SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma.

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined in vitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

Metallothionein-1 as a biomarker of altered redox metabolism in hepatocellular carcinoma cells exposed to sorafenib.

BACKGROUND: Sorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking. METHODS: We used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used. RESULTS: We observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival. CONCLUSION: These findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells.

[Ferroptosis, a new form of cell death relevant to the medical treatment of cancer]. / La ferroptose, une nouvelle forme de mort cellulaire applicable au traitement médical des cancers.

Ferroptosis is a form of cell death that has recently been reported during exposure to erastin, a chemical compound identified in a screen for molecules able to kill cancer cells carrying an active Ras oncogene. In cells exposed to inducers of ferroptosis, a catastrophic alteration of the cellular redox metabolism occurs, resulting in massive lipid peroxidation in the plasma membrane and loss of cell viability. We present our recent observations suggesting that sorafenib, the only medical treatment with proven efficacy against hepatocellular carcinoma, induces ferroptosis, a new anti-oncogenic mode of action of this drug. The discovery of ferroptosis sheds light on the critical adaptations of the redox metabolism in cancer cells. It might also foster the discovery of new biomarkers and innovative approaches for the treatment of cancer.

Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib.

The multikinase inhibitor sorafenib is currently the treatment of reference for advanced hepatocellular carcinoma (HCC). In our report, we examined the cytotoxic effects of sorafenib on HCC cells. We report that the depletion of the intracellular iron stores achieved by using the iron chelator deferoxamine (DFX) strikingly protects HCC cells from the cytotoxic effects of sorafenib. The protective effect of the depletion of intracellular iron stores could not be explained by an interference with conventional forms of programmed cell death, such as apoptosis or autophagic cell death. We also found that DFX did not prevent sorafenib from reaching its intracellular target kinases. Instead, the depletion of intracellular iron stores prevented sorafenib from inducing oxidative stress in HCC cells. We examined the possibility that sorafenib might exert a cytotoxic effect that resembles ferroptosis, a form of cell death in which iron-dependent oxidative mechanisms play a pivotal role. In agreement with this possibility, we found that pharmacological inhibitors (ferrostatin-1) and genetic procedures (RNA interference against IREB-2) previously reported to modulate ferroptosis, readily block the cytotoxic effects of sorafenib in HCC cells. Collectively, our findings identify ferroptosis as an effective mechanism for the induction of cell death in HCC. Ferroptosis could potentially become a goal for the medical treatment of HCC, thus opening new avenues for the optimization of the use of sorafenib in these tumors.

The Tumor Coagulome as a Transcriptional Target and a Potential Effector of Glucocorticoids in Human Cancers.

BACKGROUND: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. METHODS: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. RESULTS: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-ß response. CONCLUSION: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME.

EGFR activation is a potential determinant of primary resistance of hepatocellular carcinoma cells to sorafenib.

Sorafenib is currently the medical treatment of reference for hepatocellular carcinoma (HCC), but it is not known whether sorafenib is equally active in all HCC. Here, our aim was to explore intrinsic differences in the response of HCC cells to sorafenib, to identify potential mechanisms leading to primary resistance to this treatment. We analyzed a panel of six human HCC cell lines and compared the activity of the main oncogenic kinase cascades, their clonogenic potential, proliferation and apoptosis upon exposure to sorafenib. We report that HCC cells present important differences in their response to sorafenib, and that some cell lines are more resistant to the actions of sorafenib than others. We identify the activated epidermal growth factor receptor (EGFR) as a parameter that promotes the resistance of HCC cells to sorafenib. In resistant cells, the efficacy of sorafenib was increased when EGFR was inhibited, as was demonstrated using two chemical inhibitors (erlotinib or gefitinib), a monoclonal antibody directed against EGFR (cetuximab), and RNA interference directed against EGFR. A combination of EGFR inhibitors and sorafenib affords a better control over HCC proliferation, most likely through an improved blockade of the RAF kinases. Our findings therefore confirm the importance of RAF kinases as therapeutic targets in HCC, and identify EGFR as a determinant of the sensitivity of HCC cells to sorafenib. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC, and might be useful for the identification of predictive biomarkers in this context.

Upregulation of BAD, a pro-apoptotic protein of the BCL2 family, in vascular smooth muscle cells exposed to uremic conditions.

Chronic kidney disease (CKD) has recently emerged as a major risk factor for cardiovascular pathology. CKD patients display accelerated atherosclerotic process, leading to circulatory complications. However, it is currently not clear how uremic conditions accelerate atherosclerosis. Apoptosis is an important homeostatic regulator of vascular smooth cells under pathological conditions. In the present study, we explored the regulation of apoptosis in cells of the vascular wall in the uremic context. We analysed the expression and regulation of the proteins of the BCL2 family that play an essential role in apoptosis. Our results, obtained in mice and primary human smooth muscle cells exposed to two uremic toxins, point to the existence of an alteration in expression and function of one pro-apoptotic member of this family, the protein BAD. We explore the regulation of BAD by uremic toxins and report the sensitization of vascular smooth muscle cells to apoptosis upon BAD induction.

The Bcl-2 homology domain 3 (BH3) mimetic ABT-737 reveals the dynamic regulation of bad, a proapoptotic protein of the Bcl-2 family, by Bcl-xL.

The proteins of the B-cell lymphoma 2 (Bcl-2) family are important regulators of apoptosis under normal and pathological conditions. Chemical compounds that block the antiapoptotic proteins of this family have been introduced, such as 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide (ABT-737), a BH3-mimetic that neutralizes Bcl-2 and Bcl-xL. In this study, we used ABT-737 to explore the dynamic regulation of Bcl-2 proteins in living cells of different origins. Using ABT-737 as well as RNA interference or the application of growth factors, we examined the impact of the functional availability of the antiapoptotic proteins Bcl-2 and Bcl-2-extra large (Bcl-xL) on the Bcl-2 network. We report that ABT-737 increases the expression of Bcl-2-associated death promoter (Bad), a proapoptotic partner of the proteins Bcl-2 and Bcl-xL. Our observations indicate that Bad overexpression induced by ABT-737 results from the control of its normally rapid protein turnover, leading to the stabilization of this protein. We demonstrate the relevance of Bad post-translational regulation by Bcl-xL to the physiological setting using RNA interference against Bcl-xL as well as the application of epidermal growth factor, a growth factor that promotes the dissociation of Bad from Bcl-xL. Our results highlight a new facet of the mode of action of the antiapoptotic proteins Bcl-2 and Bcl-xL consisting of the regulation of the stability of the protein Bad. Finally, our results shed light on the mode of action of ABT-737, currently the best characterized inhibitor of the antiapoptotic proteins of the Bcl-2 family, and bear important implications regarding its use as an anticancer drug.

Analysis of Mesencephalic Astrocyte-derived Neurotrophic Factor in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM) is characterized by high production of immunoglobulins resulting in a constant source of endoplasmic reticulum (ER)-stress. Mesencephalic astrocyte-derived neurotrophic factor (MANF) was identified as a possible circulating biomarker that could help in monitoring ER-stress mediated diseases. MATERIALS AND METHODS: To assess the relevance of MANF in MM, we performed in silico and in vitro analysis in malignant cell lines including the myeloma cell line RPMI 8226. Serum MANF concentration was compared between healthy subjects (n=60), patients with MM (n=68), or those with monoclonal gammopathy of undetermined significance (MGUS) (n=73). RESULTS: MANF mRNA expression was upregulated in the RPMI 8226 cell line, and higher secretion of MANF was measured in RPMI 8226 supernatant. Serum MANF levels were not significantly different between MM or MGUS patients and those in age- and sex-matched healthy controls. CONCLUSION: MANF was not validated as a biomarker of interest in MM patients. Its potential implication in myeloma pathogenesis should be investigated.

DNA damage response- and JAK-dependent regulation of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) cells exposed to 5-fluorouracil (5-FU).

OBJECTIVES: The immune checkpoint molecule PD-L1 (CD274) is a crucial regulator of the tumor immune response. Its expression has been reported in the therapeutic context in Head and Neck Squamous Cell Carcinoma (HNSCC), but it remains unclear how therapeutically approved molecules regulate PD-L1 expression in HNSCC cells. MATERIALS AND METHODS: Three HNSCC cell lines (BICR6, PE/CA-PJ34 and PE/CA-PJ41) were used to analyze PD-L1 expression by immunoblotting, immunofluorescence and QPCR. Freely-available single cell RNAseq data from HNSCC were also used. RESULTS: 5-Fluorouracil (5-FU) increased the expression of PD-L1 with high efficacy in HNSCC cells. Single cell RNAseq data suggested the specificity of the regulation of PD-L1 in this context. The effect of 5-FU on PD-L1 expression was related to its genotoxic effect and was prevented by extracellular application of thymidine or using a chemical inhibitor of the DNA damage Response kinases ATM/ATR. We found that the effect of 5-FU was additive or synergistic with IFN-γ, the canonical inducer of PD-L1 in epithelial cells. QPCR analysis confirmed this finding and identified JAK-dependent transcriptional activation of PD-L1/CD274 as the underlying mechanism. The induction of PD-L1 by 5-FU was partially prevented by Epidermal Growth Factor Receptor (EGFR) inhibition with cetuximab. CONCLUSION: Our study highlights the specific DNA Damage Response- and JAK- dependent induction of PD-L1 by 5-FU in HNSCC cells. This induction is regulated by the cytokine context and is potentially therapeutically actionable.

Early decrease in serum amphiregulin or vascular endothelial growth factor levels predicts sorafenib efficacy in hepatocellular carcinoma.

Sorafenib is the standard of care for the treatment of advanced hepatocellular carcinoma (HCC). However, identifying secreted biomarkers that predict sorafenib efficacy in all HCC patients remains challenging. It was recently reported that sorafenib interferes with protein homeostasis and inhibits global translation in tumour cells. A likely consequence of this inhibition would be the interruption of autocrine loops. The aim of the present study was to investigate the effect of sorafenib on two growth factors implicated in autocrine loops and HCC tumour invasion: amphiregulin (AREG) and vascular endothelial growth factor (VEGF). ELISA, quantitative polymerase chain reaction analysis, western blotting and a cytokine array were performed on HCC cell lines and the prognostic role of these two biomarkers in HCC patients was evaluated. Serum AREG and VEGF levels were assayed by ELISA in 55 patients with advanced HCC treated with sorafenib. It was observed that sorafenib decreased AREG, VEGF and cytokine expression at the transcriptional and post­transcriptional levels. All HCC patients in our cohort had detectable concentrations of AREG and VEGF both at baseline and after sorafenib treatment. The decreased serum levels of AREG and VEGF after 15 days of sorafenib treatment were significantly associated with better overall and progression­free survival. The results of the multivariate analysis demonstrated that a decrease in AREG was an independent prognostic indicator of overall survival (hazard ratio, 0.208; 95% confidence interval, 0.173­0.673; P=0.0003). These results suggest that sorafenib inhibits auto-crine loops and that early decrease in serum AREG or VEGF levels predicts sorafenib efficacy in HCC patients.

ERK1/2 signaling regulates the immune microenvironment and macrophage recruitment in glioblastoma.

The tumor microenvironment is an important determinant of glioblastoma (GBM) progression and response to treatment. How oncogenic signaling in GBM cells modulates the composition of the tumor microenvironment and its activation is unclear. We aimed to explore the potential local immunoregulatory function of ERK1/2 signaling in GBM. Using proteomic and transcriptomic data (RNA seq) available for GBM tumors from The Cancer Genome Atlas (TCGA), we show that GBM with high levels of phosphorylated ERK1/2 have increased infiltration of tumor-associated macrophages (TAM) with a non-inflammatory M2 polarization. Using three human GBM cell lines in culture, we confirmed the existence of ERK1/2-dependent regulation of the production of the macrophage chemoattractant CCL2/MCP1. In contrast with this positive regulation of TAM recruitment, we found no evidence of a direct effect of ERK1/2 signaling on two other important aspects of TAM regulation by GBM cells: (1) the expression of the immune checkpoint ligands PD-L1 and PD-L2, expressed at high mRNA levels in GBM compared with other solid tumors; (2) the production of the tumor metabolite lactate recently reported to dampen tumor immunity by interacting with the receptor GPR65 present on the surface of TAM. Taken together, our observations suggest that ERK1/2 signaling regulates the recruitment of TAM in the GBM microenvironment. These findings highlight some potentially important particularities of the immune microenvironment in GBM and could provide an explanation for the recent observation that GBM with activated ERK1/2 signaling may respond better to anti-PD1 therapeutics.

A pan-cancer study of the transcriptional regulation of uricogenesis in human tumours: pathological and pharmacological correlates.

Uric acid (UA) is the end product of the catabolism of purines, and its serum levels are commonly increased in cancer patients. We aimed to explore the transcriptional regulation of tumour uricogenesis in human tumours, and relate uricogenesis with tumour pathological and pharmacological findings. Using data from The Cancer Genome Atlas (TCGA), we analysed the expression levels of xanthine dehydrogenase (XDH) and adenine phosphoribosyltransferase (APRT), two key enzymes in UA production and the purine salvage pathway, respectively. We found large differences between tumour types and individual tumours in their expression of XDH and APRT Variations in locus-specific DNA methylation and gene copy number correlated with the expression levels of XDH and APRT in human tumours respectively. We explored the consequences of this differential regulation of uricogenesis. Tumours with high levels of XDH mRNA were characterised by higher expression of several genes encoding pro-inflammatory and immune cytokines, and increased levels of tumour infiltration with immune cells. Finally, we studied cancer drug sensitivity using data from the National Cancer Institute-60 (NCI-60) database. A specific correlation was found between the expression levels of APRT and cell sensitivity to the chemotherapeutic agent 5-fluorouracil (5-FU). Our findings underline the existence of great differences in uricogenesis between different types of human tumours. The study of uricogenesis offers promising perspectives for the identification of clinically relevant molecular biomarkers and for tumour stratification in the therapeutic context.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.

Mathematical modelling unveils the essential role of cellular phosphatases in the inhibition of RAF-MEK-ERK signalling by sorafenib in hepatocellular carcinoma cells.

The RAS-RAF-MEK-ERK cascade is a key oncogenic signal transduction pathway activated in many types of tumours in humans. Sorafenib, the medical treatment of reference against advanced stages of hepatocellular carcinoma (HCC), inhibits the RAF-MEK-ERK cascade in HCC cells. Based on previous studies suggesting that this cascade is an important target of sorafenib in HCC cells, we explored its regulation using mathematical modelling and ordinary differential equations. We analysed the dynamic regulation of the core components of the RAF-MEK-ERK cascade in three human HCC cell lines (Huh7, Hep3B and PLC/PRF5) with heterogeneous responses to sorafenib. In silico predictions derived from our mathematical model suggested that the disappearance of phosphorylated MEK and ERK proteins catalysed by cellular phosphatases is an essential mechanism underlying the anti-ERK efficacy of sorafenib in HCC cells. This prediction was experimentally validated using specific inhibitors of the phosphatases PP2A (Protein Phosphatase 2A) and DUSP1/6 (Dual-specificity phosphatases 1/6). These findings highlight an unexpected mode of action of sorafenib on the kinome of HCC cells, and open new perspectives regarding the therapeutic targeting of the RAF-MEK-ERK cascade in this context.

Targeting the Unfolded Protein Response as a Potential Therapeutic Strategy in Renal Carcinoma Cells Exposed to Cyclosporine A.

BACKGROUND/AIM: Organ transplant patients treated with the immunosuppressive drug cyclosporine A often present malignant kidney tumors. Cyclosporine A can promote oncogenesis in a cell-intrinsic manner by increasing the production of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: We explored the impact of cyclosporine A and the role of the unfolded protein response (UPR) on three human renal cell carcinoma (RCC) cell lines under normoxic and hypoxic (1% O2) conditions. RESULTS: Cyclosporine A regulated the expression of VEGF at the post-transcriptional level. Cyclosporine A induced the inositol requiring enzyme-1α (IRE1α) arm of the UPR and stabilized neosynthesized proteins in RCC cells. Toyocamycin, an inhibitor of IRE1α, abolished the clonogenic growth of RCC cells and reduced induction of VEGF by cyclosporine A under hypoxia. CONCLUSION: Our findings highlight the impact of cyclosporine A on the proteostasis of RCC cells, and suggest the potential therapeutic interest of targeting the UPR against tumors arising in the context of organ transplantation.

Personalization of the medical treatment of solid tumours using patient-derived tumour explants (Review).

Improving the pre-clinical characterization of therapeutic approaches and developing new biological assays that will enable treatment personalization for individual patients are promising developments in oncology. Here we describe a new approach consisting of culturing human tumour explants. This approach involves the preparation of slices from freshly-obtained, surgically-resected material that can be maintained ex vivo for several days. Recent studies have provided proof of principle that this approach can be easily implemented in order to explore the mode of action of various anticancer drugs and the responses of 'real' tumours at the individual patient level. We present the practical aspects and highlight the versatility of this approach, which allows for the analysis of the susceptibility of any individual tumour to multiple anticancer drugs in parallel. We discuss its potential as a companion assay in the design of optimal clinical trials and as a guide for the prescription of medical treatment. We discuss which future clinical and biological studies are needed to validate the information gathered from cultured tumour explants, and to integrate this information with that gathered from other assays in order to optimize the medical treatment of cancer.

Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting.

Oxidized low-density lipoprotein elicits an intracellular calcium rise and increases the binding activity of the transcription factor NFAT.

Oxidized low-density lipoprotein (OxLDL) plays a key role in the generation and progression of atherosclerosis, which might be considered as an inflammatory disease. The transcription factor NFAT(Nuclear Factor of Activated T cells) plays an important role in the control of cytokine genes involved in the inflammatory response. The effect of copper-oxidized LDL (CuLDL) and monocyte-oxidized LDL (M-LDL) on the DNA-binding activity of NFAT was investigated in the T lymphocyte cell line Jurkat. Both OxLDL increased NFAT-binding activity in a dose-dependent manner within the range of 25-75 microg LDL protein/ml. This effect reached a maximum 1 h after the introduction of OxLDL in the medium. CuLDL and M-LDL both induce an intracellular calcium rise in a dose-dependent manner, with a maximum increase 15 min after the addition of OxLDL. The CuLDL-induced NFAT-binding activity was abolished in the presence of the calcium chelator EGTA or of the intracellular calcium trapping drug BAPTA, further indicating the involvement of calcium ions in the effect of OxLDL. In addition, cyclosporin A and FK 506, two inhibitors of calcineurin, a calcium-dependent phosphatase upstream of NFAT, also prevented the CuLDL-induced NFAT-binding activity, thus demonstrating the role of calcineurin. CuLDL and M-LDL also induced an increase in the intracellular level of reactive oxygen species (ROS), which reached a maximum 30 min after the addition of OxLDL. Finally, a pretreatment of cells with the antioxidant vitamin E blocked the CuLDL-induced increase in reactive oxygen species, in intracellular calcium rise and the CuLDL-induced NFAT-binding activity. The lipid extract of CuLDL, which includes the lipid peroxidation products, reproduced the effect of the CuLDL itself. These results suggest that the effect of OxLDL on NFAT is initiated by an oxidative stress, which then in turn activates the calcium-calcineurin signaling pathway of the transcription factor NFAT. This effect of OxLDL might be involved in the inflammatory process observed in atherosclerotic lesions.

Low UVA doses activate the transcription factor NFAT in human fibroblasts by a calcium-calcineurin pathway.

UVA radiation induces an inflammatory response as observed in erythema, and the cytokine genes involved in this response are under the control of the transcription factor NFAT (nuclear factor of activated T lymphocytes). The effects of UVA on NFAT DNA binding activity were investigated in cultured human fibroblasts. A dose-dependent increase was observed within the range of 0.6-4.5 J/cm2 UVA. Beyond this value, the activity decreased and a value of 60% of control was found at 13.5 J/cm2. The enhancement of NFAT activity was transient and peaked 45 min after irradiation. Furthermore, immunoblot analysis demonstrated a nuclear translocation of NFAT under low UVA doses. Concomitantly, as assessed by the fluorescent probe Fluo3, UVA induced an increase in intracellular free calcium, with a maximum increase found at 9 J/cm2. The UVA-induced activation of NFAT was prevented by the intracellular calcium trapping drug BAPTA, whereas the extracellular calcium chelator EGTA had no significant effect. In addition, the calcineurin inhibitors cyclosporin A and FK506 both prevented the UVA-induced NFAT activation. Furthermore, the antioxidant vitamin E prevented the UVA-induced increase in both intracellular free calcium and NFAT binding activity. Finally, the cytotoxicity of UVA was enhanced in the presence of the inhibitors cyclosporin and FK506, suggesting that the activation of NFAT might play a protective role after the UVA-induced oxidative stress. These results demonstrate that UVA activates the calcium-calcineurin signaling pathway of NFAT activation, that the calcium ions are mainly released from intracellular stores, and that the increase in calcium is, at least partially, due to the oxidative stress generated under UVA. Because NFAT regulates several genes implicated in the inflammatory response, the enhancement of NFAT activity by low UVA doses might be interpreted in view of the proinflammatory action of solar radiation.