Jean-Claude Mazière

Activation of transcription factors and gene expression by oxidized low-density lipoprotein

It is well recognized that oxidized LDL (OxLDL) plays a crucial role in the initiation and progression of atherosclerosis. Many biological effects of OxLDL are mediated through signaling pathways, especially via the activation of transcription factors, which in turn stimulate the expression of genes involved in the inflammatory and oxidative stress response or in cell cycle regulation. In this review, we will discuss the various transcription factors activated by OxLDL, the studied cell types, the active compounds of the OxLDL particle, and the downstream genes when identified. Identification of the transcription factors and some of the downstream genes regulated by OxLDL has helped us understand the molecular mechanism involved in generation of the atherosclerotic plaque.

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.

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.

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.

Anti‐ and pro‐oxidant effects of urate in copper‐induced low‐density lipoprotein oxidation

We recently reported that, depending on its concentration, urate is either a pro- or an antioxidant in Cu(2+)-induced low-density lipoprotein (LDL) oxidation. We also previously demonstrated an antioxidant synergy between urate and some flavonoids in the Cu(2+)-induced oxidation of diluted serum. As a result, the effect of the flavonoid quercetin on the Cu(2+)-induced oxidation of isolated LDL has been studied either in the presence or absence of urate. We demonstrate that, like urate, quercetin alone, at low concentration, exhibits a pro-oxidant activity. The pro-oxidant behavior depends on the Cu(2+) concentration but it is not observed at high Cu(2+) concentration. When compared with urate, the switch between the pro- and the antioxidant activities occurs at much lower quercetin concentrations. As for urate, the pro-oxidant character of quercetin is related to its ability to reduce Cu(2+) with the formation of semioxidized quercetin and Cu(+) with an expected yield larger than that obtained with urate owing to a more favorable redox potential. It is also shown that the pro-oxidant activity of urate can be inhibited by quercetin. An electron transfer between quercetin and semioxidized urate leading to the repair of urate could account for this observation as suggested by recently published pulse radiolysis data. It is anticipated that the interactions between quercetin-Cu(2+)-LDL and urate, which are tightly controlled by their respective concentration, determine the balance between the pro- and antioxidant behaviors. Moreover, as already observed with other antioxidants, it is demonstrated that quercetin alone behaves as a pro-oxidant towards preoxidized LDL.

Biphasic effect of UVA radiation on STAT1 activity and tyrosine phosphorylation in cultured human keratinocytes.

The effect of ultraviolet A (UVA) radiation on the DNA-binding activity of the transcription factor STAT1 was studied by electromobility shift assay in the human keratinocyte cell line NCTC 2544. The STAT1-binding activity exhibited a biphasic pattern as a function of UVA doses. For UVA doses lower than 0.6 J/cm(2), a dose-dependent increase in STAT1 activity was observed. In a second phase, with higher UVA doses (1.5 to 9 J/cm(2)), the activity decreased and reached control value at 6 J/cm2. The enhancement of STAT1 activity was transient, peaked at 1 h after UV irradiation, and regularly decreased to control value 24 h after UV. Genistein, a tyrosine kinase inhibitor, H7, a serine/threonine kinase inhibitor, and PD 98059, a MEK inhibitor, prevented the UVA-induced enhancement of STAT1-binding activity, suggesting the involvement of Tyr, Ser/Thr kinases, and MEK in the observed effect. Immunoblot analysis directly demonstrated that the amount of Tyr-phosphorylated STAT1 was parallel to its DNA-binding activity. Immunoblot analysis also demonstrated the nuclear transport of STAT1 after UVA irradiation at low doses. At high doses, a decrease in the STAT1 level was observed both in the cytoplasmic and the nuclear compartments, suggesting that the inactivation was due to a degradation process. UVA irradiation initiated a dose-dependent increase in lipid peroxidation products and reactive oxygen species. Furthermore, the involvement of the oxidative stress in the UVA-induced effect on STAT1 activity is suggested by the protective action of the antioxidants alpha-tocopherol and N-acetylcysteine on both the activation phase (UVA doses lower than 1.5 J/cm(2)) and the inhibitory phase. By contrast, the pro-oxidant drug buthionine sulfoximine enhanced the effect of UVA on STAT1-binding activity. Since STATs are known as transducers of cytokine action, the enhancement of STAT1 activity by low doses of UVA might be related to the proinflammatory effect of solar radiations at the skin level.

Impairment of the EGF signaling pathway by the oxidative stress generated with UVA.

Ultraviolet A (UVA) is a component of sunlight reaching the surface of the earth and involved in photodegenerescence and photocarcinogenesis. The effect of UVA was investigated on the EGF-induced activation of the signaling kinase ERK and the transcription factors AP1, NFkappaB, and STAT1. UVA prevented the Epidermal Growth Factor (EGF)-induced stimulation of ERK in a dose-dependent manner within the range of 1.5-9 J/cm(2). Concomitantly, the DNA binding activity of AP1, NFkappaB, and STAT1 under EGF were markedly inhibited by UVA within the same dose range. UVA by itself induced an activation of ERK activity, and a stimulation of AP1, NFkappaB, and STAT1 binding activity. UVA decreased EGF binding in a dose-dependent manner. Furthermore, the highest dose of UVA (9 J/cm(2)) prevented the EGF-induced Tyr-phosphorylation of the EGF-receptor (EGF-R). The generation of reactive oxygen species (ROS), as assessed by the fluorescent probe dichloro-fluorescein, showed an additive effect of EGF and UVA, within the studied range of UVA doses. Finally, the antioxidant Vitamin E prevented the inhibitory effect of UVA on ERK, AP1, NFkappaB, and STAT1. These results demonstrate that an overproduction of ROS, initiated by two different and successive triggering agents such as UVA and EGF, leads to inactivation of the EGF signaling pathway. This inhibition of gene expression control by EGF might play a role in the photodegenerative processes observed after exposition of skin cells to solar radiation.

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.

Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts.

Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1–0.3 U/ml SMase induced a dose‐dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose‐dependent Tyr‐phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose‐dependent manner. Concomitantly, SMase enhanced the Tyr‐phosphorylation of Janus kinase (JAK) 2, a Tyr‐kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr‐kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr‐phosphorylation, together with STAT1 and STAT3 Tyr‐phosphorylation and binding activity. The SMase‐induced increase in STAT1/3 binding activity was prevented by methyl‐β‐cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER‐activated intracellular kinases.

Oxidized low density lipoprotein enhanced RANKL expression in human osteoblast-like cells. Involvement of ERK, NFkappaB and NFAT

BACKGROUNDnReceptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand/osteoprotegerin ratio is of crucial importance in osteoclast differentiation and thus in bone dysregulation diseases.nnnMETHODSnReceptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand and osteoprotegerin were determined under oxidized low density lipoprotein treatment of human osteoblast-like cells. The involvement of oxidative stress, of the extracellular signal regulated kinase and of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and nuclear factor of activated T cells was demonstrated.nnnRESULTSnCu(2+)-oxidized low density lipoprotein increased cell-associated and extracellular receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand levels whereas osteoprotegerin levels were not affected. The increase in receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand was parallel to the generation of reactive oxygen species provoked by Cu(2+)-oxidized low density lipoprotein. The lipid extract of Cu(2+)-oxidized low density lipoprotein, together with other forms of oxidized low density lipoproteins such as smooth muscle cell-oxidized low density lipoprotein and myeloperoxidase-oxidized low density lipoprotein, also induced an increase in reactive oxygen species and cell-associated receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand. The effect of Cu(2+)-oxidized low density lipoprotein was prevented by the antioxidant vitamin E, and mimicked by the prooxidant compounds hydrogen peroxide and buthionine sulfoximine. Inhibitors of mitogen activated protein kinase/extracellular signal regulated kinase (PD 98059), nuclear factor kappa-light-chain-enhancer of activated B cells (Ro 106-9920) and nuclear factor of activated T cells (Vivit) reduced the effect of Cu(2+)-oxidized low density lipoprotein on receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cell ligand expression. Cu(2+)-oxidized low density lipoprotein signaling was also reduced by vitamin E.nnnGENERAL SIGNIFICANCEnThis work describes a new molecular mechanism and elucidates the signaling pathway whereby oxidized low density lipoprotein, by means of its lipid moiety, can modulate the crosstalk between osteoblasts/osteoclasts and bone remodeling, leading to an eventual risk of osteoporosis.