Acral peeling in Nagashima type palmo-plantar keratosis patients reveals the role of serine protease inhibitor B 7 in keratinocyte adhesion.

Acral peeling skin syndrome (APSS) is a heterogenous group of genodermatoses, manifested by peeling of palmo-plantar skin and occasionally associated with erythema and epidermal thickening. A subset of APSS is caused by mutations in protease inhibitor encoding genes, resulting in unopposed protease activity and desmosomal degradation and/or mis-localization, leading to enhanced epidermal desquamation. We investigated two Arab-Muslim siblings with mild keratoderma and prominent APSS since infancy. Genetic analysis disclosed a homozygous mutation in SERPINB7, c.796C > T, which is the founder mutation in Nagashima type palmo-plantar keratosis (NPPK). Although not previously formally reported, APSS was found in other patients with NPPK. We hypothesized that loss of SERPINB7 function might contribute to the peeling phenotype through impairment of keratinocyte adhesion, similar to other protease inhibitor mutations that cause APSS. Mis-localization of desmosomal components was observed in a patient plantar biopsy compared with a biopsy from an age- and gender-matched healthy control. Silencing of SERPINB7 in normal human epidermal keratinocytes led to increased cell sheet fragmentation upon mechanical stress. Immunostaining showed reduced expression of desmoglein 1 and desmocollin 1. This study shows that in addition to stratum corneum perturbation, loss of SERPINB7 disrupts desmosomal components, which could lead to desquamation, manifested by skin peeling.

Epidermolytic Ichthyosis Sine Epidermolysis.

Epidermolytic ichthyosis (EI) is a rare disorder of cornification caused by mutations in KRT1 and KRT10, encoding two suprabasal epidermal keratins. Because of the variable clinical features and severity of the disease, histopathology is often required to correctly direct the molecular analysis. EI is characterized by hyperkeratosis and vacuolar degeneration of the upper epidermis, also known as epidermolytic hyperkeratosis, hence the name of the disease. In the current report, the authors describe members of 2 families presenting with clinical features consistent with EI. The patients were shown to carry classical mutations in KRT1 or KRT10, but did not display epidermolytic changes on histology. These observations underscore the need to remain aware of the limitations of pathological features when considering a diagnosis of EI.

Severe dermatitis, multiple allergies, and metabolic wasting syndrome caused by a novel mutation in the N-terminal plakin domain of desmoplakin.

BACKGROUND: Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported. OBJECTIVE: We studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis. METHODS: Histopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents. RESULTS: No mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide. CONCLUSIONS: SAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

PP2A-B55alpha controls keratinocyte adhesion through dephosphorylation of the Desmoplakin C-terminus.

Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP's C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart.

Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAFV600E-induced cellular senescence in human melanocytes.

Melanoma arises from transformation of melanocytes in the basal layer of the epidermis where they are surrounded by keratinocytes, with which they interact through cell contact and paracrine communication. Considerable effort has been devoted to determining how the accumulation of oncogene and tumor suppressor gene mutations in melanocytes drive melanoma development. However, the extent to which alterations in keratinocytes that occur in the developing tumor niche serve as extrinsic drivers of melanoma initiation and progression is poorly understood. We recently identified the keratinocyte-specific cadherin, desmoglein 1 (Dsg1), as an important mediator of keratinocyte:melanoma cell crosstalk, demonstrating that its chronic loss, which can occur through melanoma cell-dependent paracrine signaling, promotes behaviors that mimic a malignant phenotype. Here we address the extent to which Dsg1 loss affects early steps in melanomagenesis. RNA-Seq analysis revealed that paracrine signals from Dsg1-deficient keratinocytes mediate a transcriptional switch from a differentiated to undifferentiated cell state in melanocytes expressing BRAFV600E, a driver mutation commonly present in both melanoma and benign nevi and reported to cause growth arrest and oncogene-induced senescence (OIS). Of ~220 differentially expressed genes in BRAFV600E cells treated with Dsg1-deficient conditioned media (CM), the laminin superfamily member NTN4/Netrin-4, which inhibits senescence in endothelial cells, stood out. Indeed, while BRAFV600E melanocytes treated with Dsg1-deficient CM showed signs of senescence bypass as assessed by increased senescence-associated ß-galactosidase activity and decreased p16, knockdown of NTN4 reversed these effects. These results suggest that Dsg1 loss in keratinocytes provides an extrinsic signal to push melanocytes towards oncogenic transformation once an initial mutation has been introduced.

Transcriptional profiling of rare acantholytic disorders suggests common mechanisms of pathogenesis.

Darier, Hailey-Hailey, and Grover diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions, we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover diseases were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared increase in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover diseases. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Furthermore, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared with normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover diseases, and identify decreases in actin organization pathways as a unique signature present in these conditions.

Melanoma cells repress Desmoglein 1 in keratinocytes to promote tumor cell migration.

Melanoma is an aggressive cancer typically arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment remains understudied. We previously showed that temporary loss of the keratinocyte-specific cadherin, Desmoglein 1 (Dsg1), controls paracrine signaling between normal melanocytes and keratinocytes to stimulate the protective tanning response. Here, we provide evidence that melanoma cells hijack this intercellular communication by secreting factors that keep Dsg1 expression low in the surrounding keratinocytes, which in turn generate their own paracrine signals that enhance melanoma spread through CXCL1/CXCR2 signaling. Evidence suggests a model whereby paracrine signaling from melanoma cells increases levels of the transcriptional repressor Slug, and consequently decreases expression of the Dsg1 transcriptional activator Grhl1. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction contributes to melanoma cell movement associated with tumor progression.

Organotypic Human Skin Cultures Incorporating Primary Melanocytes.

Three-dimensional (3D) human organotypic skin cultures provide a physiologically relevant model that recapitulates in vivo skin features. Most commonly, organotypic skin cultures are created by seeding isolated epidermal keratinocytes onto a collagen/fibroblast plug and lifting to an air liquid interface. These conditions are sufficient to drive stratification and differentiation of the keratinocytes to form an epidermal-like sheet with remarkable similarities to human epidermis. Coupled with genetic or pharmacological treatments, these cultures provide a powerful tool for elucidating keratinocyte biology. Recent focus has been placed on increasing the utility of organotypic skin cultures by incorporating other cell types that are present in the skin, such as melanocytes, immune cells, and other cells. Here we describe a step-by-step protocol for the isolation of neonatal human epidermal keratinocytes and melanocytes from foreskins, and the creation of organotypic skin cultures that include both cell types. We also describe methods that can be used to assess melanocyte behavior in these organotypic cultures, including methods for whole mount staining, measurement of melanocyte dendricity, staining for pigment, and 5-bromo-2'-deoxyuridine (BrdU) labeling for identification of proliferating cells. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary cells Alternate Protocol: Isolation of primary cells using differential trypsinization Basic Protocol 2: Organotypic culture protocol Support Protocol 1: Culture and maintenance of NHEKs and melanocytes Support Protocol 2: Lentiviral transduction of melanocytes Support Protocol 3: Retroviral transduction of NHEKs Support Protocol 4: Whole mount immunostaining protocol Support Protocol 5: Measuring melanocyte dendricity Support Protocol 6: Fontana-Masson staining protocol Support Protocol 7: BrdU labeling and staining.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Desmosomes polarize and integrate chemical and mechanical signaling to govern epidermal tissue form and function.

The epidermis is a stratified epithelium in which structural and functional features are polarized across multiple cell layers. This type of polarity is essential for establishing the epidermal barrier, but how it is created and sustained is poorly understood. Previous work identified a role for the classic cadherin/filamentous-actin network in establishment of epidermal polarity. However, little is known about potential roles of the most prominent epidermal intercellular junction, the desmosome, in establishing epidermal polarity, in spite of the fact that desmosome constituents are patterned across the apical to basal cell layers. Here, we show that desmosomes and their associated intermediate filaments (IFs) are key regulators of mechanical polarization in epidermis, whereby basal and suprabasal cells experience different forces that drive layer-specific functions. Uncoupling desmosomes and IF or specific targeting of apical desmosomes through depletion of the superficial desmosomal cadherin, desmoglein 1, impedes basal stratification in an in vitro competition assay and suprabasal tight junction barrier functions in 3D reconstructed epidermis. Surprisingly, disengaging desmosomes from IF also accelerated the expression of differentiation markers, through precocious activation of the mechanosensitive transcriptional regulator serum response factor (SRF) and downstream activation of epidermal growth factor receptor family member ErbB2 by Src family kinase (SFK)-mediated phosphorylation. This Dsg1-SFK-ErbB2 axis also helps maintain tight junctions and barrier function later in differentiation. Together, these data demonstrate that the desmosome-IF network is a critical contributor to the cytoskeletal-adhesive machinery that supports the polarized function of the epidermis.

Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ.

Biochemical methods can reveal stable protein-protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein-protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay® (PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein-protein interactions, but also can reveal post-translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin-embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma-Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Proximity ligation assay Support Protocol 1: Antigen retrieval method for formalin-fixed, paraffin-embedded tissues Support Protocol 2: Creation of custom PLA probes using the Duolink™ In Situ Probemaker Kit when commercially available probes are not suitable Basic Protocol 2: Imaging, quantification, and analysis of PLA signals.

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling.

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte-specific cell-cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1-silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1-silenced keratinocytes. Dsg1-silenced keratinocytes increased melanocyte-stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1-silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1-silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1-deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.

The Role of Desmoglein 1 in Gap Junction Turnover Revealed through the Study of SAM Syndrome.

An effective epidermal barrier requires structural and functional integration of adherens junctions, tight junctions, gap junctions (GJ), and desmosomes. Desmosomes govern epidermal integrity while GJs facilitate small molecule transfer across cell membranes. Some patients with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, caused by biallelic desmoglein 1 (DSG1) mutations, exhibit skin lesions reminiscent of erythrokeratodermia variabilis, caused by mutations in connexin (Cx) genes. We, therefore, examined whether SAM syndrome-causing DSG1 mutations interfere with Cx expression and GJ function. Lesional skin biopsies from SAM syndrome patients (n = 7) revealed decreased Dsg1 and Cx43 plasma membrane localization compared with control and nonlesional skin. Cultured keratinocytes and organotypic skin equivalents depleted of Dsg1 exhibited reduced Cx43 expression, rescued upon re-introduction of wild-type Dsg1, but not Dsg1 constructs modeling SAM syndrome-causing mutations. Ectopic Dsg1 expression increased cell-cell dye transfer, which Cx43 silencing inhibited, suggesting that Dsg1 promotes GJ function through Cx43. As GJA1 gene expression was not decreased upon Dsg1 loss, we hypothesized that Cx43 reduction was due to enhanced protein degradation. Supporting this, PKC-dependent Cx43 S368 phosphorylation, which signals Cx43 turnover, increased after Dsg1 depletion, while lysosomal inhibition restored Cx43 levels. These data reveal a role for Dsg1 in regulating epidermal Cx43 turnover.

Polyethylene glycol-mediated colorectal cancer chemoprevention: roles of epidermal growth factor receptor and Snail.

Polyethylene glycol (PEG) is a clinically widely used agent with profound chemopreventive properties in experimental colon carcinogenesis. We reported previously that Snail/beta-catenin signaling may mediate the suppression of epithelial proliferation by PEG, although the upstream events remain unclear. We report herein the role of epidermal growth factor receptor (EGFR), a known mediator of Snail and overexpressed in approximately 80% of human colorectal cancers, on PEG-mediated antiproliferative and hence antineoplastic effects in azoxymethane (AOM) rats and HT-29 colon cancer cells. AOM rats were randomized to either standard diet or one with 10% PEG-3350 and euthanized 8 weeks later. The colonic samples were subjected to immunohistochemical or Western blot analyses. PEG decreased mucosal EGFR by 60% (P < 0.001). Similar PEG effects were obtained in HT-29 cells. PEG suppressed EGFR protein via lysosmal degradation with no change in mRNA levels. To show that EGFR antagonism per se was responsible for the antiproliferative effect, we inhibited EGFR by either pretreating cells with gefitinib or stably transfecting with EGFR-short hairpin RNA and measured the effect of PEG on proliferation. In either case, PEG effect was blunted, suggesting a vital role of EGFR. Flow cytometric analysis revealed that EGFR-short hairpin RNA cells, besides having reduced membrane EGFR, also expressed low Snail levels (40%), corroborating a strong association. Furthermore, in EGFR silenced cells, PEG effect on EGFR or Snail was muted, similar to that on proliferation. In conclusion, we show that EGFR is the proximate membrane signaling molecule through which PEG initiates antiproliferative activity with Snail/beta-catenin pathway playing the central intermediary function.

Antiproliferative effect of sulindac in colonic neoplasia prevention: role of COOH-terminal Src kinase.

Although the nonsteroidal anti-inflammatory drugs (NSAID) protection against colorectal cancer is well established, the molecular mechanisms remain unclear. We show herein that induction of the tumor suppressor gene COOH-terminal Src kinase (Csk) by NSAID is important for their antiproliferative and hence chemopreventive effects. In the azoxymethane-treated rat model of experimental colon carcinogenesis, sulindac treatment markedly induced Csk with a corresponding increase in inhibitory phosphorylation of Src (Tyr(527)). Sulindac-mediated Csk induction was replicated in the human colorectal cancer cell line HT-29, with a corresponding suppression of both Src kinase activity (63% of vehicle; P < 0.05) and E-cadherin tyrosine phosphorylation (an in vivo Src target). To determine the importance of Csk in NSAID antiproliferative activity, we stably transfected a Csk-specific short hairpin RNA (shRNA) vector into HT-29 cells, thereby blunting the sulindac-mediated Csk induction. These transfectants were significantly less responsive to the antiproliferative effect of sulindac sulfide (suppression of proliferating cell nuclear antigen was 21 +/- 2.3% in transfectants versus 45 +/- 4.23% in wild-type cells), with a corresponding mitigation of the sulindac-mediated G(1)-S-phase arrest (S-phase cells 48 +/- 3.6% versus 14 +/- 2.8% of vehicle respectively). Importantly, the Csk shRNA cells had a marked decrease in the cyclin-dependent kinase inhibitor p21(cip/waf1), a critical regulator of G(1)-S-phase progression (49% of wild-type cells). Moreover, although sulindac-mediated induction of p21(cip/waf1) was 113% in wild-type HT-29, this induction was alleviated in the Csk shRNA transfectants (65% induction; P < 0.01). Thus, this is the first demonstration that the antiproliferative activity of NSAID is modulated, at least partly, through the Csk/Src axis.

Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination.

The epidermis is a multi-layered epithelium that serves as a barrier against water loss and environmental insults. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes during which basal cells commit to differentiate and move towards the skin's surface. Here, we reveal an unexpected role for the vertebrate cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into the suprabasal layer through a process of delamination, one mechanism of epidermal stratification. Actin remodeling requires the interaction of Dsg1 with the dynein light chain, Tctex-1 and the actin scaffolding protein, cortactin. We demonstrate that Tctex-1 ensures the correct membrane compartmentalization of Dsg1-containing desmosomes, allowing cortactin/Arp2/3-dependent perijunctional actin polymerization and decreasing tension at E-cadherin junctions to promote keratinocyte delamination. Moreover, Dsg1 is sufficient to enable simple epithelial cells to exit a monolayer to form a second layer, highlighting its morphogenetic potential.

Filaggrin 2 Deficiency Results in Abnormal Cell-Cell Adhesion in the Cornified Cell Layers and Causes Peeling Skin Syndrome Type A.

Peeling skin syndromes form a large and heterogeneous group of inherited disorders characterized by superficial detachment of the epidermal cornified cell layers, often associated with inflammatory features. Here we report on a consanguineous family featuring noninflammatory peeling of the skin exacerbated by exposure to heat and mechanical stress. Whole exome sequencing revealed a homozygous nonsense mutation in FLG2, encoding filaggrin 2, which cosegregated with the disease phenotype in the family. The mutation was found to result in decreased FLG2 RNA levels as well as almost total absence of filaggrin 2 in the patient epidermis. Filaggrin 2 was found to be expressed throughout the cornified cell layers and to colocalize with corneodesmosin that plays a crucial role in maintaining cell-cell adhesion in this region of the epidermis. The absence of filaggrin 2 in the patient skin was associated with markedly decreased corneodesmosin expression, which may contribute to the peeling phenotype displayed by the patients. Accordingly, using the dispase dissociation assay, we showed that FLG2 downregulation interferes with keratinocyte cell-cell adhesion. Of particular interest, this effect was aggravated by temperature elevation, consistent with the clinical phenotype. Restoration of corneodesmosin levels by ectopic expression rescued cell-cell adhesion. Taken together, the present data suggest that filaggrin 2 is essential for normal cell-cell adhesion in the cornified cell layers.

Inducible nitric oxide synthase (iNOS) mediates the early increase of blood supply (EIBS) in colon carcinogenesis.

We have recently demonstrated that dramatic alteration in mucosal microvascular blood content termed early increase in blood supply (EIBS) is a hallmark of early colon carcinogenesis. In the current study, we elucidate the mechanism of EIBS by assessing iNOS/nitric oxide axis in the histologically normal colonic mucosa of rats treated with the colon-specific carcinogen, azoxymethane. We demonstrate that there was a strong temporal correlation between EIBS and iNOS expression/activity. Importantly, we also observed that short-term treatment with nitric oxide inhibitor abrogated EIBS. These data indicate that iNOS induction may have a critical role in augmenting the predysplastic mucosal blood supply and thereby fostering colon carcinogenesis.

Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/beta-catenin signaling.

Polyethylene glycol (PEG) is one of the most potent chemopreventive agents against colorectal cancer; however, the mechanisms remain largely unexplored. In this study, we assessed the ability of PEG to target cyclin D1-beta-catenin-mediated hyperproliferation in the azoxymethane-treated rat model and the human colorectal cancer cell line, HT-29. Azoxymethane-treated rats were randomized to AIN-76A diet alone or supplemented with 5% PEG-8000. After 30 weeks, animals were euthanized and biopsies of aberrant crypt foci and uninvolved crypts were subjected to immunohistochemical and immunoblot analyses. PEG markedly suppressed both early and late markers of azoxymethane-induced colon carcinogenesis (fractal dimension by 80%, aberrant crypt foci by 64%, and tumors by 74%). In both azoxymethane-treated rats and HT-29 cells treated with 5% PEG-3350 for 24 hours, PEG decreased proliferation (45% and 52%, respectively) and cyclin D1 (78% and 56%, respectively). Because beta-catenin is the major regulator of cyclin D1 in colorectal cancer, we used the T-cell factor (Tcf)-TOPFLASH reporter assay to show that PEG markedly inhibited beta-catenin transcriptional activity. PEG did not alter total beta-catenin expression but rather its nuclear localization, leading us to assess E-cadherin expression (a major determinant of beta-catenin subcellular localization), which was increased by 73% and 71% in the azoxymethane-rat and HT-29 cells, respectively. We therefore investigated the effect of PEG treatment on levels of the negative regulator of E-cadherin, SNAIL, and observed a 50% and 75% decrease, respectively. In conclusion, we show, for the first time, a molecular mechanism through which PEG imparts its antiproliferative and hence profound chemopreventive effect.