RNA Interference Approaches to Study Epidermal Cell Adhesion.

In this chapter, we provide a method for silencing target genes in epidermal cells via RNA interference. Specifically, we describe a protocol for transfection-mediated delivery of small interfering RNA oligonucleotides (siRNA). Functional assays are indispensable to characterize the biological consequences of gene knockdowns, and we also provide a method to analyze alterations in cell adhesion properties, consequent to knockdown of genes involved in this process.

Pannexin 1 crosstalk with the Hippo pathway in malignant melanoma.

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, Yes-associated protein 1 [YAP], Transcriptional coactivator with PDZ-binding motif [TAZ], and Hippo scaffold, Ras GTPase-activating-like protein IQGAP1 [IQGAP1], in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels.

Pannexin 1 and pannexin 3 differentially regulate the cancer cell properties of cutaneous squamous cell carcinoma.

Pannexin (PANX) channels are present in skin and facilitate the movement of signalling molecules during cellular communication. PANX1 and PANX3 function in skin homeostasis and keratinocyte differentiation but were previously reduced in a small cohort of human cutaneous squamous cell carcinoma (cSCC) tumours compared to normal epidermis. In our study we used SCC-13 cells, limited publicly available RNA-seq data and a larger cohort of cSCC patient-matched samples to analyse PANX1 and PANX3 expression and determine the association between their dysregulation and the malignant properties of cSCC.

Pannexin 1 crosstalk with the Hippo pathway in malignant melanoma.

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, YAP, TAZ, and Hippo scaffold, IQGAP1, in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels.

Analysis of Proliferation, Apoptosis, and Motility in Mouse Embryonic Melanocytic Precursor Cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

Global pannexin 1 deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8 T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1) mice with the inducible melanoma model Braf, Pten, Tyr::CreER (BPC).

Isolation, Purification, and Culture of Embryonic Melanoblasts from Green Fluorescent Protein-expressing Reporter Mice.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence.

Ubiquitylated histone H2A: a molecular Jekyll and Hyde in the epidermis.

The epidermis of the skin provides a barrier between the organism and the external environment. It is constantly subjected to physical and chemical insults, and thus susceptible to wounding and to neoplastic transformation. Long-lasting epigenetic modifications in epidermal stem cells are now shown to link responses to skin injuries with cell priming for carcinoma development, through regulation of histone H2A ubiquitylation.

PANX3 Channels Regulate Architecture, Adhesion, Barrier Function, and Inflammation in the Skin.

The channel-forming glycoprotein PANX3 functions in cutaneous wound healing and keratinocyte differentiation, but its role in maintaining skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3-knockout (KO) mice and found that KO dorsal skin showed sex differences at different ages but generally had reduced dermal and hypodermal areas compared with age-matched controls.

Transforming growth factor-β in tumour development.

Transforming growth factor-β (TGFβ) is a ubiquitous cytokine essential for embryonic development and postnatal tissue homeostasis. TGFβ signalling regulates several biological processes including cell growth, proliferation, apoptosis, immune function, and tissue repair following injury. Aberrant TGFβ signalling has been implicated in tumour progression and metastasis.

Pannexin 1 binds β-catenin to modulate melanoma cell growth and metabolism.

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear.

DRM02, a novel phosphodiesterase-4 inhibitor with cutaneous anti-inflammatory activity.

Chronic inflammatory skin disorders are frequently associated with impaired skin barrier function. Selective phosphodiesterase-4 (PDE4) inhibition constitutes an effective therapeutic strategy for the treatment of inflammatory skin diseases. We now report the pharmacological anti-inflammatory profile of DRM02, a novel pyrazolylbenzothiazole derivative with selective inhibitory activity toward PDE4 isoforms A, B and D.

Integrin-linked kinase regulates melanosome trafficking and melanin transfer in melanocytes.

Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood.

Essential Role for Integrin-Linked Kinase in Melanoblast Colonization of the Skin.

Melanocytes are pigment-producing cells found in the skin and other tissues. Alterations in the melanocyte lineage give rise to a plethora of human diseases, from neurocristopathies and pigmentation disorders to melanoma. During embryogenesis, neural crest cell subsets give rise to two waves of melanoblasts, which migrate dorsolaterally, hone to the skin, and differentiate into melanocytes.

Targeting FER Kinase Inhibits Melanoma Growth and Metastasis.

Melanoma is one of the most aggressive types of tumors and exhibits high metastatic potential. Fes-related (FER) kinase is a non-receptor tyrosine kinase that has been implicated in growth and metastasis of various epithelial tumors. In this study, we have examined the role that FER kinase plays in melanoma at the molecular level.

NUMB regulates the endocytosis and activity of the anaplastic lymphoma kinase in an isoform-specific manner.

NUMB is an evolutionarily conserved protein that plays an important role in cell adhesion, migration, polarity, and cell fate determination. It has also been shown to play a role in the pathogenesis of certain cancers, although it remains controversial whether NUMB functions as an oncoprotein or tumor suppressor. Here, we show that NUMB binds to anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase aberrantly activated in several forms of cancer, and this interaction regulates the endocytosis and activity of ALK.

Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26 reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity.

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells.

Scaffolding proteins in the development and maintenance of the epidermal permeability barrier.

The skin of mammals and other terrestrial vertebrates protects the organism against the external environment, preventing heat, water and electrolyte loss, as well as entry of chemicals and pathogens. Impairments in the epidermal permeability barrier function are associated with the genesis and/or progression of a variety of pathological conditions, including genetic inflammatory diseases, microbial and viral infections, and photodamage induced by UV radiation. In mammals, the outside-in epidermal permeability barrier is provided by the joint action of the outermost cornified layer, together with assembled tight junctions in granular keratinocytes found in the layers underneath.

A reporter mouse model for tracing and molecular studies of melanocytic lineage cells and their diseases.

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas.

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases.

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions.

E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair.

Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1.