The ?-opioid receptor affects epidermal homeostasis via ERK-dependent inhibition of transcription factor POU2F3.
ABSTRACT: Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that ?-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.
Project description:POU domain class 2 transcription factor 3 (POU2F3) plays an important role in keratinocyte proliferation and differentiation. Our previous study identified four sheep POU2F3 transcript variants (POU2F3-1, POU2F3-2, POU2F3-3, and POU2F3-4), encoding three POU2F3 protein isoforms (POU2F3-1, POU2F3-2, and POU2F3-3). However, the functional differences among the three POU2F3 isoforms remain unknown. The objective of this study was to determine the tissue expression pattern of the four POU2F3 transcript variants in sheep and to investigate the functional differences in cell proliferation among the three POU2F3 isoforms. Quantitative RT-PCR analysis showed that the four POU2F3 transcripts were ubiquitously expressed in all tested adult sheep tissues, and POU2F3-1 exhibited higher expression level than the other three POU2F3 transcript variants in skin (P?<?0.05). Cell proliferation assay showed that overexpression of any one of the three POU2F3 isoforms significantly inhibited the proliferation of sheep fetal fibroblasts and HaCaT cells at 48 and 72 h after transfection (P?<?0.05). POU2F3-3 had less inhibitory effect on cell proliferation than POU2F3-1 and POU2F3-2 (P?<?0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P?>?0.05). Dual luciferase reporter assays demonstrated that overexpression of any one of the three POU2F3 isoforms significantly inhibited the promoter activities of keratin 14 (KRT14) and matrix metalloproteinase 19 (MMP19) genes (P?<?0.05). POU2F3-3 had less inhibitory effect on the promoter activities of KRT14 and MMP19 genes than POU2F3-1 and POU2F3-2 (P?<?0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P?>?0.05). These results suggest three sheep POU2F3 isoforms have similar functional effects, but to a different extent.
Project description:The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.
Project description:INTRODUCTION:Epidermal keratinocytes are increasingly recognized as active participants in the sensory transduction of itch and pain, processes known to involve primary afferent glutamatergic neurons. However the role of keratinocyte glutamate signaling in sensory functioning is not fully understood. Here, we present the observation of ?-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid type glutamate receptors (AMPAR) in epidermal keratinocytes. METHODS:Immunohistochemical and in situ hybridization analyses were conducted to assess the expression of AMPAR subunits in epidermal keratinocytes in mouse and human skin samples, and in organotypic cultures of human keratinocytes. In addition, RTPCR further confirmed the expression of GluA4-containing AMPAR in epidermal keratinocytes. RESULTS:We found prominent immunolabeling (IL) for the GluA4 subunit of AMPAR in keratinocytes of glabrous and hairy skin of mouse epidermis, as well as in human epidermal keratinocytes. RTPCR confirmed Gria4 transcript expression in epidermal mouse keratinocytes. In addition, expression of GRIA4 mRNA was confirmed in epidermal human keratinocytes by in situ hybridization. Immunohistochemical studies conducted in human skin biopsies from patients with atopic dermatitis (AD) and postherpetic neuralgia (PHN) demonstrate that keratinocyte expression of GluA4 can be altered under pathological conditions. Moreover, a decrease of GluA4 expression was observed in organotypic cultures of human keratinocytes after direct application of algogenic agents. CONCLUSIONS:We provide evidence that GluA4-containing AMPAR are expressed in epidermal keratinocytes, that human pruritic and painful dermatopathologies have alterations in the keratinocyte expression levels of GluA4-containing AMPAR, and that itch and pain producing substances can directly regulate their production in keratinocytes.
Project description:In addition to its analgesic functions, the peripheral opioid receptor system affects skin homeostasis by influencing cell differentiation, migration and adhesion; also, wound healing is altered in ?-opioid receptor knockout mice (DOPr(-/-) ). Hence, we investigated ?-opioid receptor effects on the expression of several proteins of the desmosomal junction complex and on the migratory behaviour of keratinocytes.Expression levels of desmosomal cadherins in wild-type and DOPr(-/-) mice, and the morphology of intercellular adhesion in human keratinocytes were analysed by immunofluorescence. To investigate the ?-opioid receptor activation pathway, protein expression was studied using Western blot and its effect on cellular migration determined by in vitro live cell migration recordings from human keratinocytes.Expression of the desmosomal cadherins, desmogleins 1 and 4, was up-regulated in skin from DOPr(-/-) mice, and down-regulated in ?-opioid receptor-overexpressing human keratinocytes. The localization of desmoplakin expression was rearranged from linear arrays emanating from cell borders to puncta in cell periphery, resulting in less stable intercellular adhesion. Migration and wound recovery were enhanced in human keratinocyte monolayers overexpressing ?-opioid receptors in vitro. These ?-opioid receptor effects were antagonized by specific PKC?/? inhibition indicating they were mediated through the PKC signalling pathway. Finally, cells overexpressing ?-opioid receptors developed characteristically long but undirected protrusions containing filamentous actin and ?-opioid receptors, indicating an enhanced migratory phenotype.Opioid receptors affect intercellular adhesion and wound healing mechanisms, underlining the importance of a cutaneous neuroendocrine system in wound healing and skin homeostasis.This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
Project description:Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant.
Project description:Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.
Project description:The nuclear hormone receptor PPAR?/? is integral to efficient wound re-epithelialization and implicated in epidermal maturation. However, the mechanism underlying the latter process of epidermal differentiation remains unclear. We showed that ligand-activated PPAR?/? indirectly stimulated keratinocyte differentiation, requiring de novo gene transcription and protein translation. Using organotypic skin cultures constructed from PPAR?/?- and angiopoietin-like 4 (ANGPTL4)-knockdown human keratinocytes, we showed that the expression of ANGPTL4, a PPAR?/? target gene, is essential for the receptor mediated epidermal differentiation. The pro-differentiation effect of PPAR?/? agonist GW501516 was also abolished when keratinocytes were co-treated with PPAR?/? antagonist GSK0660 and similarly in organotypic skin culture incubated with blocking ANGPTL4 monoclonal antibody targeted against the C-terminal fibrinogen-like domain. Our focused real-time PCR gene expression analysis comparing the skin biopsies from wildtype and ANGPTL4-knockout mice confirmed a consistent down-regulation of numerous genes involved in epidermal differentiation and proliferation in the ANGPTL4-knockout skin. We further showed that the deficiency of ANGPTL4 in human keratinocytes and mice skin have diminished expression of various protein kinase C isotypes and phosphorylated transcriptional factor activator protein-1, which are well-established for their roles in keratinocyte differentiation. Chromatin immunoprecipitation confirmed that ANGPTL4 stimulated the activation and binding of JUNB and c-JUN to the promoter region of human involucrin and transglutaminase type 1 genes, respectively. Taken together, we showed that PPAR?/? regulates epidermal maturation via ANGPTL4-mediated signalling pathway.
Project description:Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.
Project description:Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin. Lysosomal activity was required for mechanistic target of rapamycin signaling and mitochondrial oxidative metabolism. In turn, mitochondrial reactive oxygen species, produced as a natural byproduct of oxidative phosphorylation, were necessary for keratinocyte differentiation. Finally, treatment with exogenous reactive oxygen species rescued the differentiation defect in lysosome-inhibited keratinocytes. These findings highlight a reciprocal relationship between lysosomes and mitochondria, in which lysosomes support mitochondrial metabolism and the associated production of mitochondrial reactive oxygen species. The mitochondrial reactive oxygen species released to the cytoplasm in suprabasal keratinocytes triggers autophagy and lysosome-mediated degradation necessary for epidermal differentiation. As defective lysosome-dependent autophagy is associated with common skin diseases including psoriasis and atopic dermatitis, a better understanding of the role of lysosomes in epidermal homeostasis may guide future therapeutic strategies.
Project description:We recently identified Grainyhead-like 2 (GRHL2), a mammalian homolog of Grainyhead in Drosophila, to be a novel transcription factor that regulates hTERT gene expression and enhances proliferation of normal human epidermal keratinocytes (NHEK). In the current study, we show that GRHL2 impairs keratinocyte differentiation through transcriptional inhibition of the genes clustered at the epidermal differentiation complex (EDC), located at chromosome 1q21. Gene expression profiling and subsequent in vitro assays revealed consistent downregulation of EDC genes, for example, IVL, KRT1, FLG, LCEs, and SPRRs, in NHEK expressing exogenous GRHL2. In vivo binding assay by chromatin immunoprecipitation revealed GRHL2 association at the promoter regions of its target genes, many of which belong to EDC. Exogenous GRHL2 expression also inhibited recruitment of histone demethylase Jmjd3 to the EDC gene promoters and enhanced the level of histone 3 Lys 27 trimethylation enrichment at these promoters. Survey of GRHL2 expression in human skin tissues demonstrated enhanced protein and mRNA levels in chronic skin lesions with impaired keratinocyte differentiation, for example, atopic dermatitis and psoriasis, compared with normal epidermis. These data indicate that GRHL2 impairs epidermal differentiation by inhibiting EDC gene expression through epigenetic mechanisms and support its role in the hyperproliferative skin diseases.