Levels In Keratinocytes Research Articles

Keratin 17 as a therapeutic target for the treatment of psoriasis

Keratin 17 (K17) is the only ectopically expressed keratin in psoriatic lesional epidermis. This review focuses mainly on reports that have addressed the mechanism of K17 up-regulation and its biological role in psoriasis. In addition to IFN-γ, IL-17A and IL-22, which are derived from Th17 and Th22 cells, could up-regulate K17 mRNA and protein levels in keratinocytes in a dose-dependent manner. Moreover, these effects are partially blocked with STAT1- and STAT3-specific inhibitors, as well as small interfering RNA (siRNA) targeting STAT1 and STAT3. On the other hand, the HLA DRB1*04 and/or *07 positive patients show significant T cell responses to two peptides from K17 protein selected on the basis of predicted HLA DRB1*04 and/or *07 bindings. One peptide contains the ALEEAN sequence, while the other peptide has an amino acid sequence that has not been previously reported. Analysis of these processes led us to propose the existence of a K17/T cells/cytokine autoimmune loop, in which ectopically expressed K17 impacts on the maintenance of psoriasis by activating autoreactive T cells. Furthermore, it has been found that altered peptide ligands, which are produced through single alanine residue substitutions at a critical TCR contact position, abolish the T cell proliferation and IFN-γ production induced by K17 pathogenic peptides. K17-specific antisense ODNs and RNAi suppress K17 mRNA and protein expression in psoriatic skin in vivo, which coincides with marked clinical and histological improvement. These findings highlight K17 as an attractive target for novel therapies aimed at curtailing psoriasis driven by chronic inflammation.

The Extent of Desmoglein 3 Depletion in Pemphigus Vulgaris Is Dependent on Ca 2+-Induced Differentiation : A Role in Suprabasal Epidermal Skin Splitting?

Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes and is characterized by development of autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 and formation of intraepidermal suprabasal blisters. Depletion of Dsg3 is a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer periods under high-Ca(2+) conditions, we hypothesized that Dsg depletion depends on Ca(2+)-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in skin of patients with PV and in an organotypic raft model of human epidermis incubated using IgG fractions from patients with PV. In addition, Dsg3 depletion and loss of Dsg3 staining were prominent in cultured primary keratinocytes and in HaCaT cells incubated in high-Ca(2+) medium for 3 days, but were less pronounced in HaCaT cultures after 8 days. These effects were dependent on protein kinase C signaling because inhibition of protein kinase C blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, protein kinase C inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Considered together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca(2+)-induced differentiation. Furthermore, prominent depletion in basal epidermal layers may contribute to the suprabasal cleavage plane observed in PV.

Galvanic zinc–copper microparticles produce electrical stimulation that reduces the inflammatory and immune responses in skin

The human body has its own innate electrical system that regulates the body's functions via communications among organs through the well-known neural system. While the effect of low-level electrical stimulation on wound repair has been reported, few studies have examined the effect of electric potential on non-wounded, intact skin. A galvanic couple comprised of elemental zinc and copper was used to determine the effects of low-level electrical stimulation on intact skin physiology using a Dermacorder device. Zn-Cu induced the electrical potential recorded on intact skin, enhanced H(2)O(2) production and activated p38 MAPK and Hsp27 in primary keratinocytes. Treatment with Zn-Cu was also found to reduce pro-inflammatory cytokines, such as IL-1α, IL-2, NO and TNF-α in multiple cell types after stimulation with PHA or Propionibacterium acnes bacteria. The Zn-Cu complex led to a dose-dependent inhibition of TNF-α-induced NF-κB levels in keratinocytes as measured by a dual-luciferase promoter assay, and prevented p65 translocation to the nucleus observed via immunofluorescence. Suppression of NF-κB activity via crosstalk with p38 MAPK might be one of the potential pathways by which Zn-Cu exerted its inflammatory effects. Topical application of Zn-Cu successfully mitigated TPA-induced dermatitis and oxazolone-induced hypersensitivity in mice models of ear edema. Anti-inflammatory activity induced by the Zn-Cu galvanic couple appears to be mediated, at least in part, by production of low level of hydrogen peroxide since this activity is reversed by the addition of Catalase enzyme. Collectively, these results show that a galvanic couple containing Zn-Cu strongly reduces the inflammatory and immune responses in intact skin, providing evidence for the role of electric stimulation in non-wounded skin.

The extend of desmoglein 3 depletion in pemphigus vulgaris is dependent on Ca2+‐induced differentiation – a role in suprabasal epidermal skin splitting?

Pemphigus vulgaris (PV) is an autoimmune disease of skin and mucous membranes characterized by formation of intraepidermal blisters. Patients develop autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and 1, leading to suprabasal keratinocyte detachment. Depletion of Dsg3 was reported to be a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer time periods in high Ca2+ conditions, we hypothesized that Dsg depletion was dependent on Ca2+-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in PV patient skin and an organotypic raft model of human epidermis. Also, depletion was prominent in cultured primary keratinocytes as well as in HaCaT cells incubated in high Ca2+ media for 3d but is less pronounced in HaCaT cultures after 8d. These effects were dependent on protein kinase C alpha (PKCα) signaling because inhibition of PKCα blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, PKCα inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Taken together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca2+-induced differentiation, which may contribute to the suprabasal cleavage plane observed in PV.

Expression of a Constitutively Active Calcineurin Encoded by an Intron-Retaining mRNA in Follicular Keratinocytes

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca2+ and dephosphorylated NFATc2 under low Ca2+ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.

Retinoid Induces the Degradation of Corneodesmosomes and Downregulation of Corneodesmosomal Cadherins: Implications on the Mechanism of Retinoid-induced Desquamation

BackgroundTopical retinoids induce skin fragility. As corneodesmosomes are important adhesion structures in the epidermal cohesion, an effect of retinoids on corneodesmosomes has been suspected.ObjectiveThe aim of this study was to investigate the effect of retinoid on the expression of corneodesmosomal components including desmoglein (DSG) 1, desmocollin (DSC) 1, corneodesmosin (CDSN) and kallikrein (KLK)s.Methods2% all-trans-retinol or ethanol was applied to the back of hairless mice for five days, and the structure of the stratum corneum was examined by transmission electron microscopy. The cultured human keratinocytes were treated with all-trans-retinoic acid (RA) in low or high calcium media for 24 hours.ResultsTopical retinol increased corneocyte detachment and degradation of corneodesmosomes. RA significantly decreased DSG1 and DSC1 expression at the mRNA and protein levels in keratinocytes that were cultured in both low- and high-calcium media. On the other hand, CDSN mRNA levels did not decrease in low-calcium media or increase in high-calcium media after RA treatment. KLK5 and KLK7 expression did not increase after RA treatment.ConclusionOur results indicate that DSG1 and DSC1 downregulation by RA could be related to the increased degradation of corneodesmosomes and consequent desquamation induced by retinoids.

Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) plays an important role in maintaining skin homeostasis, and expression of IKKalpha was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKalpha in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkalpha(+/+) and Ikkalpha(+/-) mice. Ikkalpha(+/-) mice were found to develop a twofold greater number of skin tumors than Ikkalpha(+/+) mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkalpha(+/-) than in Ikkalpha(+/+) mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkalpha(+/-) compared with those in Ikkalpha(+/+) skin. Also, reduction of IKKalpha levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFalpha, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKalpha expression orchestrates UVB carcinogen, accelerating tumorigenesis.

Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.

Enhancing effects of a chromone glycoside, eucryphin, isolated from Astilbe rhizomes on burn wound repair and its mechanism

We previously reported that three compounds isolated from Astilbe thunbergii rhizomes accelerated burn wound healing. Among the three substances, eucryphin enhanced burn wound healing most strongly; however, the biological mechanisms of eucryphin are not yet well understood and here we examined the effects on chemokine, growth factor, and cytokine productions in in vivo and in vitro experiments. We have set burn wounds on the backs of mice and topically applied either vehicle alone or vehicle containing low doses of eucryphin to the burn wound. We examined the effects of eucryphin on chemokine, cytokine, and growth factor production at the wound site and in various cells. Eucryphin (10(-4) to 10(-8)% ointment) facilitated burn wound repair compared to the vehicle control. Eucryphin (100 ng per wound) increased IL-1beta, MCP-1, VEGF, and TGF-beta1 levels in the exudates from the wound area. Eucryphin increased VEGF, TGF-beta1, and HIF-1alpha expression levels in keratinocytes. These findings suggest that the enhancement of burn wound healing by eucryphin might be due to promotional angiogenesis during skin wound repair as a result of the stimulation of VEGF and TGF-beta1 production caused by the increase in HIF-1alpha expression in keratinocytes.

Einfluss von Melatonin auf die Physiologie des Haares

Melatonin, the pineal gland hormone and a strong antioxidant, has long been known, particularly in animal-experiment based research and the wool-producing industry, to be a potent regulatory neuroendocrine substance in relation to hair growth, hair color and hair cycle, depending on light periods, seasonal rhythms, environmental factors and reproductive rhythms. Nevertheless, the biological mechanisms of this extremely versatile hormone, especially with regard to human hair follicles, are not fully understood. In recent years, however, essential knowledge has been gained on the melatoninergic system of the skin, melatonin levels in keratinocytes and hair follicles, extrapineal intrafollicular melatonin synthesis and noradrenalin-induced increase in synthesis, as well as hair cycle-dependent expression of the membrane-bound melatonin receptor MT2 and the nuclear receptor RORalpha. Functional data on the growth of human hair both in vitro and in vivo show that melatonin might play an essential role in hair physiology.

Association of a genetic polymorphism (-44 C/G SNP) in the human DEFB1 gene with expression and inducibility of multiple β-defensins in gingival keratinocytes

BackgroundHuman β-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression.MethodsTransfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression.ResultsTransfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1β, TNFα, and interferon-γ (IFNγ). In contrast to constitutive expression levels, IFNγ-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression.ConclusionThe DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNγ inducibility of these β-defensins and is likely to be more protective in conditions that enhance IFNγ expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.

Gingko biloba extract reduces VEGF and CXCL-8/IL-8 levels in keratinocytes with cumulative effect with epigallocatechin-3-gallate

In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8/IL-8 play an important role and are produced by activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF and CXCL8/IL-8 by normal human keratinocytes (NHKs) activated by tumor necrosis factor alpha (TNFalpha). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFalpha-activated NHKs, we also tested its effect in association with GBE. Our results showed that GBE exerted a potent inhibition on VEGF and CXCL8/IL-8 levels in activated cells. In association with EGCG, GBE down-regulated VEGF and CXCL8/IL-8 levels in a cumulative manner in TNFalpha-stimulated NHKs. These results suggest that GBE, alone or in association with EGCG may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.

Aldo‐keto reductase 1C subfamily genes in skin are UV‐inducible: possible role in keratinocytes survival

Please cite this paper as: Aldo-keto reductase 1C subfamily genes in skin are UV-inducible: possible role in keratinocytes survival. Experimental Dermatology 2009; 18: 611-618.Abstract: Human skin is endowed with the capacity to synthesize and metabolize steroid hormones, a function of importance in skin physiology and pathology. It is the hormone-regulatory enzymes, including the aldo-keto reductase 1C subfamily (AKR1Cs) that are largely responsible for the local levels of active steroid hormones. AKR1C1 and AKR1C2 inactivate progesterone and 5alpha-dihydrotestosterone, respectively, whereas AKR1C3 activates oestradiol and testosterone. Here, we show that AKR1C1-3 are expressed in keratinocytes and fibroblasts, with marginal expression in melanocytes. In human primary keratinocytes, AKR1C1 and -2 were UVB-inducible in a dose-dependent manner, as shown by quantitative PCR and Western blot analyses. The induction of AKR1C1 by UVB was concomitant with the presence of an apoptotic marker, the cleavage product of poly-ADP ribose polymerase. Similarly, the activation of AKR1C1 and -2 upon UVB exposure was demonstrated in swine skin in vivo and in human skin explants. As expected, hydrogen peroxide-derived reactive oxygen species also induced AKR1C1 and -2 mRNA and protein levels in keratinocytes in a dose-dependent manner. Furthermore, down-regulation of AKR1Cs by small interfering ribonucleic acid led to significantly reduced cell viability. Based on the combined evidence of the presence of an apoptotic marker in the UVB-exposed keratinocytes with increased AKR1Cs expression and reduced cell viability in down-regulated AKR1Cs, we suggest that AKR1C subfamily genes are stress-inducible and might function as survival factors in keratinocytes.

Glucocorticoids Enhance Toll-Like Receptor 2 Expression in Human Keratinocytes Stimulated with Propionibacterium acnes or Proinflammatory Cytokines

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.

Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease.

Autoinhibition of IL-15 Expression in KC Cells is ERK1/2 and PI3K Dependent

Interleukin (IL)-15 is a proinflammatory cytokine and plays a key role in many diseases, including psoriasis. Although its signal transduction pathways in keratinocytes (KC) have been partially elucidated, the effects of IL-15 on expression of IL-15, IL-6 and TNF-alpha in KC are unknown. We have investigated the effects of IL-15 on the expression of the three genes in primary culture of KC by the real-time PCR, Western blot and ELISA. We observed that exogenous IL-15 suppressed the endogenous expression of IL-15, decreased the expression of IL-6 at mRNA and protein levels in KC. The inhibition was blocked by anti-IL-15 monoclonal antibody and by inactive IL-15, I50D mutant IL-15. In contrast, IL-15 increased TNF-alpha transcription in these cells. Mechanistic studies demonstrated that the auto-regulation of IL-15 expression was dependent on activity of ERK1/2 and PI3K. Our studies suggest that there is an auto-inhibitory mechanism controlling cellular IL-15 levels.

Erk5 Controls Slug Expression and Keratinocyte Activation during Wound Healing

Reepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell-cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF.

SOX9 and the tanning response: something new under the sun

SOX9 and the tanning response: something new under the sun

Keratinocyte growth factor receptor (KGF-R) in cholesteatoma tissue

Conclusion. Distribution of the receptor for epidermal growth factor (EGF-R) and of the receptor for the keratinocyte growth factor (KGF-R) in cholesteatoma was found to differ in analogy with other epithelial tissues and accordingly to epidermal differentiation and intensity of paracrine stimulation. Moreover, both EGF-R and KGF-R expression was increased, suggesting a fair correlation with aggressiveness and recurrence rate of this pathology. Objectives. To obtain information on the biological behaviour of cholesteatoma by assessing the expression and localization of EGF-R and KGF-R and correlating their tissue distribution with that of cytokeratins as a marker of differentiation. Materials and methods. Cholesteatoma tissue was taken during tympanoplasty surgery and processed for indirect immunofluorescence. Murine monoclonal antibodies were tested for the different growth factor receptors and pancytokeratins analysed. Fluorescence intensity signal was measured on randomly captured digital images, using FISH 2000/HI software, with a pseudocolours generation module. Results. EGF-R was mostly expressed at the level of keratinocytes of the basal layer, while KGF-R signal was mainly distributed on the spinous and granular suprabasal layers that were also highly positive for cytokeratins. Significant correlation between the immunofluorescence signals was found for KGF-R and cytokeratins only, demonstrating that KGF-R expression is increased in more differentiated areas of the cholesteatoma tissue, while EGF-R is associated with proliferative and migratory portions of the lesion.

Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

Backgroundp63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (ΔNp63). Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes.ResultsTo better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets.ConclusionThis unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.