H4K20me3 marks distal intergenic and repetitive regions in human mature spermatozoa.

Sperm histones represent an essential part of the paternally transmitted epigenome, but uncertainty exists about the role of those remaining in non-coding and repetitive DNA. We therefore analyzed the genome-wide distribution of the heterochromatic marker H4K20me3 in human sperm and somatic (K562) cells. To specify the function of sperm histones, we compared all H4K20me3-containing and -free loci in the sperm genome. Sperm and somatic cells possessed a very similar H4K20me3 distribution: H4K20me3 peaks occurred mostly in distal intergenic regions and repetitive gene clusters (in particular genes encoding odorant-binding factors and zinc-finger antiviral proteins). In both cell types, H4K20me3 peaks were enriched in LINEs, ERVs, satellite DNA and low complexity repeats. In contrast, H4K20me3-free nucleosomes occurred more frequently in genic regions (in particular promoters, exons, 5'-UTR and 3'-UTR) and were enriched in genes encoding developmental factors (in particular transcription activators and repressors). H4K20me3-free nucleosomes were also detected in substantial quantities in distal intergenic regions and were enriched in SINEs. Thus, evidence suggests that paternally transmitted histones may have a dual purpose: maintenance and regulation of heterochromatin and guidance towards transcription of euchromatin.

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase.

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis.

Synergistic therapeutic vascular cytoprotection against complement-mediated injury induced via a PKCα-, AMPK-, and CREB-dependent pathway.

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

Protein kinase Cε activity induces anti-inflammatory and anti-apoptotic genes via an ERK1/2- and NF-κB-dependent pathway to enhance vascular protection.

Vascular endothelial injury predisposes to endothelial dysfunction and atherogenesis. We have investigated the hypothesis that PKCε (protein kinase Cε) is an important upstream regulator of cytoprotective pathways in vascular ECs (endothelial cells). Depletion of PKCε in human ECs reduced expression of the cytoprotective genes A1, A20 and Bcl-2. Conversely, constitutively active PKCε expressed in human ECs increased mRNA and protein levels of these cytoprotective genes, with up-regulation dependent upon ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. Furthermore, inhibition of NF-κB (nuclear factor κB) by the pharmacological antagonist BAY 11-7085 or an IκB (inhibitor of NF-κB) SuperRepressor prevented cytoprotective gene induction. Activation of PKCε enhanced p65 NF-κB DNA binding and elevated NF-κB transcriptional activity. Importantly, although NF-κB activation by PKCε induced cytoprotective genes, it did not up-regulate pro-inflammatory NF-κB targets [E-selectin, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1)]. Indeed, PKCε exhibited cytoprotective and anti-inflammatory actions, including inhibition of TNFα (tumour necrosis factor α)-induced JNK (c-Jun N-terminal kinase) phosphorylation and ICAM-1 up-regulation, a response attenuated by depletion of A20. Thus we conclude that PKCε plays an essential role in endothelial homoeostasis, acting as an upstream co-ordinator of gene expression through activation of ERK1/2, inhibition of JNK and diversion of the NF-κB pathway to cytoprotective gene induction, and propose that PKCε represents a novel therapeutic target for endothelial dysfunction.

Enhanced anti-inflammatory effect of resveratrol and EPA in treated endotoxin-activated RAW 264.7 macrophages.

Macrophages play an important role in immunogenic challenges by producing reactive oxygen species, NO and proinflammatory cytokines that can aggravate and propagate local inflammation. Multiple mechanisms regulate these inflammatory processes. NF-κB and activator protein 1 pathways are crucial in the expression of proinflammatory genes, such as TNF-α, IL-1 (α or ß) and -6. Some polyphenols, which are present in beverages, vegetables and fruits, and PUFA, which are present in marine oils and fish food, possess anti-inflammatory effects in vivo and in vitro. Our aim in the present study was to assess whether polyphenols and PUFA have synergistic anti-inflammatory effects in murine macrophages in vitro. Inflammation in RAW 264.7 macrophages was induced by lipopolysaccharide at 100 ng/ml. The treatments with molecules were performed by co-incubation for 19 h. A NO production assay by Griess reaction, a phosphoprotein assay by Pathscan ELISA kit and gene expression analysis using the TaqMan® Low-density Array for ninety-one genes related to inflammation, oxidative stress and metabolism were performed to assess the synergistic anti-inflammatory effects of polyphenols, epigallocatechin gallate and resveratrol (Res; 2·5 µg/ml), and the PUFA, DHA and EPA (30 µm). Adding Res+EPA had an enhanced anti-inflammatory effect, in comparison with EPA and Res alone, leading to decreased NO levels; modulating the phospho-stress activated protein kinase/Jun N-terminal kinase (P-SAPK/JNK) level; down-regulating proinflammatory genes, such as IL, chemokines, transcription factors; and up-regulating several antioxidant genes. Therefore, this combination has a stronger anti-inflammatory effect than either of these molecules separately in RAW macrophages.

Glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase: glycosylation is required for optimal enzymatic activity.

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO.

Upregulation of pentraxin-3 in human endothelial cells after lysophosphatidic acid exposure.

OBJECTIVE: The earliest event in atherogenesis appears to be endothelium dysfunction. Lysophosphatidic acid (LPA), one of the major bioactive lipid components of oxidized low-density lipoproteins (oxLDL), can cause the activation of endothelial cells (ECs), which start to secrete multiple proinflammatory polypeptides/proteins. The purpose of this study was to better document the proatherogenic properties of LPA using a subproteomic approach focused on the secretome of LPA-treated ECs. METHODS AND RESULTS: The secretome of LPA-treated ECs was analyzed using the 2D-DIGE approach. Among the 20 spots displaying significant variations of abundance compared with the control cells, we identified pentraxin-3 by mass spectrometry. Pentraxin-3 upregulation was confirmed at the mRNA and protein level, both on immortalized and primary ECs. LPA- but also oxLDL-induced pentraxin-3 upregulation was reduced in the presence of an antagonist of the LPA-receptors and largely dependent on NFkappaB activation. Finally, we demonstrated, for the first time, the chemotactic activity of pentraxin-3 on human THP-1 monocytes by using a chemotaxis assay. CONCLUSIONS: Our findings favor the proatherogenic role of LPA, a bioactive lipid produced by activated platelets and present in oxLDL, because it enhances pentraxin-3 secretion that could contribute to the accumulation of monocytes in the atherosclerotic lesion.

Celecoxib exerts protective effects in the vascular endothelium via COX-2-independent activation of AMPK-CREB-Nrf2 signalling.

Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000 ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1ß-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs.

Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis.

Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1(-/-) mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.

PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis.

AIMS: Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε. METHODS AND RESULTS: Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1((Ser133)) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. CONCLUSIONS: PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury.

The multifunctional role and therapeutic potential of HO-1 in the vascular endothelium.

SIGNIFICANCE: Heme oxygenases (HO-1 and HO-2) catalyze the degradation of the pro-oxidant heme into carbon monoxide (CO), iron, and biliverdin, which is subsequently converted to bilirubin. In the vasculature, particular interest has focused on antioxidant and anti-inflammatory properties of the inducible HO-1 isoform in the vascular endothelium. This review will present evidence that illustrates the potential therapeutic significance of HO-1 and its products, with special emphasis placed on their beneficial effects on the endothelium in vascular diseases. RECENT ADVANCES: The understanding of the molecular basis for the regulation and functions of HO-1 has led to the identification of a variety of drugs that increase HO-1 activity in the vascular endothelium. Moreover, therapeutic delivery of HO-1 products CO, biliverdin, and bilirubin has been shown to have favorable effects, notably on endothelial cells and in animal models of vascular disease. CRITICAL ISSUES: To date, mechanistic data identifying the downstream target genes utilized by HO-1 and its products to exert their actions remain relatively sparse. Likewise, studies in man to investigate the efficacy of therapeutics known to induce HO-1 or the consequences of the tissue-specific delivery of CO or biliverdin/bilirubin are rarely performed. FUTURE DIRECTIONS: Based on the promising in vivo data from animal models, clinical trials to explore the safety and efficacy of the therapeutic induction of HO-1 and the delivery of its products should now be pursued further, targeting, for example, patients with severe atherosclerotic disease, ischemic limbs, restenosis injury, or at high risk of organ rejection.

Effects of copper sulfate-oxidized or myeloperoxidase-modified LDL on lipid loading and programmed cell death in macrophages under hypoxia.

Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia.

Additive, antagonistic, and synergistic effects of procyanidins and polyunsaturated fatty acids over inflammation in RAW 264.7 macrophages activated by lipopolysaccharide.

OBJECTIVE: Macrophages play an important role in immunogenic challenges and can aggravate and propagate local inflammation. Nuclear factor-kappa B (NF-κB) and activator protein 1 pathways can regulate these inflammatory processes by modulating expression of proinflammatory genes. Bioactive molecules present in food, such as procyanidins and polyunsaturated fatty acids, possess antiinflammatory effects in vivo and in vitro. Our aim was to assess whether they have synergistic antiinflammatory effects in murine macrophages. METHODS: A nitric oxide production assay, a phosphoprotein assay, and a low-density array for 91-gene expression related to inflammation, oxidative stress, and metabolism were performed to assess the synergistic antiinflammatory effects of dimeric procyanidins (B1, B2, B3, B4) (5 µg/mL), and the polyunsaturated fatty acids, docosahexaenoic acid, and eicosapentaenoic acid (30 µM) coincubated with lipopolysaccharide for 19 h to mimic inflammation in RAW 264.7 macrophages (mouse leukaemic monocyte macrophage cell line). RESULTS: Adding eicosapentaenoic acid plus B3 had synergistic effects leading to decreased nitric oxide levels; the modulation of phosphoprotein levels, such as P-nuclear factor-[kappa] B p65 and P-stress-activated protein kinase/Jun-amino-terminal kinase; the down-regulation of proinflammatory genes, such as interleukins, chemokines, transcription factors; and up-regulation of antioxidant genes. CONCLUSION: This combination has a stronger antiinflammatory effect than either of these molecules separately in RAW macrophages. These results could lead to in vivo studies that may yield novel preventive or palliative nutritional treatments for obesity, atherosclerosis, and cardiovascular diseases.

The peroxynitrite donor 3-morpholinosydnonimine activates Nrf2 and the UPR leading to a cytoprotective response in endothelial cells.

Endothelial dysfunction is associated with the formation of peroxynitrite, described to be toxic. Recent data also suggests that peroxynitrite is able to activate the protective Nrf2 pathway and/or the unfolded protein response (UPR). The aim of our work was to study the response of human endothelial cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, and to highlight the possible protective roles of Nrf2 or the UPR pathway in this response. Immortal and primary human umbilical vein endothelial cells were exposed to SIN-1. SIN-1 incubation led to Nrf2 activation and to the overexpression of Nrf2-regulated genes, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1. We also demonstrated that this defensive response protected cells against cell death induced by serum starvation, by reducing apoptosis (monitored by caspase-3 activity and DNA fragmentation) and favoring autophagosome formation, as evidenced by LC3-II accumulation. Interestingly, we observed an activation of the UPR, with a rapid and significant overexpression of CHOP in serum starved cells stimulated with SIN-1. While siRNA mediated knockdown of CHOP had no effect on DNA fragmentation, the invalidation of Nrf2 or HO-1 by siRNA strongly increased DNA fragmentation, but also reinforced the SIN-1-induced LC3-II accumulation. This study shows that peroxynitrite, at least at sublethal concentrations and within a narrow concentration range, could exert protective effects on endothelial cells by modulating the balance between autophagy and apoptosis, through Nrf2-dependent pathways.

Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes.

Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect PI3K binding to its main target, the epidermal growth factor receptor, nor its ability to convert phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate but impaired Akt phosphorylation at the regulatory sites Thr(308) and Ser(473). Diminished Akt activity resulted in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid rafts is required for the activity of Akt and cell survival and may serve as a potential pharmacological target in the treatment of epidermal cancers.

Copper and myeloperoxidase-modified LDLs activate Nrf2 through different pathways of ROS production in macrophages.

Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROS-producing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggest that results obtained until now with copper oxidized-LDLs should be carefully reevaluated, taking into consideration physiologically more relevant oxidized LDLs.

TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: role of the PI3K-Akt pathway.

Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and FoxO3a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher proportion of UVB-treated HaCaT cells containing unrepaired cyclobutane pyrimidine dimers (CPDs) escaped the G2/M cell cycle checkpoint in the presence of TNF-alpha (9.5+/-3.3 vs 4.8+/-2.2%). After treatment with the PI3K inhibitor LY294002, only 1.2+/-0.7% of CPD-containing HaCaT cells were actively cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter the cell cycle. The effect of TNF-alpha seems to be dependent on Akt activation and may constitute a relevant mechanism enhancing mutagenesis and tumor development.