Formation Of Adherens Junctions Research Articles

Vav Proteins in Development of the Brain: A Potential Relationship to the Pathogenesis of Congenital Zika Syndrome?

Zika virus (ZIKV) infection during pregnancy can result in a significant impact on the brain and eye of the developing fetus, termed congenital zika syndrome (CZS). At a morphological level, the main serious presentations of CZS are microcephaly and retinal scarring. At a cellular level, many cell types of the brain may be involved, but primarily neuronal progenitor cells (NPC) and developing neurons. Vav proteins have guanine exchange activity in converting GDP to GTP on proteins such as Rac1, Cdc42 and RhoA to stimulate intracellular signaling pathways. These signaling pathways are known to play important roles in maintaining the polarity and self-renewal of NPC pools by coordinating the formation of adherens junctions with cytoskeletal rearrangements. In developing neurons, these same pathways are adopted to control the formation and growth of neurites and mediate axonal guidance and targeting in the brain and retina. This review describes the role of Vavs in these processes and highlights the points of potential ZIKV interaction, such as (i) the binding and entry of ZIKV in cells via TAM receptors, which may activate Vav/Rac/RhoA signaling; (ii) the functional convergence of ZIKV NS2A with Vav in modulating adherens junctions; (iii) ZIKV NS4A/4B protein effects on PI3K/AKT in a regulatory loop via PPI3 to influence Vav/Rac1 signaling in neurite outgrowth; and (iv) the induction of SOCS1 and USP9X following ZIKV infection to regulate Vav protein degradation or activation, respectively, and impact Vav/Rac/RhoA signaling in NPC and neurons. Experiments to define these interactions will further our understanding of the molecular basis of CZS and potentially other developmental disorders stemming from in utero infections. Additionally, Vav/Rac/RhoA signaling pathways may present tractable targets for therapeutic intervention or molecular rationale for disease severity in CZS.

Cortical tension initiates the positive feedback loop between cadherin and F-actin

Adherens junctions physically link two cells at their contact interface via extracellular binding between cadherin molecules and intracellular interactions between cadherins and the actin cytoskeleton. Cadherin and actomyosin cytoskeletal dynamics are regulated reciprocally by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechanochemical cross talk that regulates adherens junction formation and homeostasis. The model couples a two-dimensional lattice-based simulation of cadherin dynamics with a reaction-diffusion representation of the reorganising actomyosin network through its regulation by Rho signalling at the intracellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis-less-dependent manner. We then recapitulate the process of cell-cell contact formation. Our model suggests that cortical tension applied on the contact rim can explain the ring distribution of cadherin and actin filaments (F-actin) on the cell-cell contact of the cell doublet. Furthermore, we propose and test the hypothesis that cadherin and F-actin interact like a positive feedback loop, which is necessary for formation of the ring structure. Different patterns of cadherin distribution were observed as an emergent property of disturbances of this positive feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.

Non-homogeneous dispersion of graphene in polyacrylonitrile substrates induces a migrastatic response and epithelial-like differentiation in MCF7 breast cancer cells

BackgroundRecent advances from studies of graphene and graphene-based derivatives have highlighted the great potential of these nanomaterials as migrastatic agents with the ability to modulate tumor microenvironments. Nevertheless, the administration of graphene nanomaterials in suspensions in vivo is controversial. As an alternative approach, herein, we report the immobilization of high concentrations of graphene nanoplatelets in polyacrylonitrile film substrates (named PAN/G10) and evaluate their potential use as migrastatic agents on cancer cells.ResultsBreast cancer MCF7 cells cultured on PAN/G10 substrates presented features resembling mesenchymal-to-epithelial transition, e.g., (i) inhibition of migratory activity; (ii) activation of the expression of E-cadherin, cytokeratin 18, ZO-1 and EpCAM, four key molecular markers of epithelial differentiation; (iii) formation of adherens junctions with clustering and adhesion of cancer cells in aggregates or islets, and (iv) reorganization of the actin cytoskeleton resulting in a polygonal cell shape. Remarkably, assessment with Raman spectroscopy revealed that the above-mentioned events were produced when MCF7 cells were preferentially located on top of graphene-rich regions of the PAN/G10 substrates.ConclusionsThe present data demonstrate the capacity of these composite substrates to induce an epithelial-like differentiation in MCF7 breast cancer cells, resulting in a migrastatic effect without any chemical agent-mediated signaling. Future works will aim to thoroughly evaluate the mechanisms of how PAN/G10 substrates trigger these responses in cancer cells and their potential use as antimetastatics for the treatment of solid cancers.Graphical

Vascular Endothelial (VE)-cadherin-mediated adherens junctions involvement in cardiovascular progenitor cell specification.

Vascular Endothelial cadherin, a type II classical cadherin, is the major cadherin molecule participating in homotypic cell-cell adhesion structures between endothelial cells. It associates with cytoplasmic and membrane cytoskeletal elements to form endothelial adherens junctions (AJs), pivotal in regulating endothelial barrier function in the adult. VE-cadherin-mediated AJs are also involved in signaling via direct or indirect associations with receptors. The generation of mutant animals, especially mice and zebrafish, revealed many details concerning the role of VE-cadherin-mediated AJs in cardiovascular development. In general, VE-cadherin knockout (KO) in mice is embryonic lethal due to severe cardiovascular defects, and major signaling pathways as well as vascular formation cues were discovered in developing endothelium. However, there is little information regarding AJs formation and their components in cardiovascular progenitors. We have characterized in detail the activation pattern of mouse VE-cadherin promoter (Pvec) in a mouse embryonic stem cells (ESCs) differentiation system in vitro. Surprisingly, we found that it is activated transiently in cardiac progenitors that belong to the second heart field. Based on Pvec activation, we isolated this population in vitro and found that it can self-renew by induction of the Wnt/β-catenin pathway. Next, we successfully established cell culture conditions that allowed self-renewal of this population that consists of endothelial and cardiac progenitors. Transplantation in rat hearts showed that they can survive and differentiate to cardiomyocytes and endothelial cells. Although further characterization is needed, these cells can be used in cell-based therapies as well as in drug screening.

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Significance Proteins are modified by many posttranslational modifications (PTMs) with crucial regulatory functions. A PTM attracting increasing interest is ADP-ribosylation, capable of altering crucial cellular targets. We show that mono-ADP-ribosylation by PARP12 of the protein Golgin-97 regulates transport to the plasma membrane of a specific group of functionally crucial cargo proteins. PARP12 is shown to be part of a regulatory cascade initiated by PKD and involving the direct phosphorylation and activation of PARP12. These events define, through Golgin-97 mono-ADP-ribosylation, a Golgi sorting mechanism for specific basolateral cargoes, including E-cadherin. Defects in this cascade impair E-cadherin transport and formation of cell–cell contacts, with potential consequences for the formation of adherens junctions, polarization of epithelial cells, and development of epithelial–mesenchymal transformation.

Generating Monocyte-Derived Endothelial-like Cells for Vascular Regeneration.

A major limitation in engineering vascular grafts is the lack of proper endothelium to prevent thrombosis and subsequent graft failure. Obtaining endothelial cells from patients' vasculature is intrusive and requires extensive culture time. Here we present an alternative strategy wherein abundant and easily accessible monocytes from peripheral blood are cultured and differentiated towards an endothelial-like state capable of preventing thrombosis through production of nitric oxide and formation of endothelial adherens junctions. Considering the plethora of monocytes present within peripheral blood, this method provides a robust alternative to generating endothelial cells required for vascular graft production.

Association of the ubiquitin specific peptidase 9X -linked and Afadin expression patterns with sexual maturation in boar testis.

Closely correlated expression patterns between ubiquitin specific peptidase 9X-linked (USP9X) and adherens junction formation factor (Afadin) in mouse testis development suggests that Usp9x regulates the deubiquitination of Af-6 (also known as Afadin, AFDN), and subsequently, the cell adhesion dynamics during gametogenesis. However, this relationship has not yet been tested in other domestic animals. The study was examined the temporal and spatial expression patterns of porcine USP9X and AFDN from the pre-pubertal to adult stages using real time-PCR and immunohistochemistry. Furthermore, we detected the transcripts of USP9X and AFDN in the testis of 1-, 6- and 12-months old boar, respectively. USP9X and AFDN were found to have similar expressions patterns, with basal expression after 1 month followed by a significant up-regulation from 6 months (puberty) onwards. In addition, neither the AFDN or USP9X proteins were detected in spermatogenic cells but they were expressed in the leydig cells and sertoli cells. USP9X was detected around the basal lamina during pre-puberty, and predominantly expressed in the leydig cells at puberty. Finally, in adult testis, USP9X was increased at the sertoli cell-cell interface and the sertoli cell-spermatid interface. In summary, closely correlated expression patterns between USP9X and AFDN in boar testis supports the previous findings in mice. Furthermore, the junction connections between the sertoli cells may be regulated by the ubiquitination process mediated via USP9X.

Identification of lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver for invasion and migration of oral cancer by tumor heterogeneity exploitation

BackgroundCancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy.MethodsWe used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis.ResultsWe identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation.ConclusionAnalysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Analysis of RAGE Proteome and Interactome in Lung Adenocarcinoma Using PANTHER and STRING Databases.

Adenocarcinoma accounts for about 40% of all lung cancers. Histological studies indicate a loss of expression of the Receptor for Advanced Glycation End-products (RAGE) in lung adenocarcinoma cells compared to neighboring non-malignant tissue. Gene silencing of RAGE in human lung adenocarcinoma cells was performed and then cells were subjected to LC-MS/MS (n = 3, FDR < 1%). Differentially expressed proteins were analyzed using the PANTHER Classification System and STRING Interactome, identifying functions and protein-protein interaction networks. We observed expression of dominant-negative (DN-) RAGE, an isoform lacking the critical intracellular signaling tail observed in the full length (FL-) RAGE. Proteomic analysis suggests DN-RAGE likely plays a crucial role in cell polarity, metastases, and in cell-cell or cell-matrix complexes through focal adhesion or adherens junction formation. DN-RAGE may also regulate the expression of FL-RAGE and may provide a "switch" that could transition from a pro-inflammatory to a migratory cell as vimentin expression increased along with a reduction in cell polarity proteins. STRING interactome analysis identified seven protein-protein interaction networks involved in the regulation of gene expression, cell organization, cytoskeletal changes, sub-membrane plaque formation, as well as cytokinesis, cell shape, and motility. Suggesting expression of DN-RAGE may contribute to metastases and the development of advanced cancer.

Dbnl and β-catenin promote pro-N-cadherin processing to maintain apico-basal polarity.

The neural tube forms when neural stem cells arrange into a pseudostratified, single-cell-layered epithelium, with a marked apico-basal polarity, and in which adherens junctions (AJs) concentrate in the subapical domain. We previously reported that sustained β-catenin expression promotes the formation of enlarged apical complexes (ACs), enhancing apico-basal polarity, although the mechanism through which this occurs remained unclear. Here, we show that β-catenin interacts with phosphorylated pro-N-cadherin early in its transit through the Golgi apparatus, promoting propeptide excision and the final maturation of N-cadherin. We describe a new β-catenin-dependent interaction of N-cadherin with Drebrin-like (Dbnl), an actin-binding protein that is involved in anterograde Golgi trafficking of proteins. Notably, Dbnl knockdown led to pro-N-cadherin accumulation and limited AJ formation. In brief, we demonstrate that Dbnl and Drebrin-like β-catenin assist in the maturation of pro-N-cadherin, which is critical for AJ formation and for the recruitment AC components like aPKC and, consequently, for the maintenance of apico-basal polarity.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Hydrodynamic forces influence the gene transcription of mechanosensitive intercellular junction associated genes in corneal endothelial cells

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium. Native and tissue-engineered (TE) corneal endothelium were cultured in a corneal bioreactor for 7 days with 16 mmHg IOP and 5 μl/ml of medium flow. RNA was harvested, and gene expression was quantified. Cells that were used to reconstruct the TE corneal endothelia were also seeded on plastic to characterize their morphology by calculating their circularity index. For native endothelia, hydrodynamic forces increased gene expression of GJA1 (connexin 43), CDH2 (N-cadherin), TJP1 (ZO-1), ITGAV (integrin subunit αv), ITGB5 (integrin subunit β5) and CTNND1 (p120-ctn) by 1.68 ± 0.40, 1.10 ± 0.27, 3.80 ± 0.56, 1.82 ± 0.33, 1.32 ± 0.21 and 3.04 ± 0.63, respectively. For TE corneal endothelium, this fold change was 1.72 ± 0.31, 1.58 ± 0.41, 6.18 ± 1.03, 1.80 ± 0.71, 1.77 ± 0.55, 2.42 ± 0.71. Furthermore, gene transcription fold changes (hydrodynamic/control) increased linearly with TE corneal endothelium cells population morphology with r = 0.83 for TJP1 (ZO-1) and r = 0.58 for CTNND1 (p120-ctn). In fact, the more elongated the cells populations were, the greater hydrodynamic conditions increased the transcription of TJP1 (ZO-1) and CTNND1 (p120-ctn). These results suggest that hydrodynamic forces contribute to the maintenance of tight and adherens junctions of native corneal endothelial cells, as well as to the formation of tight and adherens junctions of corneal endothelial cells that are in the process of forming a functional endothelial barrier.

PCB and TCDD derived embryonic cardiac defects result from a novel AhR pathway

Polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contaminants known to impact cardiac development, a key step in the embryonic development of most animals. To date, little is understood of the molecular mechanism driving the observed cardiac defects in exposed fishes. The literature shows PCB & TCDD derived cardiac defects are concurrent with, but not caused by, expression of cyp1A, due to activation of the aryl hydrocarbon receptor (AhR) gene activation pathway. However, in this study, detailed visualization of fish hearts exposed to PCBs and TCDD show that, in addition to a failure of cardiac looping in early heart development, the inner endocardial lining of the heart fails to maintain proper cell adhesion and tissue integrity. The resulting gap between the endocardium and myocardium in both zebrafish and Atlantic sturgeon suggested functional faults in endothelial adherens junction formation. Thus, we explored the molecular mechanism triggering cardiac defects using immunohistochemistry to identify the location and phosphorylation state of key regulatory and adhesion molecules. We hypothesized that PCB and TCDD activates AhR, phosphorylating Src, which then phosphorylates the endothelial adherens junction protein, VEcadherin. When phosphorylated, VEcadherin dimers, found in the endocardium and vasculature, separate, reducing tissue integrity. In zebrafish, treatment with PCB and TCDD contaminants leads to higher phosphorylation of VEcadherin in cardiac tissue suggesting that these cells have reduced connectivity. Small molecule inhibition of Src phosphorylation prevents contaminant stimulated phosphorylation of VEcadherin and rescues both cardiac function and gross morphology. Atlantic sturgeon hearts show parallels to contaminant exposed zebrafish cardiac phenotype at the tissue level. These data suggest that the mechanism for PCB and TCDD action in the heart is, in part, distinct from the canonical mechanism described in the literature and that cardiac defects are impacted by this nongenomic mechanism.

Cadherin-mediated host-pathogen interactions.

Cell adhesion molecules mediate cell-to-cell and cell-to-matrix adhesions and play an immense role in a myriad of physiological processes during the growth and development of a multicellular organism. Cadherins belong to a major group of membrane-bound cell surface proteins that, in coordination with nectins, drive the formation and maintenance of adherens junctions for mediating cell to cell adhesion, cellular communication and signalling. Alongside adhesive function, the involvement of cadherins in mediating host-pathogen interactions has been extensively explored in recent years. In this review, we provide an in-depth understanding of microbial pathogens and their virulence factors that exploit cadherins for their strategical invasion into the host cell. Furthermore, macromolecular interactions involving cadherins and various microbial factors such as secretory toxins and adhesins lead to the disintegration of host cell junctions followed by the entry of the pathogen or triggering downstream signalling pathways responsible for successful invasion of the pathogenic microbes are discussed. Besides providing a comprehensive insight into some of the structural complexes involving cadherins and microbial factors to offer the mechanistic details of host-pathogen interactions, the current review also highlights novel constituents of various cell signalling events such as endocytosis machinery elicited upon microbial infections.

Glycolysis is integral to histamine-induced endothelial hyperpermeability.

Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis.

Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves

Endothelial cells (ECs) have gained an increased scientific focus since they were reported to provide guidance for Schwann cells and subsequently following axons after nerve injuries. However, previous protocols for the isolation of nerve-derived ECs from human nerves are ineffective regarding time and yield. Therefore, we established a novel and efficient protocol for the isolation of ECs from human peripheral nerves by means of immunomagnetic CD31-antibody conjugated Dynabeads and assessed the purity of the isolated cells. The easy-to-follow and time-effective isolation method allows the isolation of > 95% pure ECs. The isolated ECs were shown to express highly specific EC marker proteins and revealed functional properties by formation of CD31 and VE-cadherin positive adherens junctions, as well as ZO-1 positive tight-junctions. Moreover, the formation of capillary EC-tubes was observed in-vitro. The novel protocol for the isolation of human nerve-derived ECs allows and simplifies the usage of ECs in research of the human blood-nerve-barrier and peripheral nerve regeneration. Additionally, a potential experimental application of patient-derived nerve ECs in the in-vitro vascularization of artificial nerve grafts is feasible.

Cell Migration Guided by Cell-Cell Contacts in Innate Immunity.

The directed migration of leukocytes to sites of damage or infection is necessary for a productive immune response. There is substantial evidence supporting a key role for chemoattractants in directed migration, however, less is known about how cell-cell contacts affect the migratory behavior of leukocytes in innate immunity. Here, we explore how cell-cell contacts can affect the directed migration of innate immune cells, including their role in attracting, repelling, or stopping cell motility. Further investigation of cell contact dynamics as guidance cues may yield new insights into the regulation of innate immunity.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence.In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed.In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls.Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.