Biology subjects

Green, K. J.

Publications and source records attributed to Green, K. J..

17 recordsLinked to original sources

bioRxiv2026

Evolution of immune infiltration and immune triad formation across early melanoma progression

Tumor-infiltrating lymphocytes (TIL) in melanoma are a clinically important prognostic marker and are routinely used to inform patient care. However, when immune recognition of transformed melanocytes begins and how it evolves during early melanoma progression is not well understood. Using spatial transcriptomics and a unique cohort of patient-matched lesions representing multiple stages of early evolution, we investigate T cells, dendritic cells (DCs) and multi-cell immune "triads" during melanoma initiation. We find that T cell and DC density, triad density, and triad proximity to melanoma cells are elevated in regions of Stage I invasive melanoma as compared to Stage 0 in situ lesions and nevi. Further, we find that triad abundance correlates with both T cell and DC density, but is not predicted by clustering patterns of these cell types over larger spatial scales. We conclude that immune recognition of melanoma and engagement with the lesion significantly increases with progression from Stage 0 to Stage I disease. Further, using nevi that are depth-matched in the dermis to Stage I invasive lesions, we demonstrate this increased immune recognition is not a consequence of dermal invasion alone. This points to a paradigm where immune engagement of invasive melanoma reflects recognition of melanoma cell-intrinsic changes that emerge in Stage I disease.

cancer biology↗

bioRxiv2026

Desmoglein-1 coordinates cell proliferation and actin dynamics to promote epidermal morphogenesis.

Skin development depends on coordinated biochemical and mechanical signaling to form a multilayered epidermal barrier. Our previous work demonstrated that the stratified tissue-specific desmosomal cadherin Desmoglein-1 (Dsg1) promotes epidermal differentiation in human in vitro models. Dsg1 first appears in the developing mouse epidermis around E14, coinciding with a developmental transition in stratification and the onset of terminal differentiation, raising the question of how Dsg1 contributes to epidermal morphogenesis in vivo. We generated a wild-type (WT) single-cell RNA sequencing (scRNA-seq) atlas of murine skin and integrated scRNA-seq data from Dsg1 knockout (Dsg1 KO) embryos spanning E14.5, E15.5, E16.5, and E18.5. Six keratinocyte subpopulations were annotated, including a Transitional subpopulation co-expressing basal and suprabasal markers, positioned between basal and differentiated states. Dsg1 KO epidermis exhibited reduced thickness at E14.5-E15.5, but recovered by E16.5-E18.5, suggesting compensatory adaptation. Differential gene expression analysis revealed enrichment of cell-cycle and actin-associated programs in Dsg1 KO during late development. EdU incorporation and PCNA staining confirmed increased proliferation in superficial layers accompanied by altered actin organization and changed cell morphology. Notably, actin and the Arp2/3 actin nucleation-promoting factor N-WASP exhibited marked changes in distribution during early and late development, respectively. Mechanistically, Dsg1 associates with Nck1, an adaptor linking N-WASP to Arp2/3-dependent actin polymerization. Silencing Nck1 or N-WASP reduced Dsg1-mediated stratification significantly, while a Nck1-binding-deficient Dsg1 mutant failed to rescue stratification in Dsg1-deficient keratinocytes in vitro. Together, these findings identify a Dsg1-Nck1-N-WASP axis linking desmosomal adhesion to cytoskeletal remodeling and support a model in which Dsg1 coordinates cell-cycle, cell morphology, and actin dynamics to drive epidermal morphogenesis.

cell biology↗

bioRxiv2025

Molecular prosthetics for CFTR designed for anion selectivity outperform amphotericin B in cultured cystic fibrosis airway epithelia

The ion channel-forming natural product amphotericin B (AmB) can serve as a molecular prosthetic for the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and thereby restore host defenses in cultured cystic fibrosis (CF) airway epithelia. This is despite the fact that the permeability of AmB-based channels favors cations, and these channels lose their capacity to increase airway surface liquid (ASL) pH in CF airway epithelia at high concentrations. We hypothesize that modifying such channels to favor anion permeability would make them more CFTR-like and thus increase their potential therapeutic effects compared to AmB. Here we show that a synthetic derivative of AmB, AmB-AA, which has an added positively charged appendage and forms ion channels with an improved relative permeability to anions, outperformed AmB in increasing the ASL pH in CF airway epithelia at both low and high concentrations. Further modifications led to another AmB derivative, C2epiAmB-AA, that also minimized cholesterol binding and thus toxicity to cultured CF airway epithelia and was an effective surrogate for CFTR in primary cultured airway epithelia from people with CF.

pharmacology and toxicology↗

bioRxiv2025

Association of RhoGEF Ect2 with Desmoplakin Supports RhoA Activity at Intercellular Junctions: Implications for Carvajal Disease

Desmoplakin (DP) is an essential component of the desmosomal adhesion complex, tethering intermediate filaments to sites of intercellular adhesion to confer mechanical integrity to tissues. As a frequent target for mutation in cardiocutaneous syndromes that vary widely in phenotype, DPs roles as a signaling hub are rapidly emerging. Here, we identify the RhoGEF Ect2 as a previously unappreciated binding partner of the desmosomal protein DP. DP is required for the localization of Ect2 to keratinocyte desmosomes and cardiac intercalated discs in vitro and in vivo, where it maintains active RhoA (Rho-GTP) at the membrane. We demonstrate further that Ect2 activity is supported by PKC in a DP-dependent manner in cardiac myocytes. Finally, a truncated form of DP expressed in patients with Carvajal syndrome associated with severe cardiocutaneous defects is impaired in its ability to bind and localize Ect2 to cell junctions in cardiomyocytes and keratinocytes isolated from patients. Our findings delineate an important relationship between a component of the desmosome and a critical regulator of actin cytoskeletal remodeling that could have widespread implications for understanding cardiac and cutaneous health and disease pathogenesis.

cell biology↗

bioRxiv2025

Failed Metabolic Adaptation to Stress Underlies Pathogenesis in a Heterozygous Genetic Disorder

Disorders linked to heterozygous variants occupy a continuum in terms of the timing and severity of phenotypic emergence. An important question regarding this variability entails the effect stress has on the residual protein function. Using Darier disease (DD), caused by heterozygous variants of the SERCA2 calcium pump, as a model, we uncovered a potential connection between extrinsic stress and pathogenesis. The skin lesions characteristic of DD entail loss of intercellular adhesion and rarely appear pre-adolescence, suggesting that factors beyond heterozygosity contribute to disease pathogenesis. Testing whether age-related stressors contribute to DD, we show that DD patient-derived keratinocytes subjected to stress yield twice the reactive oxygen species of controls, accompanied by greater disruption of intercellular adhesion. Metabolic analysis of DD cells revealed perturbation of the pentose phosphate pathway (PPP), a stress response system responsible for regenerating antioxidants like glutathione. At baseline, DD cells had less free glutathione but an increase in protective glutathione-based modifications of SERCA2, a reversible form of protein oxidation. With stress, DD cells form an aberrant, heavily glutathionylated perinuclear halo consisting of keratin and the intercellular adhesion component, desmoplakin. We propose a model whereby SERCA2 heterozygosity causes mild oxidative stress that under homeostatic conditions can be buffered by glutathionylation. When stressed, the depleted glutathione store is shunted towards the desmoplakin-intermediate filament system at the expense of SERCA2, rendering it vulnerable to damage. A lesional flare, then, would represent a case of more complete SERCA2 inhibition and a novel example of how heterozygous disorders interact with stress to disrupt intercellular adhesion.

cell biology↗

bioRxiv2023

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis

Epithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP) 1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ) 3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening 4-10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself 11,12, or proximate to the junctions 13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ 12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

cell biology↗

bioRxiv2023

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 {beta}-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.

cell biology↗

bioRxiv2022

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that govern their size and functions remains unclear. We show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation and cell cycle re-entry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actin contractility, decreases the stiffness of the bulge and HG, and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

genetics↗

bioRxiv2022

Transcriptional profiling of rare acantholytic disorders suggests common mechanisms of pathogenesis

BackgroundDarier, Hailey-Hailey, and Grovers diseases are rare non-autoimmune acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. ObjectiveTo better understand the underlying mechanisms leading to disease in these conditions we performed RNA-seq on lesional skin samples from Darier, Hailey-Hailey, and Grovers disease patients. MethodsRNA-seq and bioinformatics analyses were performed on banked paraffin embedded diagnostic samples from each disease. For detailed Methods, please see the Methods section in this articles Online Repository at www.jacionline.org. ResultsThe transcriptomic profiles of Darier, Hailey-Hailey, and Grovers disease were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases, psoriasis and atopic dermatitis. Analysis of enriched pathways showed a shared upregulation in keratinocyte differentiation and Th17 inflammatory pathways, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grovers disease. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in Darier, Hailey-Hailey, and Grovers disease. Further, upstream regulator analysis suggests that a decrease in SRF/MRTF activity may be 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 to normal skin. ConclusionThese findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grovers disease, and identify decreases in actin organization pathways as a unique signature present in these conditions. Key MessagesO_LIDarier Disease, Hailey-Hailey Disease, and Grovers Disease share similar transcriptional profiles suggesting common mechanisms of pathogenesis. C_LIO_LISRF/MRTFA activity is reduced in Darier Disease, Hailey-Hailey Disease and Grovers disease, implicating actin organization in acantholysis. C_LI

genomics↗

bioRxiv2022

Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche

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 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 Snai2 (Slug), and consequently decreases the Dsg1 transcriptional activator Grhl1 (Grainyhead like 1), a known promoter of epidermal differentiation and barrier function. 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.

cancer biology↗

bioRxiv2022

PP2A-B55α 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)-B55 holoenzyme as the major serine/threonine phosphatase regulating DPs C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55 holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55 regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55 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.

cell biology↗

bioRxiv2021

Epidermal Stratification Requires Retromer-Mediated Desmoglein-1 Recycling

Sorting and trafficking transmembrane cargo is essential for tissue development and homeostasis. However, the importance of intracellular trafficking in the development and regeneration of stratified epidermis has not been investigated. Here we identify the interaction between VPS35, an essential component of the retromer endosomal trafficking complex, and the desmosomal cadherin, Desmoglein 1 (Dsg1). Dsg1 is specifically expressed in stratified tissues and when properly localized on the plasma membrane, promotes epidermal stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support keratinocyte stratification and differentiation. The retromer enhancing chaperone, R55 promotes the plasma membrane localization of Dsg1 and a Dsg1 mutant associated with Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome, enhancing the ability of SAM-Dsg1 to promote stratification. Our work provides the first evidence for retromer function in epidermal regeneration and identifies it as a potential therapeutic skin disease target.

cell biology↗

bioRxiv2021

Subtype transcriptomic profiling of myeloid cells in Alzheimer Disease brain illustrates the diversity in active microglia phenotypes

Microglia contribute to Alzheimers Disease (AD) progression and are candidate therapeutic targets. Human microglia exhibit an array of transcriptional phenotypes implying that accurate manipulation of microglial function will require clarity of their molecular states and context dependent regulation. To increase the number of microglia analyzed per subject we employed fluorescence activated nuclei sorting prior to single-nucleus RNA-seq on human prefrontal cortices. We observed microglia phenotypes previously unrecognized in human brain gene expression studies and mapped their transcriptomic relationships by trajectory inference. Three clusters were enriched for endolysosomal pathways, one of which showed differential expression of AD GWAS genes in addition to genes implicated in nucleic acid detection and interferon signaling. Analysis of the "homeostatic" microglia cluster revealed a uniquely AD subcluster. Our study demonstrates the value of deeply profiling microglia to explore the biological implications of microglia transcriptomic diversity.

neuroscience↗

bioRxiv2020

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. Dsg1s importance in epidermal integrity is underscored by genetic, autoimmune and bacterial toxin-mediated disorders interfering with Dsg1 function. Dsg1 loss-of-function mutations in humans result not only in skin lesions, but also multiple allergies, and isolated patient keratinocytes exhibit increased pro-allergic 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 three tandem Dsg1 genes in mice using CRISPR/Cas9. Whole transcriptome analysis of E18.5 Dsg1-/- skin showed changes consistent with the observed aberrant differentiation and barrier impairment. Comparing epidermal transcriptomes from E18.5 Dsg1-deficient mice and humans with Dsg1 mutations revealed a shared psoriatic-like IL-17-skewed inflammatory signature and less so a pro-allergic IL-4/13 signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from autoimmune anti-Dsg1 pemphigus foliaceus antibodies, transcriptomic analysis of pemphigus skin lesions lacks a prominent IL-17 signature. Thus, beyond impairing the physical barrier, chronic loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, possibly predisposing to skin inflammation.

immunology↗

bioRxiv2020

Early life nociception is influenced by peripheral growth hormone signaling

A number of cellular systems work in concert to modulate nociceptive processing in the periphery, but the mechanisms that regulate neonatal nociception may be distinct compared to adults. Our previous work indicated a relationship between neonatal hypersensitivity and growth hormone (GH) signaling. Here, we explored the peripheral mechanisms by which GH modulated neonatal nociception under normal and injury conditions (incision). We found that GH receptor signaling in primary afferents maintains a tonic inhibition of peripheral hypersensitivity. After injury, a macrophage dependent displacement of injury-site GH was found to modulate neuronal transcription at least in part via serum response factor regulation. A single GH injection into the injured hindpaw muscle effectively restored available GH signaling to neurons and prevented acute pain-like behaviors, primary afferent sensitization, and neuronal gene expression changes. GH treatment also inhibited long-term somatosensory changes observed after repeated peripheral insult. Results may indicate a novel mechanism of neonatal nociception. Significance statementAlthough it is noted that mechanisms of pain development in early life are unique compared to adults, little research focuses on neonatal-specific peripheral mechanisms of nociception. This gap is evident in the lack of specialized care for infants following an injury including surgeries. This report evaluates how distinct cellular systems in the periphery including the endocrine, immune and nervous systems work together to modulate neonatal-specific nociception. We uncovered a novel mechanism by which muscle injury induces a macrophage-dependent sequestration of peripheral growth hormone that effectively removes its normal tonic inhibition of neonatal nociceptors to promote acute pain-like behaviors. Results indicate a possible new strategy for treatment of neonatal post-surgical pain.

neuroscience↗

bioRxiv2020

Desmosomes pattern cell mechanics 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 classical 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 (IF) 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.

cell biology↗

bioRxiv2018

Keratinocyte desmoglein 1 regulates the epidermal microenvironment and tanning response

Coordinated responses to environmental stimuli within the keratinocyte:melanocyte niche are poorly understood. Desmoglein 1 (Dsg1), a keratinocyte-specific desmosomal cell-cell adhesion protein with emerging signaling roles, is reduced by ultraviolet light radiation. Loss-of-function Dsg1 mutations elevate keratinocyte cytokines in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. We asked whether Dsg1 regulates keratinocyte:melanocyte paracrine communication to induce the tanning response. Dsg1-silenced keratinocytes increased Pro-opiomelanocortin mRNA and cytokine secretion. Melanocytes treated with conditioned media from Dsg1-silenced keratinocytes exhibited increased Mitf and Trp1 mRNA, melanin secretion, and dendrite length. Inhibiting the melanocyte pigment-associated melanocortin 1 receptor reduced pigment secretion in response to Dsg1-deficient conditioned media. Melanocytes incorporated into Dsg1-deficient human skin equivalents relocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Dsg1 decreased in keratinocytes surrounding dysplastic nevi and early melanoma, but not benign nevi. We posit Dsg1 controls keratinocyte:melanocyte communication through paracrine signaling, which goes awry upon Dsg1 loss in melanoma development.

cell biology↗