With regards to the interaction between SIRT1 and HIF proteins, Lim et http://www.selleckchem.com/products/Axitinib.html al. demonstrated that SIRT1 binds to and deacetylates HIF-1�� at lysine 674. This interaction blocks p300 recruitment to the promoter of HIF-1 target genes and thereby represses HIF-1 transcriptional activity [27]. Conflictingly, Dioum et al. have reported that SIRT1 does not target HIF-1��, rather it deacetylates HIF-2��, and their interaction promotes HIF-2 transcriptional activity [28]. In addition, because SIRT1 is a redox sensor and dependent on the metabolic status of the cell, its regulation by hypoxia has been a point of interest. In one report, SIRT1 is down regulated in hypoxic conditions due to decreased NAD+ levels [27], while in another report it is up regulated in a HIF-dependent manner [40].
From the current literature, one can conclude that the interaction between SIRT1 and HIF-1 and the resulting outcome of their interactions is still unclear. Previously generated data from our lab were also in conflict with the above-mentioned studies. Therefore, here, we report our findings on the involvement of SIRT1 and HIF-1 in hypoxic conditions. First we looked at the influence of hypoxia on SIRT1 expression. We consistently observed that SIRT1 protein or mRNA was not modified in three different HCC cell lines cultured under hypoxic conditions. Abundant SIRT1 and HIF-1�� protein are simultaneously expressed in hypoxic cells. From this observation and considering the reported negative association between SIRT1 and HIF-1 [21], we questioned whether inhibiting SIRT1 activity would thereby augment HIF-1 function under hypoxic conditions.
Our data demonstrates that inhibition of SIRT1 activity reduces the hypoxia-induced transcriptional activity of HIF-1 gene targets and the accumulation of HIF-1�� protein itself. We show that this regulation is relevant in vivo by demonstrating that SIRT1 inhibition leads to a decreased HIF-mediated response to systemic hypoxia. In addition, SIRT1 inhibition resulted in growth inhibition in a mouse xenograft tumor model of HCC. And finally we demonstrate that endogenous SIRT1 and HIF-1�� co-immunoprecipitate and HIF-1 is a target of SIRT1 deacetylase activity. Taken together these data suggest that SIRT1 targets HIF-1�� protein and that this interaction is required for HIF-1�� activity in hypoxic conditions.
Results SIRT1 and HIF-1�� are simultaneously expressed in hypoxic cells HIF-1 is tightly regulated by oxygen availability and functions predominantly in hypoxic conditions. In order to demonstrate that SIRT1 is necessary for the accumulation of HIF-1�� protein, we first verified that SIRT1 and HIF-1�� AV-951 proteins are co-expressed in hypoxic conditions. SIRT1 protein was strongly expressed in Hep3B, HepG2 and Huh7 HCC cells lines in normal culture conditions as well as in cells incubated at 1% O2 (Figure 1A).