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

Behnam Saberi, Mie Shinohara, Maria D. Ybanez, Naoko Hanawa, William A. Gaarde, Neil Kaplowitz, Derick Han

Research output: Contribution to journalArticle

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 H 2O2 (∼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 H 2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H 2O2-induced necrosis, suggesting that a signaling "program" is important in mediating H2O2-induced necrosis in primary hepatocytes.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume295
Issue number1
DOIs
StatePublished - Jul 2008
Externally publishedYes

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AMP-Activated Protein Kinases
Hepatocytes
Necrosis
Down-Regulation
Antisense Oligonucleotides
Protein Isoforms
Up-Regulation
Apoptosis

Keywords

  • Akt
  • AMP-activated kinase
  • Hydrogen peroxide
  • Necrosis
  • Protein kinase C

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Regulation of H2O2-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes. / Saberi, Behnam; Shinohara, Mie; Ybanez, Maria D.; Hanawa, Naoko; Gaarde, William A.; Kaplowitz, Neil; Han, Derick.

In: American Journal of Physiology - Cell Physiology, Vol. 295, No. 1, 07.2008.

Research output: Contribution to journalArticle

Saberi, Behnam ; Shinohara, Mie ; Ybanez, Maria D. ; Hanawa, Naoko ; Gaarde, William A. ; Kaplowitz, Neil ; Han, Derick. / Regulation of H2O2-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes. In: American Journal of Physiology - Cell Physiology. 2008 ; Vol. 295, No. 1.
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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 H 2O2 (∼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 H 2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H 2O2-induced necrosis, suggesting that a signaling {"}program{"} is important in mediating H2O2-induced necrosis in primary hepatocytes.",
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N2 - 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 H 2O2 (∼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 H 2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H 2O2-induced necrosis, suggesting that a signaling "program" is important in mediating H2O2-induced necrosis in primary hepatocytes.

AB - 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 H 2O2 (∼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 H 2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H 2O2-induced necrosis, suggesting that a signaling "program" is important in mediating H2O2-induced necrosis in primary hepatocytes.

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