Function And Mechanisms Of Over-expression Of AMP-activated Protein Kinase In LX2 Cells

Hepatic fibrosis is a common pathologic change of some kinds of chronic liver disease (CLD), which is also the central process to aggravate to liver cirrhosis (LC), even liver cancer. Hepatic stellate cells play a vital role in the development of hepatic fibrosis. In response to injury of liver, HSCs are activated to a myofibroblast-like phenotype with enhance proliferation, fibrogenesis, and contractility. Recent evidence has shown that activated-HSCs proceeded spontaneous apoptosis during resolution of hepatic fibrosis. Therefore, inducing apoptosis of HSCs is a potential therapic strategy for hepatic fibrosis.Recent evidence showed that over-expression of AMPK could induce apoptosis of rat liver cells, neuroblastoma cell and MIN cells. But the apoptotic mechanism has not been known clearly. AMP-activated protein kinase (AMPK), a serine/threonine protein kinase, is a sensor for the cell engery state, serving as a metabolic master switch. Any increase in the intracellular AMP/ATP ratio can activate AMPK. Once activated, the system switches on catabolic pathways that generate ATP, while switching off ATP-consuming processes that are not essential for short-term cell survival. When AMPK over-expressed, it could activated JNK, which could activate transcription factor c-Jun to regulate the expression of proteins, at the end cells resulted in apoptosis through the caspase pathway.AMPK is a serine/threonine protein kinase, composed of a catalytic subunit (α) and two regulatory subunits (βandγ). The excess AMPK activity can obtain by treated with AICA-dboside (AICAR) or by infection of Ad-CA-AMPK, since AICA dboside is not a specific activator of AMPK, the results obtained in studies using AICAR cannot be taken as an unequivocal proof that AMPK was mediating the response. Therefore Ad-CA-AMPK should be a better choice. Up to now, some researchers have constructed an adenovims to express activated AMPKα1, which plays an more and more important role in studying the effect of AMPK. A truncated polypeptide containing AMPKα11-312 residues no longer associates with theβandγsubunits, but keeps significant kinase activity. Moreover, mutation of thronine 172 within theαsubunit ,the major site phosphorylated by AMPK kinase, to an aspadtic acid residue within this truncated protein prevent its inactivation by protein phosphatases. This adenovirus is a very important tool to research the effect of AMPK.The main results and conclusions are as follows:1. Constructed adenovirus mediated over-expression of constructive AMPK in LX2 cells. LX2 cells were transfected at the adenovirus tite pfu100, and were harvested at different times (24,48,72h), then western blotting was executed to detected the expression of ectogenous ampk. Ectogenous ampk could be detected at 72h but could not before 48h. It indicated that the expression of ectogenous ampk was between 48h and 72h, and the activity of AMPK could last to at least 72h. At the later experiments LX2 cells were collected at 72h.2. After infection of Ad-AMPK, distinctively apoptotic peak was detected by flow cytometer with stained with PI. This result indicated that LX2 proceeded apoptosis. And the DNA appeared distinctively apoptotic ladder, which support the result the LX2 cells proceeded apoptosis.3. After infection of Ad-AMPK, Bax was up-regulated, and pro-casepase-3 was activated, however, Bcl-2 was low-expressed in LX2 cells. It could be demonstrated that apoptosis of LX2 mediated by AMPK was closely associated with Bcl-2 family.4. After Bax was knocked down by RNAi, LX2 cells infected with Ad-AMPK no longer gathered and detached from the bottom of the culture flask, and not took place apoptosis, but had a good growth condition.5. When the Bax was knocked down, even the LX2 cells were infected with Ad-AMPK, pro-casepase-3 had hardly difference from the control group, because RNAi Bax inhibited the effect of AMPK inducing apoptosis of LX2. This also indicated that Bax was a very important element in the apoptotic pathway.Taken together, at the guide that inducing apoptosis of HSC can relieve liver fibrosis, we confirmed that over-expression of AMPK could induce apoptosis of LX2, and we hope this signaling pathway could be targeted for therapeutic intervention in liver fibrosis. With the mechanism of AMPK inducing apoptosis of LX2, the consequence first conformed that Bax is a new member of the pathway.