Plakophilin3 loss leads to an increase in lipocalin2 expression, which is required for tumour formation.

An increase in tumour formation and metastasis are observed upon plakophilin3 (PKP3) loss. To identify pathways downstream of PKP3 loss that are required for increased tumour formation, a gene expression analysis was performed, which demonstrated that the expression of lipocalin2 (LCN2) was elevated upon PKP3 loss and this is consistent with expression data from human tumour samples suggesting that PKP3 loss correlates with an increase in LCN2 expression. PKP3 loss leads to an increase in invasion, tumour formation and metastasis and these phenotypes were dependent on the increase in LCN2 expression. The increased LCN2 expression was due to an increase in the activation of p38 MAPK in the HCT116 derived PKP3 knockdown clones as LCN2 expression decreased upon inhibition of p38 MAPK. The phosphorylated active form of p38 MAPK is translocated to the nucleus upon PKP3 loss and is dependent on complex formation between p38 MAPK and PKP3. WT PKP3 inhibits LCN2 reporter activity in PKP3 knockdown cells but a PKP3 mutant that fails to form a complex with p38 MAPK cannot suppress LCN2 promoter activity. Further, LCN2 expression is decreased upon loss of p38ß, but not p38α, in the PKP3 knockdown cells. These results suggest that PKP3 loss leads to an increase in the nuclear translocation of p38 MAPK and p38ß MAPK is required for the increase in LCN2 expression.

Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.

The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell-cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.

The armadillo protein p0071 is involved in Rab11-dependent recycling.

p0071 is an intercellular junction protein of the p120 catenin family. We have identified Rab11a as a novel interaction partner of p0071. p0071 interacted preferentially with active Rab11a. Knockdown experiments revealed an interdependent regulation of both proteins. On the one hand, p0071 depletion induced a perinuclear accumulation of Rab11, suggesting a role of p0071 in the anterograde transport of Rab11 from the pericentrosomal region to the plasma membrane but not in retrograde transport. p0071 as well as Rab11 depletion increased transferrin receptor recycling indicating that p0071-induced Rab11 mislocalization interfered with Rab11 function and shifted recycling from the slow Rab11-dependent pathway to the fast Rab4-dependent pathway. When p0071 or Rab11 depletion was combined with a Rab4 knockdown the effect was reversed. On the other hand, Rab11a depletion increased p0071 recycling to cell contacts thereby identifying p0071 as a Rab11 cargo protein. This correlated with increased intercellular adhesion. Thus, we propose that p0071 has a key role in regulating recycling through the Rab11-dependent perinuclear recycling compartment, and links the regulation of adherens junctions to recycling to allow dynamic modulation of intercellular adhesion.

Plakophilin-2 and the migration, differentiation and transformation of cells derived from the epicardium of neonatal rat hearts.

During development, epicardial cells act as progenitors for a large fraction of non-myocyte cardiac cells. Expression and function of molecules of the desmosome in the postnatal epicardium has not been studied. The objective of this study was to assess the expression of desmosomal molecules, and the functional importance of the desmosomal protein plakophilin-2 (PKP2), in epicardial and epicardium-derived cells. Epicardial explants were obtained from neonatal rat hearts. Presence of mechanical junction proteins was assessed by immunocytochemistry. Explants after PKP2 knockdown showed increased abundance of alpha smooth muscle actin-positive cells, increased abundance of lipid markers, enhanced cell migration velocity and increased abundance of a marker of cell proliferation. We conclude that a population of non-excitable, cardiac-resident cells express desmosomal molecules and, in vitro, show functional properties (including lipid accumulation) that depend on PKP2 expression. The possible relevance of our data to the pathophysiology of arrhythmogenic right ventricular cardiomyopathy, is discussed.

MAL/MRTF-A controls migration of non-invasive cells by upregulation of cytoskeleton-associated proteins.

Monomeric actin regulates gene expression through serum response factor (SRF) by inhibiting its transcriptional coactivator myocardin-related transcription factor (MAL/MRTF). Many affected genes encode cytoskeletal components. We have analysed the migratory effects of actin-MAL signalling and of new target genes in non-invasive highly adherent cells. Expression of active MAL impaired migration of both fibroblasts and epithelial cells, whereas dominant-negative constructs and partial knockdown of MAL/MRTF enhanced motility. Knockdown of three newly characterised G-actin-regulated MAL targets, integrin α5, plakophilin 2 (Pkp2) and FHL1, enhanced cell migration. All three were upregulated by external stimulation through actin-MAL-SRF signalling, and MAL and SRF were inducibly recruited to cis-regulatory elements of the integrin α5 and Pkp2 genes. Finally, the reduced migration of epithelial cells stably expressing MAL was partially reversed by knockdown of Pkp2 and FHL1. We conclude that the actin-MAL pathway promotes adhesive gene expression, including integrin α5, Pkp2 and FHL1, and that this is anti-motile for non-invasive cells harbouring high basal activity.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. V. The importance of plakophilin-2 demonstrated by small interference RNA-mediated knockdown in cultured rat cardiomyocytes.

In the adult mammalian heart, the cardiomyocytes are connected by large polar arrays of closely spaced or even fused composite, plaque-bearing adhering junctions (areae compositae, ACs), in a region usually termed "intercalated disk" (ID). We have recently reported that during late embryogenesis and postnatally these polar assemblies of AC-junction structures are gradually formed as replacements of distinct embryonal junctions representing desmosomes and fasciae adhaerentes which then may amalgamate to the fused AC structures, in some regions occupying more than 90% of the total ID area. Previous gene knockout results as well as mutation analyses of specific human cardiomyopathies have suggested that among the various AC constituents, the desmosomal plaque protein, plakophilin-2, plays a particularly important role in the formation, architectural organization and stability of these junctions interconnecting mature cardiomyocytes. To examine this hypothesis, we have decided to study losses of--or molecular alterations in--such AC proteins with respect to their effects on myocardiac organization and functions. Here we report that plakophilin-2 is indeed of obvious importance for myocardial architecture and cell-cell coupling of rat cardiomyocytes growing in culture. We show that siRNA-mediated reduction of the cardiomyocyte content of plakophilin-2 but not of some other major plaque components such as desmoplakin results in progressive disintegration--and losses--of AC junction structures and that numerous variously sized vesicles appear, which are plaque protein-associated as demonstrable by immunofluorescence and immunoelectron microscopy. The importance of plakophilin-2 as a kind of "organizer" protein in the formation, stabilization and functions of the AC structure and the ID architecture is discussed in relation to other junction proteins and to causes of certain cardiomyopathies.

Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells.

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.