Marie-Alix Conte

Oxidized LDL activates STAT1 and STAT3 transcription factors: possible involvement of reactive oxygen species

The effect of cupric ion‐oxidized low density lipoprotein (Cu‐LDL) or endothelial cell‐oxidized LDL (E‐LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu‐LDL exhibited a more marked effect than E‐LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid‐reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol‐containing compound N‐acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu‐LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.

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.

Lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells

The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle. LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells. At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively. The LDL degradation by J774 macrophage-like cells was concomitantly stimulated. Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells. In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS. This effect was accompanied by a parallel increase in superoxide anion release by the cells. These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.