Mitogen-activated protein kinases (MAPKs) are key signaling molecules that respond to mitogenic stimulation or environmental stress, resulting in the expression of target proteins. c-Jun N-terminal kinase (JNK) and p38 MAPKs are activated by inflammatory cytokines or environmental stress. Specific p38 MAPK inhibitors, such as SB202190 or SB203580, are widely used to dissect p38 MAPK-related signal transduction mechanisms. While using SB202190 to inhibit p38 MAPK-related signaling, we observed that SB202190 treatment could activate JNK. Further experiments showed that treatment of cells with SB202190 could phosphorylate JNK and activating transcription factor 2 (ATF-2), and increased AP-1 DNA binding. Using multiple cell lines and primary endothelial cells, we demonstrated that specific p38 MAPK inhibitors SB202190 or SB203580 induces the activation of the JNK pathway. Further, using with RNA interference and kinase-inactive expression of intermediates of the JNK pathway, we demonstrated SB202190- or SB203580-induced JNK activation is dependent on the MLK-3-MKK4/MKK7-dependent signal transduction pathway. Finally, we demonstrate that treatment of cells with SB202190 or SB203580 induces the phosphorylation and activation of MLK3.

Post-transcriptional regulation represents a major mechanism by which eukaryotic gene expression is regulated through cis-trans interactions that serve as signals for rapid alterations of messenger RNA (mRNA) stability. Regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with a 50 kD uPAR mRNA binding protein. We purified this protein from human bronchial epithelial (Beas2B) cells and identified it as phosphoglycerate kinase (PGK). We cloned PGK cDNA by polymerase chain reaction and expressed the recombinant PGK protein, which specifically bound the uPAR mRNA coding region by gel mobility shift and Northwestern blotting. We also confirmed a direct interaction of PGK protein with uPAR mRNA by immunoprecipitation. Overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells resulted in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression. Reduced uPAR mRNA expression involved decreased stability of the uPAR mRNA. Decline in 3H-thymidine incorporation and migration occurred in H157 cells transfected with PGK cDNA. These results demonstrate that PGK regulates uPAR expression at the post-transcriptional level.

We hypothesized that lectin-like oxidized LDL receptor-1 (LOX-1) deletion may inhibit oxidative stress signals, reduce collagen accumulation and attenuate cardiac remodeling after chronic ischemia. Activation of LOX-1 plays a significant role in the development of inflammation, apoptosis and collagen signals during acute ischemia. Wild-type and LOX-1 knockout (KO) mice were subjected to occlusion of left coronary artery for 3 weeks. Markers of cardiac hypertrophy, fibrosis-related signals (collagen IV, collagen-1 and fibronectin) and oxidant load (nicotinamide adenine dinucleotide phosphate oxidase expression, activity of mitogen-activated protein kinases and left ventricular (LV) tissue thiobarbituric acid reactive substances) were analyzed. In in vitro experiments, HL-1 cardiomyocytes were transfected with angiotensin II (Ang II) type 1 receptor (AT1R) or type 2 receptor (AT2R) genes to determine their role in the cardiomyocyte hypertrophy. LOX-1 KO mice had 25% improvement in survival over the 3-week period of chronic ischemia. LOX-1 deletion reduced collagen deposition and cardiomyocyte hypertrophy (~75%) in association with a decrease in oxidant load and AT1R upregulation (all P<0.05). The LOX-1 KO mice hearts exhibited a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 17 (ADAM17) expression and matrix metalloproteinase 2 activity, and increased AT2R expression (P<0.05). Attenuation of cardiac remodeling was associated with improved cardiac hemodynamics (LV ±dp/dt and cardiac ejection fraction). In vitro studies showed that it is AT1R, and not AT2R overexpression that induces cardiomyocyte hypertrophy. We demonstrate for the first time that LOX-1 deletion reduces oxidative stress and related intracellular signaling, which leads to attenuation of the positive feedback loop involving AT1R and LOX-1. This results in reduced chronic cardiac remodeling.

Apoptosis is a major mechanism of cancer cell destruction by chemotherapy and radiotherapy. The anthracycline class of antitumor drugs undergoes redox cycling in living cells producing increased amounts of reactive oxygen species and semiquinone radical, both of which can cause DNA damage, and consequently trigger apoptotic death of cancer cells. We show here that MCF-7 cells overexpressing thioredoxin (Trx) were more apoptotic in response to daunomycin. Thioredoxin (Trx) 2 is a low molecular mass protein (12 kDa) that is widely distributed; Trx is found within the cytoplasmic, membrane, extracellular, and mitochondrial cellular fractions (1, 2). The Trx system includes Trx, Trx reductase, and peroxiredoxins. Trx reductase is an efficient protein-disulfide reductase that uses NADPH as a source of reducing equivalents. Besides being an antioxidant itself (3, 4), Trx also plays an important role in regulating the expression of other antioxidant genes such as manganese superoxide dismutase (5). Trx overexpression also enhances the expression of peroxiredoxin that could reduce peroxides to molecular oxygen and H 2 O (6). Trx has been shown to regenerate oxidatively inactivated proteins (7,8). In addition to its role as an antioxidant protein, Trx has been shown to have growth promoting properties (9). In contrast, a recent study has demonstrated that overexpression of redox-active Trx could promote cell death via activation of caspase-8 (10). Additional studies have shown that Trx reductase is critical for cell death, and a Trx-dependent mechanism has been suggested (11). Recent studies also indicate that caspases, the executioner of cell death by apoptosis, could be activated by Trx due to its disulfide reducing properties (12). Caspases are rich in cysteine motifs that are required for their catalytic activity. Therefore, oxidation could inhibit caspase activity, which could be restored by the Trx system (12). Furthermore Trx also has been shown to promote p53-DNA binding due to its reducing actions on DNA-binding cysteine motifs on p53 (14). Taken together, accumulating evidence suggests that Trx is a multifunctional protein, which can participate in proliferation as well as cell death process. The antioxidative action of Trx could be due to its manganese superoxide dismutase inducing properties (5, 15) as well as direct scavenging of hydroxyl radicals or singlet oxygen.The anthracycline class of anticancer drugs such as doxorubicin or daunomycin has been shown to induce p53-dependent apoptosis in cancer cells (16,17). Additionally anthracyclines have also been shown to cause DNA damage, which increases p53 expression (18,19). p53 is a sequence-specific transcription factor, which can induce proapoptotic or suppress antiapoptotic genes in response to DNA damage or irreparable cell cycle arrest (20). Phosphorylation of p53 on the Ser 15 residue dissociates MDM2 and activates p53 as a transcription factor, which binds to various p53-dependent genes resulting in their activation or repression (20). While evaluating ...