Figure 3. Activation of AMPK inhibits HSP70 expression in response to various stresses. HepG2 cells pretreated with or without 1 mM AICAR for 15 min were incubated with 5 mM celastrol for 1 h, 50 mM CdCl2, or 20 mM MG132 for 5 h, HSP70 mRNA levels were examined by real-time PCR (A). HSP70 protein levels were examined by Western blot after incubation with CdCl2 or MG132 for another 3 h, or replacing celastrol with fresh medium for 5 h (B). mean 6 SEM, n = 3. *P,0.05, **P,0.01 vs.\ stressor stimulated cells without AICAR pretreatment. B: experiments were performed at least three times and the representative results are shown. doi:10.1371/journal.pone.0013096.g003 induced HSP70 expression at mRNA and protein levels. Pretreatment with AICAR suppressed HSP70 expression induced by these stressors. These results suggest that the negative regulation of HSP70 expression by AMPK might be a common event under stress. Effects of AMPK activation on HSF1 activity and HSP70 mRNA stability under heat stress Heat shock transcription factor 1 (HSF1) is the major transcription factor controlling heat shock protein gene transcrip- tion [6,7]. To explore the mechanisms underlying HSP70 negative regulation by AMPK under stress, we first examined the effect of AMPK on heat stress-induced HSF1 phosphorylation and nuclear translocation, as well as HSF1 binding with the HSE in HSP70 gene by Western blot and EMSA, respectively. As shown in Figure 4A–C, pretreatment of HepG2 cells with AICAR to activate AMPK had no effect on these events driven by heat stress. We then examined whether AMPK modulate HSP70 expression AMPK Regulates HSP70 at post-transcriptional level by checking the effect of AMPK activation on HSP70 mRNA stability under heat stress. We found that after activation of AMPK with AICAR under heat stress, the half-life of HSP70 mRNA was significantly decreased from 3.8160.32 h in the control cells to 2.0060.11 h in the AICAR treated cells (P,0.01, n = 3) (Figure 4D), indicating that HSP70 mRNA was significantly stabilized by the AMPK inhibition under heat stress. This stabilization effect, at least in part, contributed to the dramatic increase of HSP70 mRNA under heat stress. Discussion In the present study, we found that heat stress inhibited AMPK activity by phosphatase 2A (PP2A)-mediated AMPK a subunit dephosphorylation, and AMPK inhibition enhanced HSP70 expression. AMPK activation had no effect on heat stress-induced HSF1 activation but significantly decreased HSP70 mRNA stability. These results demonstrate that under heat stress, PP2A mediated AMPK inhibition upregulates HSP70 at least partly through increase of HSP70 mRNA stability. AMPK is a cellular energy sensor activated by metabolic stresses that either inhibit ATP synthesis or accelerate ATP consumption. Although Corton et al. [8] reported that AMPK activity was increased under heat stress in rat primary hepatocytes, Kodiha et al. [9] found that heat stress inhibited AMPK by dephosphoryltion of AMPKa in both Hela and 293 cells. Our results showed that under heat stress, AMPKa was dephosphorylated not only in human cells (HepG2, 293T), but also in rodent cells (C2C12, Hepa1-6, bEnd3 and MIN6), suggesting that AMPK inhibition is an universal response to heat stress. ACC and PEPCK are two molecules downstream of AMPK. We found that the inhibition of AMPK under heat stress in turn led to the activation of ACC and upregulation of PEPCK, demonstrating the functional inhibition of AMPK under heat stress. We further found that PP2A activation, rather than intracellular ATP alteration, resulted in AMPKa dephosphorylation under heat stress. PP2A has been reported to be activated by ceramide [18,19]. Recently, Wu et al. [14] reported that cermaide mediated palmitate-induced PP2A activation and subsequent AMPK inhibition. As intracellular ceramide rapidly increases upon heat stress [20,21], heat stress- induced PP2A activation may be also mediated by ceramide but needs further investigation. Although PP2C has also been reported to be capable of inactivating AMPK [12], our results showed that PP2A knock-down by RNAi almost totally reversed the inhibition of AMPK by heat stress, suggesting that PP2A plays major role in AMPK inhibition in response to heat stress. By using AMPK activator and siRNA targeting AMPKa, we revealed that the inhibition of AMPK under heat stress contributed to HSP70 upregulation. Furthermore, activation of AMPK with AICAR also inhibited HSP70 induction by other HSP inducers, including celastrol, CdCl2 and MG132. Therefore, the negative regulation of HSP70 by AMPK may be a common mechanism involved in HSP70 induction under stress. As an energy sensor, AMPK is a key regulator of catabolic versus anabolic processes. Activated AMPK may modulate key metabolic enzymes via direct phosphorylation, may directly and indirectly regulate transcriptional programs through phosphoryla- tion events. How AMPK activation regulates the expression of a number of genes involved in metabolism is only beginning to be elucidated [22]. The mechanisms underlying gene regulation by AMPK inhibition remains unclear. Our results showed that under heat stress, treatment of cell with AMPK activator AICAR had no effect on HSF1 activation but reduced the HSP70 mRNA stability, suggesting that AMPK inhibition under heat stress enhances PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13096