PubMed Health⌕ Search

Biomedical subjects

Kathleen J Green

Publications and source records attributed to Kathleen J Green.

At least 19 recordsLinked to original sources

Discriminating roles of desmosomal cadherins: beyond desmosomal adhesion.

The desmosomal cadherins, which include desmogleins and desmocollins, are Ca(2+)-dependent adhesion molecules that cooperate to make up the adhesive core of intercellular junctions known as desmosomes. The roles of desmosomal cadherins in epidermal integrity and as targets in human cutaneous disease have been well established. However, the molecular basis of these disorders is still poorly understood, due in part to a lack of fundamental knowledge about the organization of the adhesive interface and molecular machinery that dictates the proper presentation of desmogleins and desmocollins on the cell surface. Further, the diversity of the desmosomal cadherin family, and their individualized expression patterns within complex tissues, suggests that these adhesion molecules may have differentiation-specific functions that transcend their roles in intercellular adhesion. Here we will review the most recent data from our own group and others that are beginning to unveil the diverse properties and functions of this complex family of adhesion molecules.

Animals↗

Plakoglobin deficiency protects keratinocytes from apoptosis.

The armadillo family protein plakoglobin (Pg) is a well-characterized component of anchoring junctions, where it functions to mediate cell-cell adhesion and maintain epithelial tissue integrity. Although its closest homolog beta-catenin acts in the Wnt signaling pathway to dictate cell fate and promote proliferation and survival, the role of Pg in these processes is not well understood. Here, we investigate how Pg affects the survival of mouse keratinocytes by challenging both Pg-null cells and their heterozygote counterparts with apoptotic stimuli. Our results indicate that Pg deletion protects keratinocytes from apoptosis, with null cells exhibiting delayed mitochondrial cytochrome c release and activation of caspase-3. Pg-null keratinocytes also exhibit increased messenger RNA and protein levels of the anti-apoptotic molecule Bcl-X(L) compared to heterozygote controls. Importantly, reintroduction of Pg into the null cells shifts their phenotype towards that of the Pg+/- keratinocytes, providing further evidence that Pg plays a direct role in regulating cell survival. Taken together, our results suggest that in addition to its adhesive role in epithelia, Pg may also function in contrast to the pro-survival tendencies of beta-catenin, to potentiate death in cells damaged by apoptotic stimuli, perhaps limiting the potential for the propagation of mutations and cellular transformation.

Animals↗

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Animals↗

Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

Animals↗

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

Actin Cytoskeleton↗

The differentiation-dependent desmosomal cadherin desmoglein 1 is a novel caspase-3 target that regulates apoptosis in keratinocytes.

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.

Apoptosis↗

Mechanisms of plakoglobin-dependent adhesion: desmosome-specific functions in assembly and regulation by epidermal growth factor receptor.

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related beta-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not beta-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.

Adenoviridae↗

Plakoglobin suppresses keratinocyte motility through both cell-cell adhesion-dependent and -independent mechanisms.

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins and has been shown to play a critical role in the organization of desmosomes and tissue integrity. Because dissolution of intercellular junctions is frequently an initial step in the onset of epithelial cell migration, we examined whether loss of PG promotes cell motility by compromising adhesive strength. Keratinocyte cultures established from PG-/-mice exhibited weakened adhesion and increased motility in transwell migration assays; both were restored by reintroducing PG through adenoviral infection. Interestingly, single PG-/- cells also exhibited increased motility, which was suppressed by reintroducing PG, but not the closely related beta-catenin. Whereas both N- and C-terminally truncated PG deletion mutants restored adhesion, only N-terminally deleted PG, but not C-terminally deleted PG, suppressed single-cell migration. Furthermore, both the chemical inhibitor PP2 and dominant-negative Src tyrosine kinase inhibited single-cell motility in PG-/- cells, whereas constitutively active Src overcame the inhibitory effect of PG. These data demonstrate that PG strengthens adhesion and suppresses motility in mouse keratinocytes, through both intercellular adhesion-dependent and -independent mechanisms, the latter of which may involve suppression of Src signaling through a mechanism requiring the PG C terminus.

Animals↗

Intermediate filament associated proteins.

Intermediate filament associated proteins (IFAPs) coordinate interactions between intermediate filaments (IFs) and other cytoskeletal elements and organelles, including membrane-associated junctions such as desmosomes and hemidesmosomes in epithelial cells, costameres in striated muscle, and intercalated discs in cardiac muscle. IFAPs thus serve as critical connecting links in the IF scaffolding that organizes the cytoplasm and confers mechanical stability to cells and tissues. However, in recent years it has become apparent that IFAPs are not limited to structural crosslinkers and bundlers but also include chaperones, enzymes, adapters, and receptors. IF networks can therefore be considered scaffolding upon which associated proteins are organized and regulated to control metabolic activities and maintain cell homeostasis.

Animals↗

Epidermal growth factor receptor inhibition promotes desmosome assembly and strengthens intercellular adhesion in squamous cell carcinoma cells.

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.

Antibodies, Monoclonal↗

Regulation of desmosome assembly and adhesion.

Desmosomes are highly organized intercellular junctions that provide mechanical integrity to tissues by anchoring intermediate filaments to sites of strong adhesion. Transcriptional regulation of desmosomal cadherins specifies their expression pattern and assembly into junctions of distinct composition, thus tailoring desmosome functions in adhesion and morphogenesis within different cells and complex tissues. Desmosome assembly and disassembly are regulated post-translationally by calcium, kinase/phosphatase activity, proteolytic processing, and cross talk with adherens junctions. Post-translational events also govern the level of non-junctional forms of plakoglobin and plakophilins. These armadillo proteins participate in various nuclear functions, in some cases transducing signals regulating cell growth and differentiation.

Animals↗

Comparative analysis of armadillo family proteins in the regulation of a431 epithelial cell junction assembly, adhesion and migration.

p0071 is an armadillo family protein related to both the adherens junction protein p120ctn and to the desmosomal proteins plakophilins 1-3. p0071 assembles into both adherens junctions and desmosomes, suggesting that this protein may regulate the balance between adherens junction and desmosome formation. Furthermore, this subfamily of proteins may also regulate cell functions directly influenced by intercellular junctions, including the strength of cell adhesion and the ability of cells to migrate. These possibilities were tested by expressing exogenous p0071 in A431 epithelial cells and monitoring the effects on adhesive junction assembly in comparison to other closely related armadillo family proteins. In this model system, p0071 specifically enhanced adherens junction assembly but dramatically compromised desmosome assembly, resulting in keratin filament retraction from regions of cell-cell contact. Protein interaction studies revealed that p0071 bound to the first 160 amino-terminal residues of desmoplakin and also interacted directly with plakoglobin, suggesting that p0071 may regulate desmosome assembly by controlling plakoglobin availability. Using an in vitro assay to measure the strength of cell-cell contacts, both plakophilin-1 and p120ctn were found to increase the strength of adhesion. Interestingly, p0071 expression caused no overall changes in adhesive strength, but dramatically inhibited the ability of A431 cells to close an in vitro wound. These results suggest that p120ctn/plakophilin family proteins interact with intercellular junction binding partners to differentially modulate the adhesive and migratory behavior of epithelial cells.

Adherens Junctions↗

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion.

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.

Animals↗

p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions.

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell-cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell-cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin.

Adherens Junctions↗

Defining desmosomal plakophilin-3 interactions.

Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

Animals↗

Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains.

p0071, a member of the armadillo protein family, is most closely related to p120(ctn) and the plakophilins 1-3. Whereas plakophilins are desmosomal plaque proteins, p120(ctn) localizes to adherens junctions and interacts with classical cadherins. In contrast, p0071 has been described as a protein with dual localization in adherens junctions and desmosomes depending on the cell type examined. Here we have analyzed the localization of p0071 and its domains in detail. Although by sequence analysis, p0071 is more closely related to the adherens junction proteins p120(ctn), ARVCF and delta-catenin, endogenous p0071 associated preferentially with desmosomes in MCF-7 epithelial cells. Overexpressed p0071 localized along cell borders and overlapped only partially with desmosomal markers but colocalized with non-desmosomal cadherins and recruited cadherins to the membrane. The head domain of p0071 was sufficient for desmosomal targeting, whereas the arm repeat domain associated with adherens junctions and enhanced membrane association of classical cadherins. The tail domain localized preferentially to the nucleus and associated with desmosomes. To examine the mechanism underlying this dual localization more closely we determined binding partners of p0071 by using yeast-two-hybrid and mom-targeting assays. These approaches show that the head domain interacted with desmosomal proteins desmocollin 3a and desmoplakin, whereas the armadillo repeat domain binds to non-desmosomal cadherins. Head and armadillo repeat domains both interacted with plakoglobin by binding to different sites. Our data suggest that, in addition to plakoglobin, p0071 is the second armadillo protein present in both types of adhesive junctions and may play a role in regulating crosstalk between adherens junctions and desmosomes.

Adherens Junctions↗