Plakophilins—hard work in the desmosome,recreation in the nucleus?

The linkage of the different types of cytoskeletal proteins to cell adhesion structures at the cytoplasmic membrane and the connection of these contact sites to corresponding sites of adjacent cells is a prerequisite for integrity and stability of cells and tissues. The structurally most prominent types of such cell-cell adhesion complexes are the desmosomes (maculae adhaerentes), which are found in all epithelia and certain non-epithelial tissues. As an element of the cytoskeleton, intermediate filaments are connected to the adhesive desmosomal transmembrane proteins by the cytoplasmic desmosomal plaque proteins. At least three different types of proteins are found in the desmosomal plaque, one of which is represented by the plakophilins, a recently described sub-family of sequence-related armadillo-repeat proteins. Consisting of three isoforms, plakophilins (plakophilin 1 to 3, PKP 1 to 3) are located in all desmosomes in a differentiation-dependent manner. While PKP 2 and PKP 3 are part of almost all desmosome-bearing cell types (PKP 2 except for differentiated cells of stratified epithelia and PKP 3 for hepatocytes and cardiomyocytes), PKP 1 is restricted to desmosomes of cells of stratified and complex epithelia. Besides the architectural function that plakophilins seem to fulfill in the desmosomes, at least PKP 1 and 2 are also localized in the nucleus independently of any differentiation-related processes and with an up to now enigmatic function in this compartment. In the following article we want to summarize the current knowledge concerning structure, function and regulation of the plakophilins that has been achieved during the last decade.     

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.     

Plakophilin 3 — a novel cell-type-specific desmosomal plaque protein

Desomosomes are cell-cell adhesion structures of epithelia and some non-epithelial tissues, such as heart muscle and the dendritic reticulum of lymph node follicles, which on their cytoplasmic side anchor intermediate filaments at the plasma membrane. Besides clusters of specific transmembrane glycoproteins of the cadherin family (desmogleins and desmocollins), they contain several desmosomal plaque proteins, such as desmoplakins, plakoglobin, and one or more plakophilins. Using recombinant DNA and immunological techniques, we have identified a novel desmosomal plaque protein that is closely related to plakophilins 1 and 2, both members of the "armadillo-repeat" multigene family, and have named it plakophilin 3 (PKP3). The product of the complete human cDNA defines a protein of 797 amino acids, with a calculated molecular weight of 87.081 kDa and an isoelectric point of pH 10.1. Northern blot analysis has shown that PKP3 mRNA has a size of approximately 2.9 kb and is detectable in the total RNA of cells of stratified and single-layered epithelia. With the help of specific poly- and monoclonal antibodies we have localized PKP3, by immunofluorescence or immunoelectron microscopy, to desmosomes of most simple and almost all stratified epithelia and cell lines derived therefrom, with the remarkable exception of hepatocytes and hepatocellular carcinoma cells. We have also determined the structure of the human PKP3 gene and compared it with that of plakophilin 1 (PKP1). Using fluorescence in situ hybridization, we have localized the human genes for the three known plakophilins to the chromosomes 1q32 (PKP1), 12p11 (PKP2) and 11p15 (PKP3). The similarities and differences of the diverse plakophilins are discussed.     

Stratifin (14-3-3 σ) Limits Plakophilin-3 Exchange with the Desmosomal Plaque

Desmosomes are prominent cell-cell adhesive junctions in stratified squamous epithelia and disruption of desmosomal adhesion has been shown to have dramatic effects on the function and integrity of these tissues. During normal physiologic processes, such as tissue development and wound healing, intercellular adhesion must be modified locally to allow coordinated cell movements. The mechanisms that control junction integrity and adhesive strength under these conditions are poorly understood. We utilized a proteomics approach to identify plakophilin-3 associated proteins and identified the 14-3-3 family member stratifin. Stratifin interacts specifically with plakophilin-3 and not with other plakophilin isoforms and mutation analysis demonstrated the binding site includes serine 285 in the amino terminal head domain of plakophilin-3. Stratifin interacts with a cytoplasmic pool of plakophilin-3 and is not associated with the desmosome in cultured cells. FRAP analysis revealed that decreased stratifin expression leads to an increase in the exchange rate of cytoplasmic plakophilin-3/GFP with the pool of plakophilin-3/GFP in the desmosome resulting in decreased desmosomal adhesion and increased cell migration. We propose a model by which stratifin plays a role in regulating plakophilin-3 incorporation into the desmosomal plaque by forming a plakophilin-3 stratifin complex in the cytosol and thereby affecting desmosome dynamics in squamous epithelial cells.     

Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia

We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.     

Expression of keratin 5 as a distinctive feature of epithelial and biphasic mesotheliomas. An immunohistochemical study using monoclonal antibody AE14

In previous biochemical analyses, keratin 5 (Mr 58,000) has been detected in most mesotheliomas with epithelial component but not in pulmonary adenocarcinomas (Blobel et al., Am J Pathol 121: 235-247, 1985). In the present study, we have characterized a monoclonal antibody, AE14, as being selectively specific for keratin 5 (apart from the reactivity with certain hair proteins) as shown by immunoblotting of gel-electrophoretically separated proteins from various tissues. Immunohistochemical screening of a variety of normal human tissues, using immunoperoxidase microscopy on cryostat sections, revealed the binding of this antibody to the basal, immature cells of stratified squamous epithelia, to basal cells of pseudostratified epithelia, to some myoepithelial cells, thymic reticulum cells, certain pancreatic duct cells, as well as a variable subpopulation of mesothelial cells of the pleura and the peritoneum. In 12/13 epithelial and biphasic mesotheliomas of the pleura, heterogeneous but extended staining with antibody AE14 was seen whereas 21 pulmonary adenocarcinomas were negative or, in six of these cases, showed staining of only a few cells. Among carcinomas from other sites, colonic adenocarcinomas and renal cell carcinomas were negative whereas limited staining was found in some pancreatic adenocarcinomas. It is suggested that antibody AE14 may be useful, as a defined polypeptide-specific reagent, in the histologic distinction between mesotheliomas and most adenocarcinomas. Furthermore, the expression patterns of keratin 5 as detected by antibody AE14 in various normal and malignant epithelial tissues are discussed, particularly their relation to processes of squamous metaplasia and their indication of phenotypic tumor heterogeneity.     

Tight junction-related structures in the absence of a lumen: occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas

Tight junctions (TJs), hallmark structures of one-layered epithelia and of endothelia, are of central biological importance as intramembranous "fences" and as hydrophobic "barriers" between lumina represented by liquid- or gas-filled spaces on the one hand and the mesenchymal space on the other. They have long been thought to be absent from stratified epithelia. Recently, however, constitutive TJ proteins and TJ-related structures have also been identified in squamous stratified epithelia, including the epidermis, where they occur in special positions, most prominently in the uppermost living epidermal cell layer, the stratum granulosum. Much to our surprise, however, we have now also discovered several major TJ proteins (claudins 1 and 4, occludin, cingulin, symplekin, protein ZO-1) and TJ-related structures in specific positions of formations of epithelium-derived tissues that lack any lumen and do not border on luminal or body surfaces. Using immunohistochemistry and electron microscopy we have localized TJ proteins and structures in peripheral cells of the Hassall's corpuscles of human and bovine thymi as well as in specific central formations of tumor nests in squamous cell carcinomas, including the so-called "horn pearls". Such structures have even been found in carcinoma metastases. In carcinomas, they often seem to separate certain tumor regions from others or from stroma. The structural significance and the possible functional relevance of the locally restricted synthesis of TJ proteins and of the formations of TJ-related structures are discussed. It is proposed to include the determination of the presence or absence of such proteins and structures in the diagnostic program of tumor pathology.     

Mucin 21 in esophageal squamous epithelia and carcinomas: analysis with glycoform-specific monoclonal antibodies

Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.     

Immunological comparison of desmosomal components from several bovine tissues

A panel of monoclonal antibodies and conventional antisera directed against desmosomal proteins from bovine muzzle epidermis was used to identify immunologically related proteins from two other bovine stratified squamous epithelia, cornea and esophagus. Desmosome-enriched tissue fractions were prepared from epidermis, cornea, and esophagus. These tissue extracts were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels, blotted onto nitrocellulose paper, and labeled using an indirect immunoperoxidase technique. Labeling with the conventional antisera demonstrates that each of the previously characterized epidermal desmosomal proteins or protein families has an immunologically cross-reacting counterpart in cornea and esophagus. However, chemical differences between homologous desmosomal proteins in these three tissues have also been detected. The corresponding proteins in the different tissues have similar but not always identical apparent molecular weights. Moreover, tissue-restricted antigenic determinants were detected in two of the desmosomal proteins families using four monoclonal antibodies, each of which recognizes a distinct antigenic determinant.     

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.     

Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2)

 In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia. Accepted: 29 June 1998     

Molecular characterization and expression of the stratification-related cytokeratins 4 and 15

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.     

Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium

The trachea and esophagus originate from the foregut endoderm during early embryonic development. Their epithelia undergo a series of changes involving the differentiation of stem cells into unique cell types and ultimately forming the mature epithelia. In this study, we monitored the expression of p63 in the esophagus and the trachea during development and examined in detail morphogenesis in p63^–/– mice. At embryonic day 15.5 (E15.5), the esophageal and tracheobronchial epithelia contain two to three layers of cells; however, only the progenitor cells express p63. These progenitor cells differentiate first into ciliated cells (p63^–/-tubulin IV⁺) and after birth into mature basal cells (p63⁺/K14⁺/K5⁺/BS-I-B4⁺). In the adult pseudostratified, columnar tracheal epithelium, K14⁺/K5⁺/BS-I-B4⁺ basal cells stain most intensely for p63, whereas ciliated and mucosecretory cells are negative. In stratified squamous esophageal epithelium and during squamous metaplasia in the trachea, cells in the basal layer stain strongest for p63, whereas p63 staining declines progressively in transient amplifying and squamous differentiated cells. Generally, p63 expression is restricted to human squamous cell carcinomas, and adenocarcinomas and Barrett's metaplasia do not stain for p63. Examination of morphogenesis in newborn p63^–/– mice showed an abnormal persistence of ciliated cells in the esophagus. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar ciliated epithelium deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny and the maintenance of basal cells. retinoic acid; stem cell; carcinoma; basal cell; differentiation     

Macrophage-specific RAM11 monoclonal antibody cross-reacts with basal cells of stratified squamous epithelia

RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular (atheromatous tissue) macrophages. This study demonstrates a cross-reaction of RAM11 with an unknown antigen in rabbit normal epithelial cells. Formalin-fixed, paraffin sections of the New Zealand White rabbit normal skin, oral mucosa, esophagus, small intestine and lung were immunostained with RAM11 antibody followed by goat anti-mouse Cy-3-conjugated antiglobulin. RAM11-positive immunofluorescence was observed in basal layer cells of stratified squamous epithelia (skin, oral mucosa, esophagus). No RAM11 immunostaining was found in any cells of simple (intestinal, bronchial) epithelia. These findings show that basal cells of stratified squamous keratinized and non-keratinized epithelia of the rabbit express an antigenic epitope which is common with that of macrophage antigen recognized by RAM11 monoclonal antibody.     

Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.     

Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque

Using antibodies and recombinant DNA techniques, we have identified plakophilin 2, a novel desmosomal plaque protein of M(r) 100,000 (estimated from SDS-PAGE), which is a member of the arm-repeat family of proteins and can occur in two splice forms (2a and 2b) because of the insertion of a 44 amino acid (aa)-encoding exon. In its aa sequence (837 and 881 aa, calculated pIs: 9.33 and 9.38, mol wts 92,750 and 97,410 kD), it is conspicuously related to the 80-kD plakophilin 1, with which it shares a central region of 9 repeats of the arm-motif, preceeded by a long head region and followed by a very short (11 aa) carboxy-terminal sequence. Plakophilin 2 and its mRNA have been detected in a wide range of tissues and cell types, including cells devoid of desmosomes. By light and electron microscopical immunolocalization, plakophilin 2 has been localized to plaques of desmosomes of one-layered ("simple") and complex epithelia, carcinomas, diverse epithelium-derived cell culture lines, as well as cardiac tissue and the dendritic reticulum cells of lymphatic germinal centers, i.e., desmosomes in which plakophilin 1 is not detected. However, plakophilin 2 has also been localized in the desmosomes of certain but not all stratified epithelia where it coexists with plakophilin 1. Remarkably, plakophilin 2 is also enriched in the karyoplasm of a wide range of cell types, including many that lack desmosomes and in which, therefore, the nuclear state is the only locally enriched form of plakophilin 2 present. We conclude that plakophilins 2a and 2b are basic nuclear proteins that in certain cell types additionally assemble with other proteins to form the desmosomal plaque and serve general nuclear functions as well as a function specific to many but not all desmosomes.     

A set of glycoproteins recognized by A2B5 antibody with major bands at 55 and 76 kDa is overexpressed in human head and neck squamous cell carcinomas

A2B5 antibody was found to strongly label frozen sections of human head and neck squamous cell carcinomas. The low amount of glycolipids (c-series gangliosides and sulfatides) purified from the same tumors and reactive with A2B5 by immunostaining on thin-layer plates could not account for the high level of tissue labeling. Proteins were extracted from both normal tissues and squamous cell carcinomas and analyzed by Western blot with A2B5 antibody on PVDF membranes. The antibody was found to stain a set of glycoproteins with two major bands at 55 and 76 kDa present in normal tissues and overexpressed in carcinomas. Staining was abolished by prior treatment of the PVDF membranes either with Arthrobacter ureafaciens neuraminidase or with a solution of 10 mM periodate that is known to destroy carbohydrates. Our results show that the carbohydrate epitope recognized by A2B5 antibody can be displayed by both glycolipids and glycoproteins.     

MMP7 Is Required to Mediate Cell Invasion and Tumor Formation upon Plakophilin3 Loss

Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation.