Regulation of H₂O₂-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

Abstract

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H2O2 is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H2O2 treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H2O2 treatment induces a “lag phase” where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H2O2 (∼80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H2O2-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H2O2 even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H2O2. Furthermore, AMPK activators protected against H2O2-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H2O2. Knockdown of PKC-α using antisense oligonucleotides also slightly protected (∼22%) against H2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H2O2-induced necrosis, suggesting that a signaling “program” is important in mediating H2O2-induced necrosis in primary hepatocytes.

Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation

“…H 2 O 2 did activate neither JNK nor caspases and induced necrosis in rat hepatocytes (11). A similar observation was recently made in mouse hepatocytes (53). However, H 2 O 2 -induced necrosis involved JNK activation and was dependent on protein kinase C (53).…”

Activation of the δ-opioid receptor inhibits serum deprivation-induced apoptosis of human liver cells via the activation of PKC and the mitochondrial pathway

“…PKC stimulation inhibits the apoptosis of different types of cells (38)(39)(40). Meanwhile, PKC also participates in the protection against hepatic ischemia reperfusion injury and is involved in the processes of cellular proliferation and apoptosis (19,41). We observed that despite δ-opioid receptor activation, the blockade of the PKC pathway increases the apoptosis of human liver cells.…”

“…Studies have found that PKC is involved in the preconditioning protection of hepatic ischemia (18). The proliferation and apoptosis of normal cells and hepatic tumor cells closely correlate with PKC (19)(20)(21)(22). Recent findings have shown that opioid receptors share a common pathway with the phosphorylation of PKC.…”

Cell death: A dynamic response concept

“…5,14 Necrosis has been described as a consequence of physical and chemical stress, which is passive and accidental. However, recent literature argues that necrosis is a result of a well-orchestrated interplay of signalling cascades, 27 with an extensive crosstalk between biochemical and molecular events. 28,29 Furthermore, some signalling molecules, which play a role in autophagy and apoptosis, also take part in this interplay.…”