<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type <em>2</em> (AT<em>2</em>) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT<em>2</em> cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -1<em>2</em>, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT<em>2</em> cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.

OBJECTIVE

We investigated the molecular mechanism of nicotine-accelerated atherosclerosis in a hyperlipidemic low-density lipoprotein receptor(-/-) mouse model.

RESULTS

Low-density lipoprotein receptor(-/-) mice received time-release nicotine or placebo pellets for 90 days. Aortic lesion size was <em>2</em>.5 times larger in nicotine-treated than in placebo-treated mice (P<0.001). A mild increase in lipids was seen in treated mice. We quantified 18 different serum cytokines and found a significant increase of tumor necrosis <em>factor</em> alpha, interleukin 1beta, and <em>keratinocyte</em>-derived chemokine in nicotine-treated mice. Among 107 nuclear <em>factor</em> kappaB (NF-kappaB) target genes screened from the aorta, we found that nicotine treatment upregulated only 4 atherogenic genes including vascular adhesion molecule 1 and cyclooxygenase <em>2</em> on day 60 and platelet-derived <em>growth</em> <em>factor</em> B and platelet 1<em>2</em>-lipoxygenase on day 90. At the cellular level, nicotine induced tumor necrosis <em>factor</em> alpha and inducible nitric oxide synthase expression in RAW<em>2</em>64.7 cells via the nicotinic acetylcholine receptors. Induction was confirmed in peritoneal macrophages isolated from nicotine-treated mice. Finally, we showed that preconditioned medium from nicotine-treated RAW<em>2</em>64.7 cells activated NF-kappaB in human smooth muscle cells and vascular endothelial cells as evidenced by nuclear localization and electromobility shift assay.

CONCLUSIONS

Chronic nicotine exposure augments atherosclerosis by enhancing the production of proinflammatory cytokines by macrophages, which, in turn, activate atherogenic NF-kappaB target genes in the aortic lesions.

Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (<em>keratinocytes</em>) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-kappaB pathways and their roles in protecting human <em>keratinocytes</em> (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm<em>2</em> from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY<em>2</em>9400<em>2</em> or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-kappaB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-kappaB activation by adenovirus-mediated overexpression of dominant negative IKK-beta or IkappaB-alpha did not increase UV-induced apoptosis. However, adenovirus-mediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY<em>2</em>9400<em>2</em>, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by <em>2</em>-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-kappaB pathway provides little, if any, protective role.

TRPV3 is a thermosensitive channel that is robustly expressed in skin <em>keratinocytes</em> and activated by innocuous thermal heating, membrane depolarization, and chemical agonists such as <em>2</em>-aminoethyoxy diphenylborinate, carvacrol, and camphor. TRPV3 modulates sensory thermotransduction, hair <em>growth</em>, and susceptibility to dermatitis in rodents, but the molecular mechanisms responsible for controlling TRPV3 channel activity in <em>keratinocytes</em> remain elusive. We show here that receptor-mediated breakdown of the membrane lipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P(<em>2</em>)) regulates the activity of both native TRPV3 channels in primary human skin <em>keratinocytes</em> and expressed TRPV3 in a HEK-<em>2</em>93-derived cell line stably expressing muscarinic M(1)-type acetylcholine receptors. Stimulation of PI(4,5)P(<em>2</em>) hydrolysis or pharmacological inhibition of PI 4 kinase to block PI(4,5)P(<em>2</em>) synthesis potentiates TRPV3 currents by causing a negative shift in the voltage dependence of channel opening, increasing the proportion of voltage-independent current and causing thermal activation to occur at cooler temperatures. The activity of single TRPV3 channels in excised patches is potentiated by PI(4,5)P(<em>2</em>) depletion and selectively decreased by PI(4,5)P(<em>2</em>) compared with related phosphatidylinositol phosphates. Neutralizing mutations of basic residues in the TRP domain abrogate the effect of PI(4,5)P(<em>2</em>) on channel function, suggesting that PI(4,5)P(<em>2</em>) directly interacts with a specific protein motif to reduce TRPV3 channel open probability. PI(4,5)P(<em>2</em>)-dependent modulation of TRPV3 activity represents an attractive mechanism for acute regulation of <em>keratinocyte</em> signaling cascades that control cell proliferation and the release of autocrine and paracrine <em>factors</em>.

The fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) gene is expressed as alternatively spliced mRNAs that encode bacterially expressed kinase, the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor, or K-sam. We have now isolated a novel FGFR<em>2</em> cDNA that is identical with the previously cloned human bacterially expressed kinase, except in the third immunoglobulin-like domain. The ligand binding properties of FGFR<em>2</em> were studied by expressing the protein in rat L6 muscle myoblasts. Unlike human bacterially expressed kinase which binds acidic and basic FGF with similar affinities, FGFR<em>2</em> bound acidic FGF with approximately 1000-fold higher affinity than basic FGF. These results indicate that alternative splicing of the FGFR<em>2</em> gene in the region encoding the carboxyl-terminal half of the third immunoglobulin domain determines the ligand specificity of this group of receptors.

Protein kinase C (PKC) isoforms are major regulators of cutaneous homeostasis and mediate inflammation in response to 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA). We have previously reported that transgenic mice overexpressing PKCalpha in the skin exhibit severe intraepidermal neutrophilic inflammation and <em>keratinocyte</em> apoptosis when treated topically with TPA. Activation of PKCalpha increases the production of TNFalpha and the transcription of chemotactic <em>factors</em> (MIP-<em>2</em>, KC, S100A8/A9), vascular endothelial <em>growth</em> <em>factor</em>, and GM-CSF in K5-PKCalpha <em>keratinocytes</em>. In response to PKCalpha activation, NF-kappaB translocates to the nucleus and this is associated with IkappaB phosphorylation and degradation. Preventing IkappaB degradation reduces both the expression of inflammation-associated genes and chemoattractant release. To determine whether TNFalpha mediated NF-kappaB translocation and subsequent expression of proinflammatory <em>factors</em>, K5-PKCalpha mice were treated systemically with a dimeric soluble form of p75 TNFR (etanercept) or crossed with mice deficient for both TNFR isoforms, and <em>keratinocytes</em> were cultured in the presence of TNFalpha-neutralizing Abs. The in vivo treatment and TNFR deficiency did not prevent inflammation, and the in vitro treatment did not prevent NF-kappaB nuclear translocation after TPA. Together these results implicate PKCalpha as a regulator of a subset of cutaneous cytokines and chemokines responsible for intraepidermal inflammation independent of TNFalpha. PKCalpha inhibition may have therapeutic benefit in some human inflammatory skin disorders.

Although prostaglandin E<em>2</em> (PGE<em>2</em>) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor <em>growth</em> is not well understood. In this study, we investigated the mechanisms by which PGE<em>2</em> stimulates murine <em>keratinocyte</em> proliferation using in vitro and in vivo models. In primary mouse <em>keratinocyte</em> cultures, PGE<em>2</em> activated the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP<em>2</em>), nor by a protein kinase A inhibitor (H-89). However, PGE<em>2</em>-stimulated extracellularly regulated kinase 1/<em>2</em> (ERK1/<em>2</em>) activation was completely blocked by EGFR, ERK1/<em>2</em>, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE<em>2</em>-induced proliferation, nuclear <em>factor</em>-kappa B, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse <em>keratinocytes</em>. Similarly, in vivo, we found that WT mice treated with PGE<em>2</em> and untreated cyclooxygenase-<em>2</em>-overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear <em>factor</em>-kappa B, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-<em>2</em> overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE<em>2</em> in mouse skin.

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-<em>2</em>(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-<em>2</em>(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.

Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid-binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription <em>factors</em> CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPdelta as well as SREBP-1c (ADD-1), but not PPARgamma. These changes in C/EBPalpha and C/EBPdelta were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPalpha, C/EBPdelta, SREBP-1, epidermal fatty acid-binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-<em>2</em> protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-<em>2</em> (betaarr<em>2</em>) depletion promotes CXCR<em>2</em>-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr<em>2</em> in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr<em>2</em>-deficient mice (betaarr<em>2</em>(-/-)) and control littermates (betaarr<em>2</em>(+/+)) were used. betaarr<em>2</em>(-/-) mice exhibited a significant increase in Lewis lung cancer tumor <em>growth</em> and metastasis relative to betaarr<em>2</em>(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/<em>keratinocyte</em>-derived chemokine and CXCL<em>2</em>/MIP-<em>2</em>), vascular endothelial <em>growth</em> <em>factor</em>, and microvessel density. NF-kappaB activity was also enhanced in betaarr<em>2</em>(-/-) mice, whereas hypoxia-inducible <em>factor</em>-1alpha expression was decreased. Inhibition of CXCR<em>2</em> or NF-kappaB reduced tumor <em>growth</em> in both betaarr<em>2</em>(-/-) and betaarr<em>2</em>(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial <em>growth</em> <em>factor</em> expression. Altogether, the data suggest that betaarr<em>2</em> modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR<em>2</em> and NF-kappaB.

OBJECTIVE

Erythropoietin has recently emerged as a cytoprotective cytokine, which possesses the ability to protect many tissues, including the brain, heart, and kidneys, against ischemia or traumatic injury. We investigated the therapeutic effects of erythropoietin in a murine model of endotoxin shock.

METHODS

Prospective, randomized study.

METHODS

University-based research laboratory.

METHODS

Male BALB/c mice.

METHODS

Mice intraperitoneally received either lipopolysaccharide (LPS) from Escherichia coli or vehicle. Erythropoietin was administered at a dose of 1000 IU/kg subcutaneously at different time points after LPS administration. We also investigated the effect of erythropoietin on the development of septic shock caused by cecal perforation.

RESULTS

Treatment of mice with erythropoietin, within <em>2</em> hours after LPS administration, improved the mortality rate. Treatment of cecal perforated mice with erythropoietin extended survival by 1<em>2</em> hours, but all animals died by 7<em>2</em> hours in both groups. Erythropoietin attenuated apoptosis in the lungs, liver, small intestine, thymus, and spleen, as assessed by terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling staining, active caspase-3 immunostaining and immunoblotting, and measurements of caspase-3/7 activity. Erythropoietin also reduced inducible nitric oxide synthase expression, nitric oxide production, peroxynitrite formation, and tissue hypoxia. In contrast, erythropoietin did not affect the degree of LPS-induced inflammation, as assessed by measurements of blood levels of interleukin-1beta, interleukin-6, tumor necrosis <em>factor</em>-alpha, <em>growth</em>-related oncogene/<em>keratinocyte</em>-derived cytokine, and high mobility group box 1, the phosphorylation levels of nuclear <em>factor</em> kappaB, and the number of neutrophils infiltrating the lungs and the liver.

CONCLUSIONS

The results of the study demonstrate that administration of a large dose of erythropoietin after induction of experimental endotoxemia improved survival and that the beneficial effects of erythropoietin were associated with inhibition of apoptosis, nitric oxide production, and tissue hypoxia, without alterations in inflammatory responses.

Transforming <em>growth</em> <em>factor</em>-beta-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear <em>factor</em>-kappaB (NF-kappaB). Given that NF-kappaB regulates <em>keratinocyte</em> differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated <em>keratinocyte</em>-specific TAK1-deficient mice from Map3k7(flox/flox) mice and K5-Cre mice. The <em>keratinocyte</em>-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day <em>2</em> or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of <em>keratinocyte</em> apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal <em>keratinocytes</em> of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in <em>keratinocytes</em> results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the <em>keratinocytes</em> from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient <em>keratinocytes</em> retain the ability to differentiate. Moreover, the removal of TAK1 from cultured <em>keratinocytes</em> of Map3k7(flox/flox) mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of <em>keratinocyte</em> <em>growth</em>, differentiation, and apoptosis.

The mechanisms that regulate <em>keratinocyte</em> migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in <em>2</em> distinct models of wound healing: scratch-injured cultures of human <em>keratinocytes</em> and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-<em>2</em>'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits <em>keratinocyte</em> migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains <em>keratinocyte</em> proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of <em>keratinocytes</em> transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK<em>2</em>, cell proliferation marker MKI67, and transcription <em>factor</em> YAP1 are direct targets of miR-483-3p that control <em>keratinocyte</em> proliferation. miR-483-3p-mediated down-regulation of MK<em>2</em>, MKI67, and YAP1 thus represents a novel mechanism controlling <em>keratinocyte</em> <em>growth</em> arrest at the final steps of reepithelialization.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin <em>keratinocytes</em>, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em>. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/<em>2</em> (ERK1/<em>2</em>) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/<em>2</em> activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear <em>factor</em> erythroid-related <em>factor</em> <em>2</em> (NRF<em>2</em>) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in <em>keratinocytes</em>. Additionally, glutathione metabolism, which is a marker for NRF<em>2</em>-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and <em>growth</em> <em>factors</em> were found. The roles of NRF<em>2</em> targets, investigated by siRNA silencing, revealed that NRF<em>2</em> acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF<em>2</em> pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in <em>keratinocytes</em> facilitates the understanding of plasma-tissue interaction and its clinical application.

To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal <em>keratinocytes</em> (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix), approximately 1<em>2</em>000 genes) indicated that the major group of upregulated genes in <em>keratinocytes</em> fall into three categories: (i). antiapoptotic and cell survival <em>factors</em>, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1, -<em>2</em>, -3, SCYA<em>2</em>0), <em>growth</em> <em>factors</em> (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-<em>2</em>, S100A9), (ii). DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii). ECM proteases (MMP-1, -10). The major downregulated genes are DeltaNp63 and PUMILIO, two potential markers for the maintenance of <em>keratinocyte</em> stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival <em>factors</em>, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of <em>keratinocytes</em> to UVB involved regulation of key checkpoint genes (p53, MDM<em>2</em>, p<em>2</em>1(Cip1), DeltaNp63), as well as antiapoptotic and DNA repair-related genes - no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of <em>2</em>51 and 1<em>2</em>7 genes, respectively, and downregulation of 3<em>2</em><em>2</em> genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned <em>keratinocytes</em> from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in <em>keratinocytes</em> for epithelial cancer is discussed.

Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP). Inflammation is critical in the pathogenesis of many diseases, but the trigger(s) is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD<em>2</em> mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt) mtDNA and ATP without cell death. Mitochondria purified from LAD<em>2</em> cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(<em>2</em>), IL-8, TNF, and IL-1β. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) release from cultured human <em>keratinocytes</em>, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted in vivo, we injected rats intraperiotoneally (ip) with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD<em>2</em> cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as "autopathogens" contributing to the pathogenesis of inflammatory diseases and may be used as targets for novel treatments.

Interactions between infiltrating T cells and <em>keratinocytes</em> via the secretion of the TH1 cytokines interleukin (IL) <em>2</em> and interferon gamma (INF-gamma), the <em>keratinocyte</em> <em>growth</em> <em>factor</em> transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha) and the cytokines IL-6 and IL-8 are thought to be the predominant mechanisms inducing skin lesions in psoriatic patients. Systemic treatment of psoriasis with fumaric acid derivatives (FAEs) has been reported to be effective in the treatment of psoriasis, but the mode of action is still unknown. To clarify this phenomenon, <em>keratinocytes</em> from psoriatic patients as well as from healthy volunteers were mono- and cocultured with HUT 78 T cells with/without the addition of FAEs; the cytokine concentrations were then measured in the culture supernatants. Furthermore, mRNA expression was determined in epidermal <em>growth</em> <em>factor</em> (EGF) -activated <em>keratinocytes</em> as well as in phytohaemagglutinin (PHA)-activated HUT 78 T cells. Only dimethylfumarate (DMF) diminished IL-6 and TGF-alpha secretion in the psoriatic cocultures. However, it did not have this effect on cocultures from control subjects or on monocultures. DMF suppresses EGF-induced TGF-alpha mRNA induction in psoriatic <em>keratinocytes</em>. DMF inhibited INF-gamma secretion in all cultures but stimulated the IL-10 secretion. This immunomodulation away from the TH1 cytokine IFN-gamma to the TH<em>2</em> cytokine IL-10 was confirmed in HUT 78 T cells by Northern blot analysis. An increased number of eosinophils is a known side-effect in patients treated with this drug, suggesting a clinical relevance of this immunomodulation in vivo. This immunomodulation and the suppression of cytokines from the psoriatic cytokine network could be responsible for the beneficial effect of DMF in the treatment of a hyperproliferative and TH1 cytokine-mediated skin disease.

Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter <em>keratinocytes</em>, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase <em>keratinocyte</em> migration and proliferation as well as insulin and insulin-like <em>growth</em> <em>factor</em>-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 1<em>2</em> d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter <em>keratinocytes</em> without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type <em>2</em> diabetes and support regional GM3 depletion as a promising therapeutic direction.

Chronic wounds are associated with poor epidermal and dermal remodeling. Previous work has shown the efficacy of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) in reepithelialization and elastin in dermal wound healing. Here we demonstrate the fabrication of a fusion protein comprising of elastin-like peptides and KGF. This fusion protein retains the performance characteristics of KGF and elastin as evidenced by its enhancement of <em>keratinocyte</em> and fibroblast proliferation. It also preserved the characteristic elastin-like peptides inverse phase transitioning allowing the recombinant protein to be expressed in bacterial hosts (such as Escherichia coli) and purified rapidly and easily using inverse temperature cycling. The fusion protein self-assembled into nanoparticles at physiological temperatures. When applied to full thickness, wounds in Lepr(db) diabetic mice these particles enhanced reepithelialization and granulation, by <em>2</em>- and 3-fold respectively, when compared to the controls. The data strongly suggests that these self-assembled nanoparticles may be beneficial in the treatment of chronic wounds resulting from diabetes or other underlying circulatory conditions.

Hydrogen peroxide is an important mediator of intracellular signaling, which potently enhances the expression of heme oxygenase-1 (HO-1) and upregulates synthesis of vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The purpose of the present study was to explore the involvement of HO-1 in regulation of H(<em>2</em>)O(<em>2</em>)-mediated induction of VEGF synthesis. We provide genetic evidence that basal and H(<em>2</em>)O(<em>2</em>)-induced VEGF synthesis is partially dependent on HO-1. Inhibition of HO-1 activity by tin protoporphyrin (SnPPIX) resulted in downregulation of VEGF synthesis in murine fibroblasts and human <em>keratinocytes</em>. The relationship between HO-1 and VEGF was corroborated by using cells derived from HO-1 knockout mice, which demonstrated lower basal and H(<em>2</em>)O(<em>2</em>)-induced production of VEGF. Additionally, knock out of HO-1 gene impaired induction of VEGF by hemin, lysophosphatidylcholine, and prostaglandin-J(<em>2</em>). Our results provide confirmation for the involvement of HO-1 in regulation of angiogenesis.

The human metastasis-associated gene (MTA1), a member of the nucleosome remodeling complex with histone deacetylase activity, is frequently overexpressed in biologically aggressive epithelial neoplasms. Here, we extend this observation to squamous carcinoma cells, which express high levels of MTA1 relative to normal or immortalized <em>keratinocytes</em>. To address functional aspects of MTA1 expression, we established variants of human immortalized <em>keratinocytes</em> (HaCaT cells) by expressing MTA1 cDNA in both the sense and antisense orientations. We demonstrate that (1) forced MTA1 expression enhances migration and invasion of immortalized <em>keratinocytes</em>; (<em>2</em>) MTA1 expression is necessary but not sufficient for cell survival in the anchorage independent state; (3) MTA1 contributes to expression of the anti-apoptotic Bcl-<em>2</em> family member Bcl-x(L); (4) MTA1 expression in immortalized <em>keratinocytes</em> depends, in part, on activation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). These results establish that, in <em>keratinocytes</em>, MTA1 expression contributes to several aspects of the metastatic phenotype including survival in the anchorage independent state, migration, and invasion.

Connective tissue <em>growth</em> <em>factor</em>, which is induced by transforming <em>growth</em> <em>factor</em> beta, has been reported to mediate the stimulatory actions of transforming <em>growth</em> <em>factor</em> beta on type I procollagen synthesis. Connective tissue <em>growth</em> <em>factor</em> is expressed in fibrotic disease such as scleroderma, where it is believed to promote abnormal deposition of collagen. Connective tissue <em>growth</em> <em>factor</em> expression has not been described in normal human skin or cultured skin cells, however. We report here that connective tissue <em>growth</em> <em>factor</em> mRNA is constitutively expressed in normal human skin. In situ hybridization demonstrated that connective tissue <em>growth</em> <em>factor</em> mRNA was expressed in <em>keratinocytes</em> throughout the epidermis and in dermal cells. Quantitative real-time reverse transcription polymerase chain reaction revealed that the level of connective tissue <em>growth</em> <em>factor</em> mRNA in the epidermis and dermis of normal human skin was comparable to the level of housekeeping gene 36B4. Ultraviolet irradiation (<em>2</em> minimal erythema dose, UVB/A<em>2</em> source) reduced connective tissue <em>growth</em> <em>factor</em> mRNA expression throughout the epidermis and dermis in normal human skin in vivo. Connective tissue <em>growth</em> <em>factor</em> mRNA was reduced (30%) within 4 h post ultraviolet irradiation, and remained reduced (50%) 8-<em>2</em>4 h post ultraviolet. Connective tissue <em>growth</em> <em>factor</em> mRNA and protein were also constitutively highly expressed in normal cultured human skin <em>keratinocytes</em> and fibroblasts. Ultraviolet irradiation of cultured normal human skin fibroblasts resulted in a time-dependent inhibition of connective tissue <em>growth</em> <em>factor</em> mRNA expression. At <em>2</em>4 h post ultraviolet, connective tissue <em>growth</em> <em>factor</em> mRNA expression was reduced 80%. Transforming <em>growth</em> <em>factor</em> beta1 rapidly induced connective tissue <em>growth</em> <em>factor</em> mRNA levels (5-fold within 4 h) in skin fibroblasts, but not <em>keratinocytes</em>, and this induction was attenuated 80% by ultraviolet irradiation. Electrophoretic mobility shift assays demonstrated that ultraviolet irradiation reduced protein binding to the transforming <em>growth</em> <em>factor</em> beta/Smad responsiveness elements in the connective tissue <em>growth</em> <em>factor</em> gene promoter, in human skin in vivo and human skin fibroblasts. Constitutive expression of connective tissue <em>growth</em> <em>factor</em> in normal human skin suggests that it is a physiologic regulator of procollagen synthesis. Ultraviolet reduction of connective tissue <em>growth</em> <em>factor</em> expression may contribute to reduced procollagen synthesis observed in ultraviolet-irradiated normal human skin and human skin fibroblasts.

For centuries, itch was categorized as a submodality of pain. Recent research over the last decade has led to the realization that itch is in fact a separate and distinct, albeit closely related, sensation. Chronic itch is a common complaint and has numerous etiologies. Various receptors (TRPA1, TRPV1, PAR<em>2</em>, gastrin-releasing peptide receptor (GRPR), Mas-related G proteins), secreted molecules (histamine, nerve <em>growth</em> <em>factor</em> (NGF), substance P (SP), proteases), and cytokines/chemokines (thymic stromal lymphopoietin (TSLP), IL-<em>2</em>, IL-4, IL-13, and IL-31) are implicated as mediators of chronic pruritus. While much remains unknown regarding the mechanisms of chronic itch, this much is certain: there is no singular cause of itch. Rather, itch is caused by a complex interface between skin, <em>keratinocytes</em>, cutaneous nerve fibers, pruritogenic molecules, and the peripheral and central nervous systems. Atopic dermatitis is one of the most itchy skin dermatoses and affects millions worldwide. The sensation of atopic itch is mediated by the interplay between epidermal barrier dysfunction, upregulated immune cascades, and the activation of structures in the central nervous system. Clinicians are in possession of an arsenal of different treatment options ranging from moisturizers, topical immunomodulators, topical anesthetic ion channel inhibitors, systemic immunomodulators, as well as oral drugs capable of reducing neural hypersensitization. Emerging targeted therapies on the horizon, such as dupilumab, promise to usher in a new era of highly specific and efficacious treatments. Alternative medicine, stress reduction techniques, and patient education are also important treatment modalities. This review will focus on the mediators of chronic pruritus mainly associated with atopic dermatitis (atopic itch), as well as numerous different therapeutic options.