Loss-of-function desmoplakin I and II mutations underlie dominant arrhythmogenic cardiomyopathy with a hair and skin phenotype.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is an inherited, frequently underdiagnosed disorder, which can predispose individuals to sudden cardiac death. Rare, recessive forms of AC can be associated with woolly hair and palmoplantar keratoderma, but most autosomal dominant AC forms have been reported to be cardiac specific. Causative mutations frequently occur in desmosomal genes including desmoplakin (DSP). OBJECTIVES: In this study, we systematically investigated the presence of a skin and hair phenotype in heterozygous DSP mutation carriers with AC. METHODS: Six AC pedigrees with 38 carriers of a dominant loss-of-function (nonsense or frameshift) mutation in DSP were evaluated by detailed clinical examination (cardiac, hair and skin) and molecular phenotyping. RESULTS: All carriers with mutations affecting both major DSP isoforms (DSPI and II) were observed to have curly or wavy hair in the pedigrees examined, except for members of Family 6, where the position of the mutation only affected the cardiac-specific isoform DSPI. A mild palmoplantar keratoderma was also present in many carriers. Sanger sequencing of cDNA from nonlesional carrier skin suggested degradation of the mutant allele. Immunohistochemistry of patient skin demonstrated mislocalization of DSP and other junctional proteins (plakoglobin, connexin 43) in the basal epidermis. However, in Family 6, DSP localization was comparable with control skin. CONCLUSIONS: This study identifies a highly recognizable cutaneous phenotype associated with dominant loss-of-function DSPI/II mutations underlying AC. Increased awareness of this phenotype among healthcare workers could facilitate a timely diagnosis of AC in the absence of overt cardiac features.

Estrogen-dependent sushi domain containing 3 regulates cytoskeleton organization and migration in breast cancer cells.

Aromatase inhibitors (AIs) are the standard endocrine therapy for postmenopausal breast cancer; however, currently used biomarkers, such as, estrogen receptor-alpha/progesterone receptor (ERα/PR), predict only slightly more than half of the potential responders to AI treatment. To identify novel markers of AI responsiveness, a genome-wide microarray analysis was performed using primary breast tumor samples from 50 postmenopausal women who later developed metastatic breast cancer. Sushi domain containing 3 (SUSD3) is a significantly differentially expressed gene, with 3.38-fold higher mRNA levels in AI-responsive breast tumors vs non-responders (P<0.001). SUSD3 was highly expressed in ERα-positive breast tumors and treatment with estradiol increased SUSD3 expression in ERα-positive breast cancer cells. Treatment with an antiestrogen or ERα knockdown abolished basal and estradiol-dependent SUSD3 expression. Recruitment of ERα upstream of the transcription start site of SUSD3 was demonstrated by chromatin immunoprecipitation-PCR. Flow cytometric analysis of SUSD3-knockdown cells revealed blunted estradiol effects on progression into S and M phases. SUSD3 was localized to the plasma membrane of breast cancer cells. SUSD3 knockdown decreased the appearance of actin-rich protrusions, stress fibers and large basal focal adhesions, while increasing the presence of cortical actin concomitant with a decrease in Rho and focal adhesion kinase activity. SUSD3-deficient cells demonstrated diminished cell spreading, cell-cell adhesion and motility. In conclusion, SUSD3 is a novel promoter of estrogen-dependent cell proliferation and regulator of cell-cell and cell-substrate interactions and migration in breast cancer. It may serve as a novel predictor of response to endocrine therapy and potential therapeutic target.

A role for caveolin-1 in desmoglein binding and desmosome dynamics.

Desmoglein-2 (Dsg2) is a desmosomal cadherin that is aberrantly expressed in human skin carcinomas. In addition to its well-known role in mediating intercellular desmosomal adhesion, Dsg2 regulates mitogenic signaling that may promote cancer development and progression. However, the mechanisms by which Dsg2 activates these signaling pathways and the relative contribution of its signaling and adhesion functions in tumor progression are poorly understood. In this study we show that Dsg2 associates with caveolin-1 (Cav-1), the major protein of specialized membrane microdomains called caveolae, which functions in both membrane protein turnover and intracellular signaling. Sequence analysis revealed that Dsg2 contains a putative Cav-1-binding motif. A permeable competing peptide resembling the Cav-1 scaffolding domain bound to Dsg2, disrupted normal Dsg2 staining and interfered with the integrity of epithelial sheets in vitro. Additionally, we observed that Dsg2 is proteolytically processed; resulting in a 95-kDa ectodomain shed product and a 65-kDa membrane-spanning fragment, the latter of which localizes to lipid rafts along with full-length Dsg2. Disruption of lipid rafts shifted Dsg2 to the non-raft fractions, leading to the accumulation of these proteins. Interestingly, Dsg2 proteolytic products are elevated in vivo in skin tumors from transgenic mice overexpressing Dsg2. Collectively, these data are consistent with the possibility that accumulation of truncated Dsg2 protein interferes with desmosome assembly and/or maintenance to disrupt cell-cell adhesion. Furthermore, the association of Dsg2 with Cav-1 may provide a mechanism for regulating mitogenic signaling and modulating the cell-surface presentation of an important adhesion molecule, both of which could contribute to malignant transformation and tumor progression.

Myringoplasty.

There is marked diversity in the reported success rates for achieving an intact tympanic membrane following myringoplasty. Controversy exists about the factors thought to influence surgical outcome. Both of these facts have important implications for obtaining informed consent prior to surgery. This study reviews the factors thought to determine the likelihood of achieving complete closure of the tympanic membrane following surgical closure.

The molecular composition and function of desmosomes.

Desmosomes are intercellular adhesive junctions that are particularly prominent in tissues experiencing mechanical stress, such as the heart and epidermis. Whereas the related adherens junction links actin to calcium-dependent adhesion molecules known as classical cadherins, desmosomes link intermediate filaments (IF) to the related subfamily of desmosomal cadherins. By tethering these stress-bearing cytoskeletal filaments to the plasma membrane, desmosomes serve as integrators of the IF cytoskeleton throughout a tissue. Recent evidence suggests that IF attachment in turn strengthens desmosomal adhesion. This collaborative arrangement results in formation of a supracellular network, which is critical for imparting mechanical integrity to tissues. Diseases and animal models targeting desmosomal components highlight the importance of desmosomes in development and tissue integrity, while the downregulation of individual protein components in cancer metastasis and wound healing suggests their importance in cell homeostasis. This chapter will provide an update on desmosome composition, function, and regulation, and will also discuss recent work which raises the possibility that desmosome proteins do more than play a structural role in tissues where they reside.

Isoform-specific differences in the size of desmosomal cadherin/catenin complexes.

Via their integration of the intermediate filament cytoskeleton into the cell membrane, desmosomes facilitate the maintenance of cell shape and tissue integrity as well as intercellular communication. The transmembrane components of the desmosome, the desmogleins and desmocollins, are members of the cadherin family of cell-cell adhesion molecules. Each of these proteins exists as three distinct isoforms, which are the products of individual genes and expressed in a cell-type and differentiation-specific manner. Previous work has suggested that desmoglein 1 binds to its catenin partner, plakoglobin, in an approximately 6:1 stoichiometry. In this study, the molecular organization of complexes formed by plakoglobin and desmoglein 1, 2, or 3 are further examined through immunoprecipitation, size exclusion chromatography and sucrose density sedimentation analysis. It is shown that the complex formed between plakoglobin and desmoglein 1 has an overall molecular weight greater than that of plakoglobin/desmoglein 2 or plakoglobin/desmoglein 3; however, the stoichiometry of the plakoglobin/desmoglein 1 complex does not appear to exceed 2:1.

Cadherin function: breaking the barrier.

Three desmoglein isoforms collaborate with desmocollins to build the adhesive core of desmosomes. A recent study has shown that altering the ratio of desmoglein isoforms influences epidermal barrier function, suggesting distinct roles for these cadherins that extend beyond adhesion.

Assembly of desmosomal cadherins into desmosomes is isoform dependent.

Desmosomes are intercellular adhesive junctions that exhibit cell- and differentiation-specific differences in their molecular composition. In complex epithelia, desmosomes contain multiple representatives of the desmosomal cadherin family, which includes three desmogleins and three desmocollins. Rules governing the assembly of desmosomal cadherin isoforms into desmosomes of different cell types are unknown. Here we compared the assembly properties of desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), which are widely expressed, with Dsg1 and Dsc1, which are expressed in the differentiated layers of complex epithelia, by introducing myc-tagged forms into simple and squamous epithelial cells that do not express Dsg1 or Dsc1. Dsc2.myc and Dsg2.myc assembled efficiently into desmosomes in every cell type in spite of significant shifts in the stoichiometric relationship between desmogleins and desmocollins. In contrast, Dsc1a.myc, Dsc1b.myc, and Dsg1.myc did not stably incorporate into desmosomes in any line. Coexpression of Dsc1a.myc or Dsc1b.myc and Dsg1.myc did not lead to their colocalization and failed to enhance incorporation of either cadherin into desmosomes. Dsg1.myc, but not Dsc1a, Dsc1b, disrupted desmosome assembly in a cell-type-specific manner, and disruption correlated with the recruitment of Dsg1.myc, but not Dsc1a or Dsc1b, into a Triton-insoluble pool. The plakoglobin:E-cadherin ratio decreased in Dsg1-expressing cells with disrupted desmosomes, but a decrease was also observed in a Dsc1a line. Thus, a modest reduction of plakoglobin associated with E-cadherin is apparently not sufficient to disrupt desmosome assembly. Our results demonstrate that desmosome assembly tolerates large shifts in cadherin stoichiometry, but is sensitive to isoform-specific differences exhibited by desmogleins and desmocollins.

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.

Tyrosine-phosphorylated plakoglobin is associated with desmogleins but not desmoplakin after epidermal growth factor receptor activation.

Tyrosine phosphorylation of junctional components has been proposed as a mechanism for modulating cell-cell adhesion. Although a correlation exists between the tyrosine phosphorylation of the adherens junction protein beta-catenin and loss of classical cadherin-mediated adhesion, the effects of tyrosine phosphorylation on the function of the adherens junction and desmosome-associated protein plakoglobin is unknown. In the present study, we investigated the effects of epidermal growth factor receptor (EGFR) tyrosine kinase activation on the subcellular distribution of plakoglobin and its association with its junctional binding partners. Long term epidermal growth factor (EGF) treatment of A431 cells revealed a modest decrease in the cytoskeleton-associated pool of plakoglobin (Pg) and a corresponding increase in the cytosolic pool of Pg. After short term EGF treatment, plakoglobin was rapidly phosphorylated, and tyrosine-phosphorylated Pg was distributed predominantly in a membrane-associated Triton X-100-soluble pool, along with a co-precipitating high molecular weight tyrosine-phosphorylated protein identified as desmoglein 2. Analysis of deletion and point mutants defined the primary EGFR-dependent targets as one or more of three C-terminal tyrosine residues. Whereas phosphorylated Pg remained associated with the desmoglein tail after both short and long term EGFR activation, no phosphorylated Pg was found associated with the N-terminal Pg-binding domain (DPNTP) of the intermediate filament-associated protein, desmoplakin. Together these results are consistent with the possibility that EGF-dependent tyrosine phosphorylation of Pg may modulate cell-cell adhesion by compromising the link between desmosomal cadherins and the intermediate filament cytoskeleton.

Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell-cell borders.

Desmosomes are adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. These junctions play an important role in providing strength to tissues that experience mechanical stress such as heart and epidermis. The basic structural elements of desmosomes are similar to those of the better-characterized adherens junctions, which anchor actin-containing microfilaments to cadherins at the plasma membrane. This linkage of actin to classic cadherins is thought to occur through an indirect mechanism requiring the associated proteins, alpha- and beta-catenin. In the case of desmosomes, both linear and lateral interactions have been proposed as playing an important role in formation of the plaque and linkage to the cytoskeleton. However, the precise nature of these interactions and how they cooperate in desmosome assembly are poorly understood. Here we employ a reconstitution system to examine the assembly of macromolecular complexes from components found in desmosomes of the differentiated layers of complex tissues. We demonstrate the existence of a Triton-soluble complex of proteins containing full length desmoplakin (DP), the arm protein plakoglobin, and the cytoplasmic domain of the desmosomal cadherin, desmoglein 1 (Dsg1). In addition, full length DP, but not an N-terminal plakoglobin binding domain of DP, co-immunoprecipitated with the Dsg1 tail in the absence of plakoglobin in HT1080 cells. The relative roles of the arm proteins plakoglobin and plakophilin 1 (PKP1) were also investigated. Our results suggest that, in the Triton soluble pool, PKP1 interferes with binding of plakoglobin to full length DP when these proteins are co-expressed. Nevertheless, both plakoglobin and PKP1 are required for the formation of clustered structures containing DP and the Dsg1 tail that ultrastructurally appear similar to desmosomal plaques found in the epidermis. These findings suggest that more than one armadillo family member is required for normal assembly and clustering of the desmosomal plaque in the upper layers of the epidermis.

Acute exercise effects on the immune system.

PURPOSE: In recent years, health professionals have placed increased attention on the benefits of physical activity for maintaining health in the general population as well as regaining health in many disease states. Conversely, reports of apparent decreases in immune cell function after acute exercise are widespread in the literature. The purpose of this article is to evaluate critically the available data and currently employed methods, with the aim of establishing whether genuine or artefactual alterations of immune function are being reported. During and immediately after exercise, the total number of white blood cells in peripheral blood samples increases, such that the relative proportions of cell types within the leukocyte pool are altered. A number of important areas of discussion arise from these shifts in the number of circulating cells after exercise, not least of which is the artefactual effects they may have on currently employed assays of immune cell function. Recent advances in methodology are beginning to call into question the assumption that acute exercise has any genuine immunosuppressive effect. CONCLUSION: At present, there is little evidence to suggest that the range of acute exercise intensities and durations recommended by ACSM has a major detrimental effect on the function of individual T- and B-lymphocytes, natural killer cells and neutrophils. Although individual cells may not be as adversely affected as previously supposed, it is unclear whether the numerical content of the circulating population is an important clinical consideration.

Are desmosomes more than tethers for intermediate filaments?

Desmosomes are intercellular adhesive junctions that anchor intermediate filaments at membrane-associated plaques in adjoining cells, thereby forming a three-dimensional supracellular scaffolding that provides tissues with mechanical strength. But desmosomes have also recently been recognized as sensors that respond to environmental and cellular cues by modulating their assembly state and, possibly, their signalling functions.

Analysis of the desmoplakin gene reveals striking conservation with other members of the plakin family of cytolinkers.

Members of the plakin family of cytolinker proteins integrate filaments into cellular networks and anchor these networks to the plasma membrane. Their importance is supported by the existence of cell and tissue fragility disorders caused by mutations in certain family members. In this study, the human gene encoding desmoplakin (DSP) was characterized and its structure compared with the related family members: plectin, bullous pemphigoid antigen 1 (BPAG1), envoplakin (EVPL) and periplakin (PPL). Sequence analysis of genomic clones was carried out in combination with a PCR-based strategy to define intron-exon borders. DSP was mapped using the GB4 radiation hybrid mapping panel to the interval between markers D6S296 and AFM043 x f2, corresponding to cytogenetic band 6p24. In addition, the murine gene (Dsp) was mapped to mouse chromosome 13 by interspecific backcross mapping. DSP encompasses approximately 45 kb organized into 24 exons and 23 introns, and the pattern of intron-exon borders bears a striking resemblance to other members of the plakin family. Notable features include the fact that a single large exon encodes the entire C-terminus of each gene. In contrast, the N-termini comprise numerous smaller exons with conservation of many intron-exon borders. Detailed characterization and mapping of these genes will facilitate their further evaluation as targets of genetic disorders and provide insights into the evolutionary relationships among molecules in this emerging gene family.

Molecular map of the desmosomal plaque.

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the 'a' form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter 'b' form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.

The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1. To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined. In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins. In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays. We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules. This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.

Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.

Desmosomes are highly organized intercellular adhesive junctions that are particularly prominent in epidermis and other tissues experiencing mechanical stress. Desmoplakin, a constitutive component of the desmosomal plaque, is the most abundant protein present in such junctions and plays a critical role in linking the intermediate filament network to the plasma membrane in these tissues. Here we report the first mutation in the gene encoding desmoplakin. The identified mutation, resulting in a null allele and haploinsufficiency, was observed in genomic DNA from a kindred with the dominantly inherited skin disorder, striate palmoplantar keratoderma. Affected individuals had a linear pattern of skin thickening on the fingers and palms and circumscribed areas of skin thickening on the soles. Affected skin demonstrated loosening of intercellular connections, disruption of desmosome-keratin intermediate filament interactions and a proportion of rudimentary desmosomal structures. The disorder mapped to chromosome 6p21 with a maximum lod score of 10.67. The mutation was a heterozygous C-->T transition in exon 4 of the desmoplakin gene and predicted a premature termination codon in the N-terminal region of the peptide. This is the first reported mutation of desmo-plakin and also the first inherited skin disorder in which haploinsufficiency of a structural component has been implicated. It identifies dosage of desmoplakin as critical in maintaining epidermal integrity.