Targets of Ephrin-A signaling in epidermal keratinocytes
ABSTRACT: Both ephrins and their receptors are membrane bound, restricting their interactions to the sites of direct cell-to-cell interfaces. They are widely expressed, often co-expressed and regulate developmental processes, cell adhesion, motility, survival, proliferation, and differentiation. Both tumor suppressor and oncogene activities are ascribed to EFNs and Ephs in various contexts. A major conundrum regarding the EFN/Eph system concerns their large number and functional redundancy, given the promiscuous cross-activation of ligands and receptors and the overlapping intracellular signaling pathways. To address this issue, we treated human epidermal keratinocytes with 5 EFNAs individually and defined the transcriptional responses in the cells. We found that a large set of genes is coregulated by all EFNAs. However, while the responses to EFNA3, EFNA 4 and EFNA 5 are identical, the responses to EFNA1 and EFNA2 are characteristic and distinctive. All EFNAs induce epidermal differentiation markers and suppress cell adhesion genes, especially integrins. Ontological analysis shows that all EFNAs induce cornification and keratin genes, while suppressing wound-healing associated, signaling, receptor and ECM associated genes. Transcriptional targets of AP1 are selectively suppressed by EFNAs. EFNA1 and EFNA2, but not the EFNA3, EFNA4, EFNA5 cluster, regulate the members of the ubiquitin-associated proteolysis genes. EFNA1 specifically induces collagen production. Our results demonstrate that the EFN-Eph interactions in the epidermis, while promiscuous, are not redundant but specific. This suggests that different members of the EFN/Eph system have specific, clearly demarcated functions. Human epidermal keratinocytes are treated with 25 ng/ml EFNA1; EFNA2; EFNA3; EFNA4 or EFNA5 (all as Fc conjugates) for 24h.
Project description:Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma). Human epidermal keratinocytes are treated with 25 ng/ml EphB2 or EFNA4 Fc conjugates in a 48hr time course.
Project description:Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. Reaching confluency did not affect adhesion and ECM proteins. Lipid metabolism and steroid metabolism were induced by confluency and by JNK inhibition, respectively. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis. Human epidermal keratinocytes are grown in standard growth medium either attached in tissue culture plates, or suspended, in the same medium, in bacteriological plates. After 48 hrs, attached and suspended cultures were harvested and their transcriptomes compared.
Project description:Epidermal keratinocytes respond to extracellular influences by activating cytoplasmic signal transduction pathways that change the transcriptional profiles of affected cells. To define responses to two such pathways, p38 and ERK, we used SB203580 and PD98059 as specific inhibitors, and identified the regulated genes after 1, 4, 24 and 48 hrs, using Affymetrix’ Hu133Av2 microarrays. Additionally, we compared genes specifically regulated by p38 and ERKs with those regulated by JNK and by all three pathways simultaneously. We find that the p38 pathway induces the expression of extracellular matrix and proliferation-associated genes, while suppressing microtubule-associated genes; the ERK pathway induces the expression of nuclear envelope and mRNA splicing proteins, while suppressing steroid synthesis and mitochondrial energy production enzymes. Both pathways promote epidermal differentiation and induce feedback inactivation of MAPK signaling. c-FOS, SRY and N-Myc appear to be the principal targets of the p38 pathway, Elk-1 SAP1 and HLH2 of ERK, while FREAC-4, ARNT and USF are common to both. The results for the first time comprehensively define the genes regulated by the p38 and ERK pathways in epidermal keratinocytes and suggest a list of targets potentially useful in therapeutic interventions. Human epidermal keratinocytes are grown in Keratinocyte Serum-Free Medium (Gibco) supplemented with 0.05 mg/ml bovine pituitary extract, 2.5 ng/ml epidermal growth factor, 0.09 mM CalCl2 and 1% penicillin/streptomycin (KGM). They are switched to Keratinocyte Serum Free-Media (Gibco) supplemented only with 1% penicillin/streptomycin (KBM) 24 h prior to commencing experiments. A set is left as controls, others treated with 5 uM JNK inhibitor SP600125, 15 uM p38 inhibitor SB203580, or 50 um ERK inhibitor PD98059. Timecourse of treated and parellel control samples over a 48 hr period was performed.
Project description:Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48 and 72 hours using large-scale microarray analyses. The majority of genes were found regulated only after 24 hours and remained regulated throughout the treatment. In addition to expected anti-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation and metabolism. GCs not only effectively block signaling by TNF-alpha and IL-1 but also by IFN-gamma, which was not previously known. Specifically, GCs suppress the expression of essentially all IFN-gamma-regulated genes, including IFN-gamma receptor and STAT-1. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs have anti-apoptotic effects in keratinocytes by inducing the expression of anti-apoptotic and repressing pro-apoptotic genes. Consequently, GCs treatment blocked UV-induced apoptosis of keratinocytes. GCs have a profound effect on wound healing by inhibiting cell motility and the expression of pro-angiogenic factor VEGF. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGF-beta-1 and -2, MMP1, 2, 9 and 10 and inducing TIMP-2. Finally, GCs promote terminal stages of epidermal differentiation while simultaneously inhibiting the early stages. These results provide new insights into the beneficial and adverse effects of GCs in epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies. Human epidermal keratinocytes are grown in delipidized, phenolphtalein-free medium and left as controls or treated with 0.1μM dexamethasone. Time course of treated and parallel control samples over a 72 hr period was performed twice with independent batches of cells.
Project description:In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.
Project description:Both ephrins and their receptors are membrane bound, restricting their interactions to the sites of direct cell-to-cell interfaces. They are widely expressed, often co-expressed and regulate developmental processes, cell adhesion, motility, survival, proliferation, and differentiation. Both tumor suppressor and oncogene activities are ascribed to EFNs and Ephs in various contexts. A major conundrum regarding the EFN/Eph system concerns their large number and functional redundancy, given the promiscuous cross-activation of ligands and receptors and the overlapping intracellular signaling pathways. To address this issue, we treated human epidermal keratinocytes with 5 EFNAs individually and defined the transcriptional responses in the cells. We found that a large set of genes is coregulated by all EFNAs. However, while the responses to EFNA3, EFNA 4 and EFNA 5 are identical, the responses to EFNA1 and EFNA2 are characteristic and distinctive. All EFNAs induce epidermal differentiation markers and suppress cell adhesion genes, especially integrins. Ontological analysis shows that all EFNAs induce cornification and keratin genes, while suppressing wound-healing associated, signaling, receptor and ECM associated genes. Transcriptional targets of AP1 are selectively suppressed by EFNAs. EFNA1 and EFNA2, but not the EFNA3, EFNA4, EFNA5 cluster, regulate the members of the ubiquitin-associated proteolysis genes. EFNA1 specifically induces collagen production. Our results demonstrate that the EFN-Eph interactions in the epidermis, while promiscuous, are not redundant but specific. This suggests that different members of the EFN/Eph system have specific, clearly demarcated functions. Overall design: Human epidermal keratinocytes are treated with 25 ng/ml EFNA1; EFNA2; EFNA3; EFNA4 or EFNA5 (all as Fc conjugates) for 24h.
Project description:The role of Eph/ephrin signaling in numerous biological processes has been established. However, Eph/ephrin signaling has been shown to have complex role in tumor progression. The role of EphB2 receptor in the progression of cutaneous squamous cell carcinoma (cSCC) has not been studied before. EphB2 is significantly overexpressed in cSCC cells compared with normal human epidermal keratinocytes (NHEKs). We used microarray based global gene expression profiling to study the effect of EphB2 knockdown on the expression of different genes in cSCC cells. cSCC cell lines (n=3) were transfected with either control or EphB2 siRNA (75 nM) and incubated for 72 hours. Total RNA was extracted and processed expression profiling with GeneChip 3’ IVT Express Kit according to manufacturer’s protocol.
Project description:The epidermal-specific ablation of Rb gene leads to increased proliferation, aberrant differentiation, and the disengagement of these processes in vivo and in vitro. These differences in phenotype are more severe with the loss of p107, demonstrating the functional compensation between pRb and p107. As p107 and p130 also exert overlapping functions in epidermis, we have generated Rb(F19/F19)K14cre;Rbl2-/- (pRb-;p130-) mice to analyze possible functional redundancies between pRb and p130. The epidermal phenotype was very similar between pRb- and pRb-;p130- mice, suggesting that pRb and p130 activities are not redundant in epidermis. Importantly, we can correlate the proliferation differences with specific changes in gene expression between pRb-, pRb-;p107- and pRb-;p130- primary keratinocytes using microarray analysis, and explain the phenotypes in the context of altered E2F expression and functionality. Our findings support a model in which the distinct retinoblastoma family members, in conjunction with E2F members, play a central role in regulating epidermal homeostasis through specific or overlapping activities. Experiment Overall Design: Total RNA from pooled cells from each genotype were analyzed, per duplicate (pRb-/-;p107-/-, or pRb-/-;p130-/-) or triplicate (wild type, or pRb-/-), in mouse microarrays. Comparison was performed between the 4 different genotypes.
Project description:Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Heterozygous mutations of TP63 encoding p63 cause a spectrum of developmental disorders. EEC syndrome is caused by point mutations in the p63 DNA-binding domain, and manifests ectodermal dysplasia with defects in the epidermis and epidermal related appendages, limb malformation and cleft lip/palate. Five hotspot mutations affecting amino acids, R204, R227, R279, R280 and R304, have been found in approximately 90% of the EEC population. Although the role of p63 in normal epidermal development and differentiation has been demonstrated, the molecular mechanism by which p63 mutations cause the epidermal phenotype in diseases is not yet understood. We previously reported that EEC patient keratinocytes cannot fully differentiate towards terminal stratification in both 2D and 3D cellular models. In this study, EEC patient keratinocytes carrying three hotspot... (for more see dbGaP study page.)
Project description:Betaine (trimethylglycine) is a non-toxic, highly water-soluble organic osmolyte widely used in skin care due to its assumed moisturizing and protective properties, but only few studies have addressed its specific effects in skin. Here, the cellular and molecular targets of betaine were analyzed by genome-wide expression analysis in organotypic cultures of rat epidermal keratinocytes (REK). In this model, we also examined whether betaine modifies the impacts of acute UVB exposure. The expression of several genes relevant to epidermal biology, proliferation/differentiation or malignancy as well as solute transport were verified by independent methods (qRT-PCR, western blotting). The data concerning changes in calcium metabolism after UVB exposure has been published separately (Bart et al., Br. J. Dermatol. 171:376-387, 2014). Organotypic cultures of rat epidermal keratinocytes (REK) were prepared by plating cells on a collagen-coated insert (submerged in medium), lifting the confluent cell layer to the air-liquid interface 3 days after plating. These 3D cultures were divided in four treatment groups with three replicates in each: control, betaine (10 mM), UVB (30 mJ/cm2) and betaine + UVB. Betaine was added to the cultures for 11 days, starting from day 4 until sample collection. UVB exposure was performed 24 h prior to sample collection for 2-week-old 3D cultures with a well-formed epidermal layer. For further details on the experimental set-up and characteristics of the UV source, please refer to Bart et al. (Br. J. Dermatol. 171:376-387, 2014) and Rauhala et al. (J. Biol. Chem. 288:17999-18012, 2013).