IKKepsilon involvement in Tax-mediated activation of INF pathway

HTLV-1 Tax de-regulates several cellular signaling pathways leading to cell transformation by altering gene expression, intracellular protein distribution and cell proliferation. Tax-1 induces persistent activation of several transcriptional factors and signal transduction pathways, including NF-\u3baB and CREB/ATF. It is known that Tax-1 constitutively activates TAK1 (transforming growth factor-\u3b2-activated kinase 1) and modifies the interferon (INF) regulatory signals by controlling the expression of INF transcription factors 3 (INF3) and INF4. We have recently reported that HTLV-1 and HTLV-2 Tax proteins interact with TAK1-binding protein 2 (TAB2) of the NF-\u3baB pathway and that both Tax proteins transactivate NF-\u3baB promoters [1]. TAB2 functions as an adaptor protein to recruit TAK1 to TRAF2 (TNF-\u3b1 receptor-associated factor) in TNF-\u3b1 signaling pathways. In the present study we have investigated Tax-1 and Tax-2 role in modifying INF and NF-\u3baB activation through the recruitment of IKKepsilon, an I\u3baB kinase homologue involved in NF-\u3baB and INF3 signaling pathways. By co-immunoprecipitation experiments, we have found that both IKKepsilon and Tax-1, but not Tax-2, are present in protein complexes in transfected cells. IKKepsilon and Tax-1 or Tax-2 role in the activation of INF responsive elements or NF-\u3baB containing promoters have been analyzed after transfecting the protein genes in 293T cells and measuring the effect by luciferase assay. Co-expression of Tax-1 and IKKepsilon resulted in an increased IRF activation mediated by IKKepsilon. Interaction of IKKepsilon with Tax-1 and Tax-2 and their possible effects in the de-regulation of the IRF3 pathways will be discussed

Comparison of Tax-1 and Tax-2B post-translational modifications using specific lysine mutants in relation to activation of NF-κB and intracellular localization

ost-translational modifications of HTLV-1 and HTLV-2 Tax-1 and Tax-2 proteins have been shown to play a critical role in their cellular localization, transactivation and protein interactions. Five of ten lysine residues were found to be major targets for Tax-1 modifications: Lys189(K4); Lys197(K5), Lys263(K6), Lys280(K7) and Lys284(K8), are essential for ubiquitination, while sumoylation takes place on Lys280 (K7) and Lys284(K8). Tax-2 contains four additional lysine residues, namely at position Lys100(K2i), Lys149(K3i), Lys185(K3ii), and Lys356(K10i).Very few studies have been so far performed on Tax-2 lysine mutants. We have previously demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1. To identify the Tax-2 lysine residues which are directly involved in post-translational modifications, we have constructed a series of Tax-2B mutants with substitutions of lysine (K) residues by arginines (R) and analyzed them for NF-kB and CREB/ATF transactivation, intracellular distribution and extent of ubiquitination and sumoylation. We have found that Tax-2 K7-8R mutant, contrary to its Tax-1 homologue, is only partially affected in its capacity to transactivate NF-\u3baB pathway, is regularly sumoylated and presents formation of nuclear bodies by confocal analysis. However, Tax-2 mutants with extended (K3ii-8R) and/or total (K1-10iR) mutation rate were severely affected for NF-kB transactivation and sumoylation. By comparing Tax-2 WT with mutants K7-8R and K3ii-8R, we observed that the reduction of NF-\u3baB activity is correlated to a parallel decrease in sumoylation. These results suggest that the target for Tax-2 ubiquitination and sumoylation differs from that described for Tax-1

Sense of smell in chronic rhinosinusitis: A multicentric study on 811 patients

Introduction: The impairment of the sense of smell is often related to chronic rhinosinusitis (CRS) with or without nasal polyps (CRSwNP, CRSsNP). CRSwNP is a frequent condition that drastically worsens the quality of life of those affected; it has a higher prevalence than CRSsNP. CRSwNP patients experience severe loss of smell with earlier presentation and are more likely to experience recurrence of their symptoms, often requiring revision surgery. Methods: The present study performed a multicentric data collection, enrolling 811 patients with CRS divided according to the inflammatory endotype (Type 2 and non-Type 2). All patients were referred for nasal endoscopy for the assessment of nasal polyposis using nasal polyp score (NPS); Sniffin' Sticks olfactory test were performed to measure olfactory function, and SNOT-22 (22-item sinonasal outcome test) questionnaire was used to assess patients' quality of life; allergic status was evaluated with skin prick test and nasal cytology completed the evaluation when available. Results: Data showed that Type 2 inflammation is more common than non-type 2 (656 patients versus 155) and patients suffer from worse quality of life and nasal polyp score. Moreover, 86.1% of patients with Type 2 CRSwNP were affected by a dysfunction of the sense of smell while it involved a lesser percentage of non-Type 2 patients. Indeed, these data give us new information about type-2 inflammation patients' characteristics. Discussion: The present study confirms that olfactory function weights on patients' QoL and it represents an important therapeutic goal that can also improve patients' compliance when achieved. In a future - and present - perspective of rhinological precision medicine, an impairment of the sense of smell could help the clinician to characterize patients better and to choose the best treatment available

Induction of cytochrome P450 enzyme activity by UVB and xenobiotics in normal human keratinocites.

The function of the skin is to provide a barrier for protection against the external environment. Relatively little is known about the overall role of CYP450 in the metabolism of xenobiotics or endogenous cellular compounds in the skin. The aim of this study was to analyse the expression and induction of several drugs metabolizing enzymes involved in either phase I or phase II reactions, in proliferanting human keratinocytes after exposure to UVB radiation and to three classical cytochrome inducers such as: β-naphthoflavone (BNF), 3-methylcholanthrene (MC), phenobarbital (PB). We investigated 7-ethoxyresorufìn O-deethylase (EROD) and 7-pentoxyresorufìn O-depenthylase activities (PROD). Normal human keratinocytes were cultured with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12. At confluency cells were incubated with inducers or irradiated with different doses of UVB. The microsomal fraction was studied by western-blot analysis. The MC-induced EROD activity was up to 4 fold higher when compared with BNF induced activity. UVB exposure resulted in a dose-dependent (25-75 mJ) and time dependent (6-24 h) induction of CYP450 1A1. Immunoblotting assay showed expression for CYP450 1B1 for both keratinocytes and melanocytes. Proadifen, an inhibitor of CYP450-monooxygenase, led to a significant decrease in EROD activity. The results of the present study clearly show that irradiation with UVB is capable of modifying the activity of CYP450 isoenzymes in keratinocytes. The phase II enzyme gluthatione S transferase activity (GST) was induced by UVB and PB. These experimental findings stress the value of epidermal cell culture for pharmaco-toxicological studies of topical agents used in dermatology

Neutral sphingomyelinase is modulated by glutathione in keratinocytes UVB-induced apoptosis

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which plays a role in intracellular signal transduction. The action of a ligand binding to a surface receptor results in early activation of the enzyme sphingomyelinase (SMases) with consequent hydrolysis of membrane sphingomyelin (SM) and generation of ceramide. Increase in intracellular ceramide level is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of apoptosis. Recent evidences have shown that reduced glutathione (GSH), but not other antioxidative agent, is able to inhibit apoptotic cell death and necrosis induced by hypoxia in PC12 cells. This protective effect is mediated by GSH direct inhibition of neutral SMase activity and ceramide formation. In the present study we report that GSH inhibits activation of neutral SMases and generation of ceramide and partially prevents apoptotic cell death induced by UVB radiation in keratinocytes. Normal human keratinocytes were cultivated with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium. At preconfluency cells were incubated with GSH for 2h and then irradiated with a UVB dose of 75mJ per cm2. At different times after UVB irradiation, cells were harvested for in vitro measurement of neutral SMaese activity, lipid extraction and Western blot analysis. In vitro measurement of neutral SMase activity showed an early induction 15 min after exposure with a subsequent decrease to control level after 2h. Exposure to UVB radiation resulted in a rapid sphingomyelin hydrolysis and generation of ceramide as measured by TLC analysis. The ceramide accumulation started at 15 min after UV exposure and progressively increased up to 24h. Addition of GSH significantly inhibited activation of neutral SMase and generation of ceramide in UVB-treated keratinocytes. Moreover UVB-induced cleavage of PARP, a marker of the apoptotic response, was partially inhibited by GSH treatment. This data indicate that GSH plays a critical role in UVB signaling pathway regulating neutral SMase activity

Induzione dell'attività degli enzimi di fase I e II nei cheratinociti umani normali.

La cute può essere considerata come un effettivo distretto di biotrasformazione di composti chimici. Lo scopo di questo lavoro è stato analizzare l'espressione dei sistemi enzimatici per il metabolismo di fase I e fase II nei cheratinociti umani in proliferazione dopo irradiazione con raggi UVB e dopo esposizione a tre classici induttori: beta-naphthoflavone (BNF), 3-methylcholantrene (MC), phenobarbital (PB). Cheratinociti umani normali sono stati coltivati con fibroblasti 3T3 mitomicinati in Dulbecco's modified Eagle's medium/Ham's F12. A subconfluenza le cellule sono state incubate con gli induttori o irradiate a differenti dosi di UVB. La fase I è stata analizzata con attività 7-ethoxyresorufin O-deethylase (EROD) e 7-pentoxyresorufin O-depenthylase (PROD). la frazione microsomiale è stata studiata mediante analisi western blot. L'attività EROD indotta dall'MC era più alta di 4 volte rispetto al BNF. L'esposizione a raggi UVB risultava dose dipendente (50-75 mJ) e tempo dipendente (6-24h). L'attività della glutatione-S-transferasi(GST), enzima di fase II determinato con cinetica enzimatica, veniva significativamente espressa con UVB e PB. Questi risultati dimostrano il ruolo della cute nel metabolismo esogeno e la possibilità di utilizzare colture di cellule epidermiche per studi farmacotossicologici di agenti topici usati in dermatologia

Induction of cytochrome P450 enzyme activity by UVB and xenobiotics in normal human keratinocytes and melanocytes

Cytochrome P450 (CYP450) play a major role in the bioactivation of procarcinogenesis in target tissue and the expression of this enzyme i san important determinant of human susceptibility to cancer. Relatively little is known about the overall role of CYP450 in the metabolism of xenobiotics or endogenous cellular compounds in the skin. The aim of this study was to analyse the expression of cytochrome enzymes in proliferanting human keratinocytes and melanocytes after exposure to UVB radiation and to three classical cytochrome inducers such as: β-naphthoflavone (BNF), 3-methylcholanthrene (MC), phenobarbital (PB). We investigated 7-ethoxyresorufìn O-deethylase (EROD) (which is CYP450 1A1 dependent) and 7-pentoxyresorufìn O-depenthylase activities (PROD) (CYP450 2B1 dependent)activities. Normal human keratinocytes were cultured with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 or with KGM serum-free medium. Melanocytes were grown in medium 154. At confluency cells were incubated with inducers or irradiated with different doses of UVB. At different times after treatments, cells were harvested for in vitro measurement of CYP450 induction. The microsomal fraction was studied by western-blot analysis. Low, but measurable levels of CYP activity were detected in both basal and differentianting keratinocytes. The MC-induced EROD activity was up to 4 fold higher when compared with BNF induced activity. UVB exposure resulted in a dose-dependent (10-75 mJ) and time dependent (4-24 h) induction of CYP450 1A1 for keratinocytes and CYP450 2B1 for melanocytes. Immunoblotting assay showed expression for CYP450 1B1 for both keratinocytes and melanocytes. Proadifen, an inhibitor of CYP450-monooxygenase, led to a significant decrease in EROD activity. The results of the present study clearly show that irradiation with UVB is capable of modifying the activity of CYP450 isoenzymes not only in keratinocytes but also in melanocytes.These experimental findings stress the value of epidermal cell culture for pharmaco-toxicological studies of topical agents used in dermatology

A new role of phase I and phase II enzyme in keratinocytes UVB induced apoptosis.

A new role of Phase I and Phase II enzymes in keratinocytes UVB induced apoptosis.UVB is the major cause for cutaneous malignancies in the human population. The skin is able to activate anti-oxidants and enzymatic detoxification reactions to neutralize reactive photochemical products. Apoptosis, removing irreversibly DNA-damaged and potentially neoplastic cells, represents a major defence mechanism towards malignant transformation. Very little is known about the role of cutaneous cytochrome P450 (CYP450) isoenzymes and glutathione S-transferase (GST) in UVB response. Although not yet fully investigated, it has been studied a possible relationship between CYP450 inductors and UVB initiated apoptosis in rat hepatocytes. Altered high levels of GST has been directly correlated to resistance to chemotherapeutic drugs suggesting that GST plays a role in prevention of apoptosis. Our study is focused on phase I and phase II enzyme activities in normal human keratinocytes. In the first part of the study we demonstrated that CYP450 (1A1 and 2B1) and GST are induced not only by classical inducers such as \u3b2-naphthoflavone, 3-methylcholanthrene, phenobarbital but also by UVB radiations (50 mJ/cm2). Differentiated keratinocytes were employed for all the experiments as confirmed by immunoblotting with Cytokeratin 10, a specific marker of the basal spinous transition. In the second part we evaluated a possible involvement of these enzyme in UVB mediated apoptosis process. Western blot analysis of Bcl2 expression and PARP cleavage showed that inhibition of CYP450 by Proadifen prevented UVB induced apoptotic cell death. In contrast, the diuretic drug, ethacrinic acid, a GST inhibitor, was able to improve UVB induced apoptosis. In order to confirm the anti-apoptotic activity displayed by GST, when GST activity was increased by Phenobarbital, UVB apoptosis was prevented.These results suggest that UVB radiations may play a critical role as tumour promoters even through the regulation of CYP450 and GST metabolising enzymes

Dose response curves of interleukin 1 alpha release by cultured normal human keratinocyte after variable exposure to different concentrations of SLS

One of the primary events or response of human keratinocyte to irritant is the release of a variety of mediators. Among these, interleukin 1 alpha (IL-1alfa) has been shown to be increased following different stimuli induced by inflammatory agents or microbial endotoxins. Furthermore, it has been shown that IL-1 alfa secretion is triggered by exposure to tensides.To study the direct effects of irritants on keratinocytes without the involvement of inflammatory cells and the secondary effects of barrier disruption, we used an in vitro culture model.In this preliminary study, we investigated the in vitro release of IL-1 alfa produced by cultured normal human keratinocytes (NHK) treated with Sodium Lauryl Sulfate (SLS), an anionic detergent, at concentrations ranging from 0,00001 to 0,005. diluited in a protein free medium (KBM).After 1 h exposure, cells were washed and the medium was changed. After 24 h the supernatant was collected for Enzyme Linked Immunoassorbent Assay (ELISA).Results presented here are the mean of three independent experiments.Our results show that extracellular secretion of IL-1 alfa has an increasing trend with increasing SLS doses and exposure times