Neoplastic Cells are the Major Source of MT-MMPs in IDH1-Mutant Glioma, Thus Enhancing Tumor-Cell Intrinsic Brain Infiltration.

Tumor-cell infiltration is a major obstacle to successful therapy for brain tumors. Membrane-type matrix metalloproteinases (MT-MMPs), a metzincin subfamily of six proteases, are important mediators of infiltration. The cellular source of MT-MMPs and their role in glioma biology, however, remain controversial. Thus, we comprehensively analyzed the expression of MT-MMPs in primary brain tumors. All MT-MMPs were differentially expressed in primary brain tumors. In diffuse gliomas, MT-MMP1, -3, and -4 were predominantly expressed by IDH1mutated tumor cells, while macrophages/microglia contributed significantly less to MT-MMP expression. For functional analyses, individual MT-MMPs were expressed in primary mouse p53-/- astrocytes. Invasion and migration potential of MT-MMP-transduced astrocytes was determined via scratch, matrigel invasion, and novel organotypic porcine spinal slice migration (OPoSSM) and invasion assays. Overall, MT-MMP-transduced astrocytes showed enhanced migration compared to controls. MMP14 was the strongest mediator of migration in scratch assays. However, in the OPoSSM assays, the glycosylphosphatidylinositol (GPI)-anchored MT-MMPs MMP17 and MMP25, not MMP14, mediated the highest infiltration rates of astrocytes. Our data unequivocally demonstrate for the first time that glioma cells, not microglia, are the predominant producers of MT-MMPs in glioma and can act as potent mediators of tumor-cell infiltration into CNS tissue. These proteases are therefore promising targets for therapeutic interventions.

Evaluation of digital image analysis as a supportive tool for the stratification of head and neck vascular anomalies.

BACKGROUND: The histological differentiation of individual types of vascular anomalies (VA), such as lymphatic malformations (LM), hemangioma (Hem), paraganglioma (PG), venous malformations (VeM), arteriovenous malformations (AVM), pyogenic granulomas (GP), and (not otherwise classified) vascular malformations (VM n.o.c.) is frequently difficult due to the heterogeneity of these anomalies. The aim of the study was to evaluate digital image analysis as a method for VA stratification METHODS: A total of 40 VA tissues were examined immunohistologically using a selection of five vascular endothelial-associated markers (CD31, CD34, CLDN5, PDPN, VIM). The staining results were documented microscopically followed by digital image analyses based quantification of the candidate-marker-proteins using the open source program ImageJ/Fiji. RESULTS: Differences in the expression patterns of the candidate proteins could be detected particularly when deploying the quotient of the quantified immunohistochemical signal values. Deploying signal marker quotients, LM could be fully distinguished from all other tested tissue types. GP achieved stratification from LM, Hem, VM, PG and AVM tissues, whereas Hem, PG, VM and AVM exhibited significantly different signal marker quotients compared with LM and GP tissues. CONCLUSION: Although stratification of different VA from each other was only achieved in part with the markers used, the results of this study strongly support the usefulness of digital image analysis for the stratification of VA. Against the background of upcoming new diagnostic techniques involving artificial intelligence and deep (machine) learning, our data serve as a paradigm of how digital evaluation methods can be deployed to support diagnostic decision making in the field of VAs.

Therapeutic Targeting of Th17/Tc17 Cells Leads to Clinical Improvement of Lichen Planus.

Lichen planus (LP) is a common, chronic relapsing inflammatory disorder of the skin and mucous membranes which often poses a major therapeutic challenge due to its refractory course. Novel pathogenesis-based therapies are urgently needed. As several studies have shown that IL-17 may contribute to LP pathogenesis, we investigated whether therapeutic targeting of IL-17+ T cells leads to clinical improvement of mucosal and cutaneous LP lesions. A total of five patients with lichen planus were treated in a compassionate use trial with either secukinumab (anti-IL-17; 3 patients with acute and chronic recalcitrant muco-cutaneous LP), ustekinumab (anti-IL-12/IL-23; 1 patient with recalcitrant oral LP) or guselkumab (anti-IL-23; 1 patient with recalcitrant oral LP). The clinical course of the patients was assessed by the Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) reflecting both extent and severity of disease and functional sequelae of oral involvement for at least 12 weeks. The inflammatory infiltrate in lesional and post-lesional skin was analyzed by immunohistochemistry before and after treatment. Furthermore, the cytokine profile of peripheral blood T cells from the treated patients was assessed by flow cytometry and/or ELISpot assay. Treatment with secukinumab induced rapid and prolonged clinical amelioration of muco-cutaneous LP. Clinical improvement was accompanied by a strong reduction of the Th1 and Th17/Tc17 cellular mucosal and cutaneous infiltrate. Moreover, long-term treatment of one patient with recalcitrant oral LP with ustekinumab led to healing of the ulcerative oral lesions and a reduction of peripheral blood and lesional IL-17+ T cells. Finally, treatment with guselkumab led to a marked clinical improvement in a patient with recalcitrant erosive oral LP. These findings show for the first time that therapeutic targeting of Th17/Tc17 cells leads to a pronounced clinical amelioration of mucosal and cutaneous LP and strongly suggests that IL-17-producing T cells are central to disease pathogenesis. Thus, therapeutic targeting of Th17/Tc17 cells opens new therapeutic avenues in the treatment of recalcitrant LP.

Thymoma-Associated Paraneoplastic Autoimmune Multiorgan Syndrome-From Pemphigus to Lichenoid Dermatitis.

Introduction: Paraneoplastic autoimmune multi-organ syndrome (PAMS) is a rare clinical condition characterized by variable and heterogeneous clinical phenotypes in the presence of neoplasias which largely depend on the activation of humoral and cellular immune responses. Clinically, these patients present with a spectrum of antibody-driven pemphigus-like lesions to graft-vs.-host-disease-like exanthemas with a lichenoid inflammatory infiltrate in the skin. PAMS is occasionally associated with thymoma, in which altered immune surveillance eventually leads to multiorgan autoimmunity which often includes variable cutaneous symptoms. This disorder is associated with a profound disturbance of peripheral immune tolerance against human autoantigens. Objectives: We here present a patient with relapsing thymoma who developed PAMS with several cutaneous and extracutaneous autoimmune disorders. Materials: Peripheral blood mononuclear cells (PBMC), sera, and lesional skin biopsies were obtained at different clinical disease stages. Peripheral T cell subsets were characterized phenotypically and the cytokine profile of the peripheral blood T cellular response against distinct epidermal and dermal autoantigens of the skin was analyzed by ELISpot assay. Serological screening was performed by ELISA and immunoblot analysis. Skin biopsies were subjected to immunohistochemical analysis of distinct T cell subsets. Thymoma tissue was analyzed for the presence of T regulatory cells and compared with adult thymus and indolent thymoma. Results and Conclusions: In the present case, thymoma was the cause of the observed multi-organ autoimmune syndromes as its recurrence and surgical removal was associated with the relapse and regression of the cutaneous symptoms, respectively. Initially, the patient presented with two autoimmune disorders with Th2/Th1 imbalance, myasthenia gravis (MG) and pemphigus foliaceus (PF), which regressed upon immunosuppressive treatment. Months later, the patient developed a lichenoid exanthema with a Th1-dominated skin infiltrate. Further clinical evaluation revealed the recurrence of the thymoma and the lichenoid exanthema gradually regressed upon thymectomy. Our contention that T cell recognition against distinct cutaneous autoantigens, such as desmoglein 1 (Dsg1), shifted from a Th2 to a Th1-dominated immune response could not be fully substantiated as the patient was on a stringent immunosuppressive treatment regimen. We could only observe a decrease of the initially present serum IgG autoantibodies against Dsg1. Phenotypic analysis of the associated thymoma showed a lower number of T regulatory cells compared to adult thymus and indolent thymoma, suggesting that impaired thymus-derived immune surveillance had a direct impact on the outcome of the observed cutaneous autoimmune disorders.

Role of Src and Cortactin in Pemphigus Skin Blistering.

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear and were studied here. In keratinocytes, cell cohesion in response to autoantibodies was reduced in Src-dependent manner by two patient-derived PV-IgG fractions as well as by AK23 but not by a third PV-IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient in a timeframe of 24 h and AK23, similar to PV-IgG, interfered with reconstitution of cell cohesion after Ca2+-switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co-localized along cell contacts and interacted with the Src substrate cortactin. In keratinocytes isolated from cortactin-deficient mice, cell adhesion was impaired and Src-mediated inhibition of AK23-induced loss of cell cohesion for 24 h was significantly reduced compared to wild-type (wt) cells. Similarly, AK23 impaired reconstitution of cell adhesion was Src-dependent only in the presence of cortactin. Likewise, Src inhibition significantly reduced AK23-induced skin blistering in wt but not cortactin-deficient mice. These data suggest that the Src-mediated long-term effects of AK23 on loss of cell cohesion and skin blistering are dependent on cortactin-mediated desmosome assembly. However, in human epidermis PV-IgG-induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition, indicating that Src may not be critical for skin blistering in intact human skin, at least when high levels of autoantibodies targeting Dsg1 are present.

The Immune Checkpoint Molecule CD200 Is Associated with Tumor Grading and Metastasis in Bladder Cancer.

BACKGROUND: We examined the expression of CD200, a ligand of immune tolerance, in transitional cell carcinoma of the human bladder (TCC). MATERIALS AND METHODS: CD200 was analyzed by immunohistochemistry (IHC) in 90 patients with suspected TCC lesions of the bladder. Expression of CD200 was exemplarily validated by quantitative reverse transcription polymerase chain reaction and western blot analysis. RESULTS: CD200 was detectable at mRNA and protein levels in TCC homogenate and TCC cell lines (T24, UMUC3). TCC tissues showed significantly higher CD200 expression (p<0.005) than normal bladder tissues. CD200 signals were also higher in metastasized compared to localized TCC (p<0.05). CD200 was significantly correlated to tumor grading (p<0.001) and was strongest in the subgroup with high-grade G2 TCC (vs. low-grade G2 p<0.05). CONCLUSION: This is the first report of CD200 expression in patients with TCC. The significant correlation between CD200 expression and tumor grading may suggest CD200 as a potential target and marker for immunotherapeutic approaches.

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.

c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress.

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs.

Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells.

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines.

Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate.

The desmosomal plaque proteins of the plakophilin family.

Three related proteins of the plakophilin family (PKP1_3) have been identified as junctional proteins that are essential for the formation and stabilization of desmosomal cell contacts. Failure of PKP expression can have fatal effects on desmosomal adhesion, leading to abnormal tissue and organ development. Thus, loss of functional PKP 1 in humans leads to ectodermal dysplasia/skin fragility (EDSF) syndrome, a genodermatosis with severe blistering of the epidermis as well as abnormal keratinocytes differentiation. Mutations in the human PKP 2 gene have been linked to severe heart abnormalities that lead to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the past few years it has been shown that junctional adhesion is not the only function of PKPs. These proteins have been implicated in cell signaling, organization of the cytoskeleton, and control of protein biosynthesis under specific cellular circumstances. Clearly, PKPs are more than just cell adhesion proteins. In this paper we will give an overview of our current knowledge on the very distinct roles of plakophilins in the cell.

Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.

BACKGROUND: The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and crucial for patterning of many aspects of the vertebrate body plan. In vertebrates, the mechanism of SHH signal transduction and its action on target genes by means of activating or repressing forms of GLI3 have been studied most extensively during limb development and the specification of the central nervous system. From these studies it has emerged, that Gli3 expression must be subject to a tight spatiotemporal regulation. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown. RESULTS: Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control. CONCLUSION: These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.

Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (complexus adhaerens).

The lymph node sinus are channel structures of unquestionable importance in immunology and pathology, specifically in the filtering of the lymph, the transport and processing of antigens, the adhesion and migration of immune cells, and the spread of metastatic cancer cells. Our knowledge of the cell and molecular biology of the sinus-forming cells is still limited, and the origin and biological nature of these cells have long been a matter of debate. Here, we review the relevant literature and present our own experimental results, in particular concerning molecular markers of intercellular junctions and cell differentiation. We show that both the monolayer cells lining the sinus walls and the intraluminal virgultar cell meshwork are indeed different morphotypes of the same basic endothelial cell character, as demonstrated by the presence of a distinct spectrum of general and lymphatic endothelial markers, and we therefore refer to these cells as sinus endothelial/virgultar cells (SEVCs). These cells are connected by unique adhering junctions, termed complexus adhaerentes, characterized by the transmembrane glycoprotein VE-cadherin, combined with the desmosomal plaque protein desmoplakin, several adherens junction plaque proteins including alpha- and beta-catenin and p120 catenin, and components of the tight junction ensemble, specifically claudin-5 and JAM-A, and the plaque protein ZO-1. We show that complexus adhaerentes are involved in the tight three-dimensional integration of the virgultar network of SEVC processes along extracellular guidance structures composed of paracrystalline collagen bundle "stays". Overall, the SEVC system might be considered as a local and specific modification of the general lymphatic vasculature system. Finally, physiological and pathological alterations of the SEVC system will be presented, and the possible value of the molecular markers described in histological diagnoses of autochthonous lymph node tumors will be discussed.

Alternative splicing of TGF-betas and their high-affinity receptors T beta RI, T beta RII and T beta RIII (betaglycan) reveal new variants in human prostatic cells.

BACKGROUND: The transforming growth factors (TGF)-beta, TGF-beta1, TGF-beta2 and TGF-beta 3, and their receptors [T beta RI, T beta RII, T beta R III (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants T beta RIB, T beta RIIB and TGF-beta 2B in human prostatic cells. RESULTS: Interestingly, a novel human receptor transcript T beta RIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant T beta RIB with four additional amino acids was identified also in human. Expression of the variant T beta RIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of T beta RIIC and TGF-beta 2B mainly in the epithelial cells with a preferential localization of TGF-beta 2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-beta ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-beta2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of T beta RII. CONCLUSION: In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants T beta RIB and the novel isoform T beta RIIC in man clearly contributes to the growing complexity of the TGF-beta family.

Desmosomes: just cell adhesion or is there more?

Desmosomes are cell adhesion structures (junctions) that are particularly abundant in cells derived from the ectodermal lineages. These junctions are required to maintain the integrity of organs subjected to mechanical stress, in particular the skin and the heart. This conclusion is partially based on tissue fragility phenotypes observed in mice with null mutations in certain desmosomal genes. Furthermore, patients have been identified that develop severe skin disorders, and even fatal heart diseases, due to impaired desmosome function. Nevertheless, desmosomes are more than cellular glue. New evidence suggests that these junctions can transmit signals from the extracellular environment to the nucleus, for example by controling the cytoplasmic pool of transcriptional co-factors that belong to the armadillo family of desmosomal proteins (i.e. plakoglobin, plakophilins). Understanding the signaling properties of desmosomes will provide new insights into developmental processes such as skin and skin appendage development. Furthermore, there is evidence to suggest that abnormal signaling through these junctions contributes to the symptoms of certain skin and heart diseases.

Differential expression of desmosomal plakophilins in various types of carcinomas: correlation with cell type and differentiation.

Plakophilins (PKPs) are a set of 3 constitutive armadillo repeat proteins of the desmosomal plaque, termed PKP 1, PKP 2, and PKP 3, which have been shown to be functionally relevant for desmosomal adhesion. We have performed a systematic immunohistochemical study of the 3 PKPs in oral and pharyngeal squamous cell carcinomas (SqCCs; n = 40); colorectal, pancreatic, and prostate adenocarcinomas (n = 31), and hepatocellular carcinomas (HCCs; n = 8). In SqCCs, PKP 1 and PKP 3 revealed common desmosome-type immunostaining, their expression level being inversely correlated with the degree of malignancy. Instead, staining for PKP 2 was limited. In contrast, all adenocarcinomas contained PKP 2 and-often abundantly-PKP 3 in desmosome-typical pattern, whereas PKP 1 was expressed only in prostate tumors. The presence of PKP 3 in adenocarcinomas was confirmed by immunoblotting. In HCCs, only PKP 2 was detected. Under certain staining conditions, focal nuclear immunoreactivity for PKP 1 was observed in some SqCCs and HCCs. Our results, which are inconsistent with previously published data to some extent, indicate a principal preservation of the cell type and differentiation-related expression patterns of PKPs in normal epithelia. For PKP 1, a suppressor function of malignant behavior seems conceivable, whereas the putative functional significance of its occurrence in tumor cell nuclei requires further studies.

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.