains provide specialized degrees of articulation in envoplakin, periplakin and desmoplakin.

Envoplakin, periplakin and desmoplakin are cytoskeletal proteins that provide structural integrity within the skin and heart by resisting shear forces. Here we reveal the nature of unique hinges within their plakin domains that provides divergent degrees of flexibility between rigid long and short arms composed of spectrin repeats. The range of mobility of the two arms about the hinge is revealed by applying the ensemble optimization method to small-angle X-ray scattering data. Envoplakin and periplakin adopt 'L' shaped conformations exhibiting a 'helicopter propeller'-like mobility about the hinge. By contrast desmoplakin exhibits essentially unrestricted mobility by 'jack-knifing' about the hinge. Thus the diversity of molecular jointing that can occur about plakin hinges includes 'L' shaped bends, 'U' turns and fully extended 'I' orientations between rigid blocks of spectrin repeats. This establishes specialised hinges in plakin domains as a key source of flexibility that may allow sweeping of cellular spaces during assembly of cellular structures and could impart adaptability, so preventing irreversible damage to desmosomes and the cell cytoskeleton upon exposure to mechanical stress.

Cholestasis induces reversible accumulation of periplakin in mouse liver.

BACKGROUND: Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in non-cutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis. METHODS: Time- and context-dependent expression of PPL in various mouse models of hepatic and renal disorders were examined by immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions. RESULTS: The hepatic expression of PPL was significantly decreased in Fxr-/- mice. In contrast, the expression was dramatically increased during cholestasis, with massive PPL accumulation observed at the boundaries of hepatocytes in wild-type mice. Interestingly, the hepatic accumulation of PPL resulting from cholestasis was reversible. In addition, similar accumulation of PPL at cellular boundaries was found in epithelial cells around renal tubules upon ureteral obstruction. CONCLUSIONS: PPL may be involved in the temporal accommodation to fluid stasis in different tissues. Further examination of the roles for PPL may lead to the discovery of a novel mechanism for cellular protection by cytolinkers that is applicable to many tissues and in many contexts.