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4-Hydroxynonenal, a significant aldehyde end product of membrane lipid peroxidation with numerous biochemical activities, has consistently been detected in various human diseases. Concentrations actually detectable in vivo (0.1-5 microM) have been shown to up-regulate different genes and modulate various enzyme activities. In connection with the latter aspect, we show here that, in isolated rat hepatocytes, 1 microM 4-hydroxynonenal selectively activates protein kinase C-delta, involved in apoptosis of many cell types; it also induces very early activation of Jun N-terminal kinase, in parallel increasing activator protein-1 DNA-binding activity in a time-dependent manner and triggering apoptosis after only 120 min treatment. These phenomena are likely protein kinase C-delta-dependent, being significantly reduced or annulled by cell co-treatment with rottlerin, a selective inhibitor of protein kinase C-delta. We suggest that 4-hydroxynonenal may induce apoptosis through activation of protein kinase C-delta and of Jun N-terminal kinase, and consequent up-regulation of activator protein-1 DNA binding.

Accumulation of advanced glycation end products (AGEs) induces alterations in the intracellular redox balance, leading cells to functional injury. Current literature reports that intracellular signaling triggered by the interaction of AGEs with their specific receptors RAGEs depends on the cell type and the state of activation/stress. In this work, NT2 human neurons were exposed for 48 h to glycated fetal serum containing 750-3000 pmol/ml pentosidine; the treatment induced an increase in apoptosis rate linear with AGE concentration up to 1500 pmol/ml, but necrotic death was elicited with the highest AGE amount employed (3000 pmol/ml pentosidine). Pentosidine at 1500 pmol/ml, which was the concentration responsible for the highest apoptotic effect (40% of apoptotic neurons), was able to determine early generation of intracellular reactive oxygen species and increase in RAGE levels. Under these conditions, protein kinase C (PKC) delta activity was increased approximately 2-fold, and DNA binding activity of redox-sensitive transcription factor activator protein-1 (AP-1) was enhanced 2.5-fold. A relationship among oxidative stress, PKCdelta activity, AP-1 activation, and apoptosis was demonstrated by pretreating neurons with 500 muM vitamin E, with 20 mug/ml Ginkgo biloba extract, or with 3 muM Rottlerin, inhibitor of PKCdelta; these pretreatments were able to protect neurons from the glycoxidation-dependent effects.

Current evidence demonstrates that protein kinase C (PKC) belongs to a group of cell-signaling molecules that are sensitive targets for redox modifications and functional alterations that mediate oxidant-induced cellular responses. Our studies have demonstrated that diminished intracellular GSH was associated to inactivation of classic isoforms and increased activity of novel PKCs, and triggered molecular signals important for cell survival. Loss of GSH and oxidative damage are probably an early signaling event in apoptotic death, which is characterized by the activation of PKC-delta. Apoptotic process consequent to GSH depletion was inhibited by rottlerin, a PKC-delta-specific inhibitor, which exerted a negative effect on oxyradical production. Therefore, it may be concluded that PKC-delta activity is related to reactive oxygen species production and is involved in the pathway leading to apoptosis and growth arrest.

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.