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.

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.

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.

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.

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.

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.

Eosinophil and airway nerve interactions in asthma.

Airway eosinophils are increased in asthma and are especially abundant around airway nerves. Nerves control bronchoconstiction and in asthma, airway hyperreactivity (where airways contract excessively to inhaled stimuli) develops when eosinophils alter both parasympathetic and sensory nerve function. Eosinophils release major basic protein, which is an antagonist of inhibitory M2 muscarinic receptors on parasympathetic nerves. Loss of M2 receptor inhibition potentiates parasympathetic nerve-mediated bronchoconstriction. Eosinophils also increase sensory nerve responsiveness by lowering neurons' activation threshold, stimulating nerve growth, and altering neuropeptide expression. Since sensory nerves activate parasympathetic nerves via a central neuronal reflex, eosinophils' effects on both sensory and parasympathetic nerves potentiate bronchoconstriction. This review explores recent insights into mechanisms and effects of eosinophil and airway nerve interactions in asthma.

Maternal high-fat diet and obesity compromise fetal hematopoiesis.

OBJECTIVE: Recent evidence indicates that the adult hematopoietic system is susceptible to diet-induced lineage skewing. It is not known whether the developing hematopoietic system is subject to metabolic programming via in utero high-fat diet (HFD) exposure, an established mechanism of adult disease in several organ systems. We previously reported substantial losses in offspring liver size with prenatal HFD. As the liver is the main hematopoietic organ in the fetus, we asked whether the developmental expansion of the hematopoietic stem and progenitor cell (HSPC) pool is compromised by prenatal HFD and/or maternal obesity. METHODS: We used quantitative assays, progenitor colony formation, flow cytometry, transplantation, and gene expression assays with a series of dietary manipulations to test the effects of gestational high-fat diet and maternal obesity on the day 14.5 fetal liver hematopoietic system. RESULTS: Maternal obesity, particularly when paired with gestational HFD, restricts physiological expansion of fetal HSPCs while promoting the opposing cell fate of differentiation. Importantly, these effects are only partially ameliorated by gestational dietary adjustments for obese dams. Competitive transplantation reveals compromised repopulation and myeloid-biased differentiation of HFD-programmed HSPCs to be a niche-dependent defect, apparent in HFD-conditioned male recipients. Fetal HSPC deficiencies coincide with perturbations in genes regulating metabolism, immune and inflammatory processes, and stress response, along with downregulation of genes critical for hematopoietic stem cell self-renewal and activation of pathways regulating cell migration. CONCLUSIONS: Our data reveal a previously unrecognized susceptibility to nutritional and metabolic developmental programming in the fetal HSPC compartment, which is a partially reversible and microenvironment-dependent defect perturbing stem and progenitor cell expansion and hematopoietic lineage commitment.

Interactions of eosinophils with nerves.

Coculture of eosinophils and nerves is a powerful tool in determining the interactions between the two cell types. We have developed methods for culture of parasympathetic ganglia and dorsal root ganglia from humans, and we have further refined the technique to coculture with eosinophils. Here we describe methods for coculturing primary parasympathetic ganglia or dorsal root ganglia with eosinophils.

Hypothalamic signaling in anorexia induced by indispensable amino acid deficiency.

Animals exhibit a rapid and sustained anorexia when fed a diet that is deficient in a single indispensable amino acid (IAA). The chemosensor for IAA deficiency resides within the anterior piriform cortex (APC). Although the cellular and molecular mechanisms by which the APC detects IAA deficiency are well established, the efferent neural pathways that reduce feeding in response to an IAA-deficient diet remain to be fully characterized. In the present work, we investigated whether 1) central melanocortin signaling is involved in IAA deficiency-induced anorexia (IAADA) and 2) IAADA engages other key appetite-regulating neuronal populations in the hypothalamus. Rats and mice that consumed a valine-deficient diet (VDD) for 2-3 wk exhibited marked reductions in food intake, body weight, fat and lean body mass, body temperature, and white adipose tissue leptin gene expression, as well as a paradoxical increase in brown adipose tissue uncoupling protein-1 mRNA. Animals consuming the VDD had altered hypothalamic gene expression, typical of starvation. Pharmacological and genetic blockade of central melanocortin signaling failed to increase long-term food intake in this model. Chronic IAA deficiency was associated with a marked upregulation of corticotropin-releasing hormone expression in the lateral hypothalamus, particularly in the parasubthalamic nucleus, an area heavily innervated by efferent projections from the APC. Our observations indicate that the hypothalamic melanocortin system plays a minor role in acute, but not chronic, IAADA and suggest that the restraint on feeding is analogous to that observed after chronic dehydration.