Aim: It has been observed that the age-related total activation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) is due to a lack of regulation by phosphorylation/dephosphorylation. Such fully activation has been related to ROS increase but the responsible mechanism is still unknown. So, aim of this work was to study the mechanism underlying ROS induced impaired regulation of HMG-CoAR using a cellular model, the HepG2 cell line. In particular, in this system with high ROS content induced and in aged rat liver, the involvement of AMP dependent kinase (AMPK) and protein phosphatase 2A (PP2A), the main modulators of reductase short term regulation, has been studied.Methods: To induce ROS content as high as in aged rat liver, HepG2 cell line were treated with 200µM H2O2. ROS content was measured by fluorescence analysis. HMG-CoAR activation state was evaluated by a radioisotopic method. The activation state of the AMPK and the level of PP2A was measured by western blotting. H2O2-induced signal transduction pathway was evaluated by western blotting and the downstream effects by using specific inhibitors. Protein association was evaluated by co-immunoprecipitation.Results: In HepG2 cell line the HMG-CoAR results completely activated by ROS. AMPK activation state is high even if the enzyme isn’t able to phosphorylate HMG-CoAR; PP2A level doesn’t change. The p38/MAPK phosphorylation rises and the use of a p38 inhibitor or some phosphatase inhibitors prevents ROS-induced HMG-CoAR full activation. Both in H2O2 treated cell and aged liver the association of PP2A with HMG-CoAR is well detectable.Conclusion: The presented data show that ROS increase is able to induce HMG-CoAR full activation enhancing the reductase-PP2A association through p38 activation and subsequent dephosphorylation of the enzyme. On the contrary the modified activity of the kinases seem to be not involved in this deregulative process.

MARTINI CHIARA, PALLOTTINI VALENTINA, & TRENTALANCE ANNA (2006). ROS increase is the cause of the age related 3-hydroxy-3-methylglutaryl coenzyme A reductase deregulation, 188 sup.652, 44.

ROS increase is the cause of the age related 3-hydroxy-3-methylglutaryl coenzyme A reductase deregulation

MARTINI, CHIARA;PALLOTTINI, Valentina;TRENTALANCE, Anna
2006

Abstract

Aim: It has been observed that the age-related total activation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) is due to a lack of regulation by phosphorylation/dephosphorylation. Such fully activation has been related to ROS increase but the responsible mechanism is still unknown. So, aim of this work was to study the mechanism underlying ROS induced impaired regulation of HMG-CoAR using a cellular model, the HepG2 cell line. In particular, in this system with high ROS content induced and in aged rat liver, the involvement of AMP dependent kinase (AMPK) and protein phosphatase 2A (PP2A), the main modulators of reductase short term regulation, has been studied.Methods: To induce ROS content as high as in aged rat liver, HepG2 cell line were treated with 200µM H2O2. ROS content was measured by fluorescence analysis. HMG-CoAR activation state was evaluated by a radioisotopic method. The activation state of the AMPK and the level of PP2A was measured by western blotting. H2O2-induced signal transduction pathway was evaluated by western blotting and the downstream effects by using specific inhibitors. Protein association was evaluated by co-immunoprecipitation.Results: In HepG2 cell line the HMG-CoAR results completely activated by ROS. AMPK activation state is high even if the enzyme isn’t able to phosphorylate HMG-CoAR; PP2A level doesn’t change. The p38/MAPK phosphorylation rises and the use of a p38 inhibitor or some phosphatase inhibitors prevents ROS-induced HMG-CoAR full activation. Both in H2O2 treated cell and aged liver the association of PP2A with HMG-CoAR is well detectable.Conclusion: The presented data show that ROS increase is able to induce HMG-CoAR full activation enhancing the reductase-PP2A association through p38 activation and subsequent dephosphorylation of the enzyme. On the contrary the modified activity of the kinases seem to be not involved in this deregulative process.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11590/269391
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