The BH3-only protein NOXA mediates hepatic stellate cell apoptosis by proteosome inhibition

IVETTE SOSA SEDA; JUSTIN L. MOTT; FERNANDO J BARREYRO; YUKO AKAZAWA; ALISAN KAHRAMAN; STEVEN F BRONK; NATHAN W. WERNEBURG; GREGORY J GORES

San Diego, USA

Congreso; DDW-2008 Annual Meeting of the American Gastroenterological Association (AGA). 2008; 2008

American Gastroenterological Association

  Background and Aims: The resolution phase of hepatic fibrosis is dependent, in part, upon induction of stellate cell apoptosis. Apoptosis is triggered by BH3-only proteins of the Bcl- 2 protein superfamily; however, the specific BH3-only proteins expressed and participating in stellate cell apoptosis remains unclear. Our aim was to mechanistically identify which BH3-only protein(s) contribute to stellate cell apoptosis during proteosome inhibition. Materials and Methods: Apoptosis was induced by treating LX-2 cells, an immortalized human hepatic stellate cell line, with the proteasome inhibitor MG-132. To ascertain the expression of BH3-only proteins, immunoblot analysis and quantitative real-time PCR were performed. Knockdown of protein expression was achieved by siRNA technology. Apoptosis was assessed by characteristic nuclear staining with DAPI/fluorescence microscopy and caspase-3/7 activity assay. Results: Under basal conditions, LX-2 cells express the BH3-only proteins Noxa, Puma, Bim, Bad and Bim, whereas they do not express Bmf, Hrk nor Bik. MG132 induced time and concentration dependent apoptosis of LX-2 cells, which was maximal (55+/-5% of total cells) after 24 hours of treatment with 10 ìM MG132. Following treatment with 10 ìM MG132, Noxa protein expression increased 22-fold and Bim 2-3 fold; expression of the other BH3-only proteins was unchanged. The striking increase in Noxa protein expression was likely transcriptionally mediated as its mRNA also increased 16-fold following proteosome inhibition. siRNA-targeted knockdown of Noxa or Bim was achieved as confirmed by immunoblot analysis. siRNA targeted knockdown of Noxa but not Bim, markedly attenuated MG132-mediated apoptosis of LX2 cells. Indeed, MG132-mediated apoptosis was reduced to 24+/-5% of total cells by Noxa siRNA as assessed by both morphologic and caspase-3/7 activity. In conclusion: Noxa, a pro-apoptotic BH3-only protein, mediates proteasome inhibitor- induced apoptosis of LX-2 cells. Both Noxa protein and mRNA levels were increased consistent with transcriptional upregulation of Noxa expression. Noxa appears to be a key BH3-only protein mediating stellate cell apoptosis. the other BH3-only proteins was unchanged. The striking increase in Noxa protein expression was likely transcriptionally mediated as its mRNA also increased 16-fold following proteosome inhibition. siRNA-targeted knockdown of Noxa or Bim was achieved as confirmed by immunoblot analysis. siRNA targeted knockdown of Noxa but not Bim, markedly attenuated MG132-mediated apoptosis of LX2 cells. Indeed, MG132-mediated apoptosis was reduced to 24+/-5% of total cells by Noxa siRNA as assessed by both morphologic and caspase-3/7 activity. In conclusion: Noxa, a pro-apoptotic BH3-only protein, mediates proteasome inhibitor- induced apoptosis of LX-2 cells. Both Noxa protein and mRNA levels were increased consistent with transcriptional upregulation of Noxa expression. Noxa appears to be a key BH3-only protein mediating stellate cell apoptosis. 10 ìM MG132, Noxa protein expression increased 22-fold and Bim 2-3 fold; expression of the other BH3-only proteins was unchanged. The striking increase in Noxa protein expression was likely transcriptionally mediated as its mRNA also increased 16-fold following proteosome inhibition. siRNA-targeted knockdown of Noxa or Bim was achieved as confirmed by immunoblot analysis. siRNA targeted knockdown of Noxa but not Bim, markedly attenuated MG132-mediated apoptosis of LX2 cells. Indeed, MG132-mediated apoptosis was reduced to 24+/-5% of total cells by Noxa siRNA as assessed by both morphologic and caspase-3/7 activity. In conclusion: Noxa, a pro-apoptotic BH3-only protein, mediates proteasome inhibitor- induced apoptosis of LX-2 cells. Both Noxa protein and mRNA levels were increased consistent with transcriptional upregulation of Noxa expression. Noxa appears to be a key BH3-only protein mediating stellate cell apoptosis. the other BH3-only proteins was unchanged. The striking increase in Noxa protein expression was likely transcriptionally mediated as its mRNA also increased 16-fold following proteosome inhibition. siRNA-targeted knockdown of Noxa or Bim was achieved as confirmed by immunoblot analysis. siRNA targeted knockdown of Noxa but not Bim, markedly attenuated MG132-mediated apoptosis of LX2 cells. Indeed, MG132-mediated apoptosis was reduced to 24+/-5% of total cells by Noxa siRNA as assessed by both morphologic and caspase-3/7 activity. In conclusion: Noxa, a pro-apoptotic BH3-only protein, mediates proteasome inhibitor- induced apoptosis of LX-2 cells. Both Noxa protein and mRNA levels were increased consistent with transcriptional upregulation of Noxa expression. Noxa appears to be a key BH3-only protein mediating stellate cell apoptosis.