HIF-1a Stabilization by Bcl-2 Figure 5. HIF-1a prolyl hydroxylation is not required for bcl-2-induced increase of HIF-1a expression and HIF-1 activity in hypoxia. (A) Western blot analysis of HIF-1a, bcl-2 and PHD2 protein expression and (B) HRE-dependent transcriptional activity in M14 cells stably expressing HA-HIF-1a wild-type (HIF1a wt) or mutated (HIF1a PP/AA), after transiently transfection with control vector (empty), bcl-2 or PHD2 expressing vectors, and then exposure to hypoxia for 24 h. (A) b-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown. (B) Relative luciferase activity of each sample were normalized to the control vector transfected cells. Results represent the mean 6 SD of 3 independent experiments performed in triplicate, * p#0.01. doi:10.1371/journal.pone.0011772.g005 regulates HIF-1a expression independently from prolyl hydroxyl- ation of HIF-1a. These results are also supported by the findings that forced expression of bcl-2 had no impact on HIF-1a stabilization when cells were treated with PHD inhibitors Cobalt Chloride and Desferoxamine, two iron antagonists known to inhibit hydroxylase activity (Figure S4). bcl-2 forms a complex with HSP90 and HIF-1a proteins, enhancing their interaction and protecting HIF-1a from degradation mediated by 17-AAG HSP90 is a molecular chaperone required for the stability and function of a number of proteins implicated in cancer cell growth and angiogenesis, including HIF-1a [28]. In particular, it binds and stabilizes HIF-1a, and it represents a critical factor in an O2/PHD/ VHL-independent degradation pathway of HIF-1a protein [2]. To evaluate a possible contribution of HSP90 to bcl-2-induced stabilization of HIF-1a, we determined whether the pharmacolog- ical inhibition of HSP90 with 17-AAG, an inhibitor that can alter the interaction of HSP90 with its clients [29], modulates HIF-1a expression (Figure 6A) and transcriptional activity (Figure 6B) in control cells and two bcl-2 transfectants cells under hypoxia. 17- AAG reduced hypoxia-induced HIF-1a accumulation in control cells, while only a very barely down-regulation of HIF-1a protein expression was evident in bcl-2 overexpressing clones after 17-AAG treatment (Figure 6A). These results suggested that bcl-2 overexpression might confer a resistance of HIF-1a protein from the degradation induced by the 17-AAG. On the functional level, 0.05 mM 17-AAG induced about 30% versus 10% inhibition of HRE-dependent transcriptional activity in control cells compared with bcl-2 transfectants. The higher dose of 2 mM completely inhibited HRE-dependent transcriptional activity in control cells, by contrast bcl-2 transfectants cells were resistant to HRE-dependent transcriptional activity inhibition induced by the same dose of 17- AAG (Figure 6B). Most importantly, as shown in Figure 6C, HSP90 protein is highly expressed in both control and bcl-2 overexpressing cells, and the impact of either bcl-2 status and either hypoxic conditions on HSP90 protein expression was not relevant. To provide evidence that the HSP90 is involved in bcl-2-induced stabilization of HIF-1a, we investigated the effect of bcl-2 on the interaction between HIF-1a and HSP90 proteins by immunopre- cipitation of HIF-1a and Western blot analysis of HSP90 protein. As depicted in Figure 6D, bcl-2 overexpression under hypoxia enhanced the ability of HIF-1a to form a complex with HSP90. To confirm the interaction between HIF-1a and HSP90 proteins, we performed a reverse immunoprecipitation experiment from total extract of hypoxic cells. Under these conditions, in spite of similar levels of immunoprecipitated HSP90, a larger amount of HIF-1a protein within the immunoprecipitate was found in total extracts of bcl-2 transfectants (Figure 6E), confirming a stronger interaction between HIF-1a and HSP90 proteins in bcl-2 transfectants. We also studied the interaction between HSP90 and bcl-2 protein under hypoxic conditions and we found that HSP90 was associated with ectopic bcl-2 protein (Figure 6E). Similar results were also observed when immunoprecipitation experiments were carried out in nuclear extracts (data not shown). These findings suggest that bcl-2 may promote stabilization of HIF-1a by increasing its ability to interact with the HSP90 chaperone complex. To gain insight to these results, we investigated whether the bcl-2/HSP90/HIF-1a binding could be reversed when exposing the cells to 17-AAG. We found that 17-AAG treatment reduced the binding between HSP90 and HIF-1a only in control cells and weakly in bcl-2 transfectants, confirming that bcl-2 overexpressing cells were more resistant to 17- AAG-induced degradation of HIF-1a. Moreover, we found that the interaction of bcl-2 protein with HIF-1a was not affected by 17- AAG treatment (Figure 6F). Because our results showed that both HSP90 and HIF-1a proteins bind to bcl-2, we investigated the potential formation of a HSP90/HIF-1a/bcl-2 tri-complex. To address this hypothesis, the cell lysates were firstly immunoprecip- itated with anti-HIF-1a antibody, then subjected to a second immunoprecipitation with anti-bcl-2 antibody, and the immuno- complexes were analyzed by Western blot analysis using antibody against HSP90 protein. As shown in Figure 6G, HSP90 could be found in complex with HIF-1a and bcl-2 protein in cells overexpressing bcl-2, demonstrating the formation of a HSP90/ PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11772