Chuan Ji

IκB Kinase, a Molecular Target for Inhibition by 4-Hydroxy-2-nonenal

In unstimulated cells, transcription factor NF-κB is retained in the cytoplasm by interaction with the inhibitory protein, IκBα. Appropriate cellular stimuli inactivate IκBα by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-κB to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-κB in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dose-dependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-κB DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IM-induced AP-1 DNA binding activity. HNE inhibits TPA/IM-induced degradation of IκBα in both H1299 and Jurkat T cells. The accumulation of IκBα parallels the inhibition of its phosphorylation. At doses that inhibit IκBα degradation, HNE inhibits IκB kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-κB activation that acts by preventing IKK activation and subsequent IκBα degradation.

15-Lipoxygenase Metabolism of 2-Arachidonylglycerol GENERATION OF A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α AGONIST

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor α agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor α agonists.

Cell cycle re-entry following chemically-induced cell cycle synchronization leads to elevated p53 and p21 protein levels.

Mimosine (MIM) and aphidicolin (APH) are two agents frequently used in tissue culture-based experiments to achieve cell synchronization at late G1 and S phases. Following MIM or APH treatment of human cancer cell lines, a reversible growth arrest in late G1 and S phases of the cell cycle was correlated with moderate increases in p53 and p21 protein levels. Both p53-dependent and -independent increases in p21 were observed following treatment with either agent. However, a striking increase in p21 protein levels and a continuous elevation in both p53 and p21 protein levels were observed over 48 h after cells re-entered the cell cycle following the chemically-induced synchronization. In addition, the increase in p21 protein levels typically seen following treatment of cells with DNA damaging agents, was enhanced when cells were treated with genotoxic agents following MIM or APH synchronization. These findings suggest that caution should be exercised when interpreting results from experiments using cell synchronization agents, in particular, studies designed to investigate p53- and p21-regulatory pathways.

Modulation of DNA fragmentation factor 40 nuclease activity by poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) influences numerous cellular processes, including DNA repair, transcriptional regulation, and caspase-independent cell death, by utilizing NAD+ to synthesize long chains of poly(ADP-ribose) (PAR) on target proteins, including itself. During the apoptotic response, caspases-3 and -7 cleave PARP-1, thereby inhibiting its activity. Here, we have examined the role of PARP-1 activation and cleavage in the latter stages of apoptosis in response to DNA fragmentation. PARP-1 poly(ADP-ribosyl)ation correlated directly with induction of apoptosis by the lipid peroxidation product, 4-hydroxy-2-nonenal. A significant decrease in PAR accumulation was observed upon caspase or DNA fragmentation factor 40 (DFF40) inhibition. Because DNA fragmentation mediated by DFF40 augmented PARP-1 modification status in apoptotic cells, we hypothesized that PARP-1 alters DFF40 function following PAR accumulation. Indeed, PARP-1, in the presence of NAD+, significantly decreased DFF40 activity on plasmid substrates. Conversely, PARP-1 enhanced the DNase activity of DFF40 in the absence of NAD+. The inhibition of DFF40 activity in the presence of NAD+ was reduced by co-incubation with poly(ADP-ribose) glycohydrolase and a PARP inhibitor. Additionally, caspase-cleaved PARP-1, in the presence of NAD+, did not inhibit DFF40 activity significantly. Our results suggest that PARP-1 poly(ADP-ribosyl)ation is a terminal event in the apoptotic response that occurs in response to DNA fragmentation and directly influences DFF40 activity.