2 to regulate HIF-1a protein expression strictly depends on oxygen availability. We further identified HIF-1a protein stabilization as a key mechanism for HIF-1 induction by bcl-2 under hypoxia. Our data demonstrated that bcl-2 under this condition affects HIF- 1a protein at the post-translational level, indeed the degradation rate of HIF-1a protein was faster in the control cells than in bcl- 2 transfectants. Although under normoxia this HIF-1a stabili- zation is not sufficient to affect the steady state levels of the protein, it becomes rate limiting during hypoxia or, in general, in conditions strictly dependent on oxygen level. In fact, we found that bcl-2 overexpression determines an increase of HIF- 1a protein half-life also in high cell density conditions, as observed under hypoxia. The stabilization of HIF-1a protein in response to changes in oxygen concentration is achieved through the impairment of HIF-1a ubiquitination and subse- quent degradation of the protein. Generally, HIF-1a is degraded in an oxygen-dependent manner through the activity of PHD2 enzyme, which hydroxylates HIF-1a on proline residues 402 and 564, and this hydroxylated form is bound by the E3 ubiquitin ligase VHL which promotes HIF-1a ubiqui- tination and its subsequent proteasomal degradation [19]. Notwithstanding, we found that bcl-2 regulates HIF-1a protein stability in a prolyl hydroxylation-independent manner since bcl-2 overexpression had similar effects on either wild type protein and the degradation resistant form of HIF-1a, which contains proline-to-alanine substitutions (P402A/P564A) trig- gering a resistance to PHD2-mediated hydroxylation. In agreement with this finding, in our experimental model PHD2 protein expression was upregulated in response to hypoxia at comparable levels in parental cells and bcl-2 overexpressing clones (data not shown). Further, bcl-2 overexpression had no impact on HIF-1a protein stabilization induced by iron antagonists known to inhibit hydroxylase activity, such as Cobalt Chloride and Desferoxamine. Some authors have reported that bcl-2 may reside, and even elicit a function, within the nucleus [21–23], modulating the transactivity of several transcription factors [35,36]. Here, we present evidence that in our experimental model the exogenous bcl-2 protein is also localized in the nucleus, beyond the cytoplasm. Of note, our results reveal, for the first time, that bcl- 2 protein interacts with HIF-1a in the nucleus, thus the pro- angiogenic effect of bcl-2 on HIF-1/VEGF axis may result from the nuclear localization of bcl-2. Since the HIF-1a/bcl-2 complex can be observed in the nucleus, we can speculate that bcl-2- mediated stabilization of HIF-1a protein occurs in this cellular compartment. By dissecting the molecular mechanism of this process, we found that bcl-2 increases HIF-1a protein stability through the involvement of the molecular chaperone HSP90, which was found to protect HIF-1a from proteasomal degrada- tion, even in VHL-deficient cells [37,38]. In this context, our data further indicate that the enhanced levels of HIF-1a protein in bcl- 2 overexpressing clones may be due to a decreased poly- ubiquitination of HIF-1a by enforcing the interaction between HIF-1a and HSP90 protein. Moreover, we have shown not only a novel association of HIF-1a with bcl-2, but we have also observed that bcl-2 is able to interact with HSP90 itself. Most importantly, we found that the interaction between bcl-2 and HIF-1a proteins was not dependent on HSP90 inhibition, because the binding of bcl-2 and HIF-1a was not reversed by the treatment with 17- AAG. In addition, sequential immunoprecipitation experiments demonstrated that bcl-2, HIF-1a and HSP90 proteins may form a tri-complex which probably contributes to enhance HIF-1a protein stability in bcl-2 overexpressing clones under hypoxia. HIF-1a Stabilization by Bcl-2 Here, we investigated the role of HSP90a and HSP90b isoforms in bcl-2-mediated HIF-1a induction under hypoxic condition. These two homologous proteins display some differences and elicit specific functions, such as differential binding to client proteins [28]. Using genetic approaches to specifically knockdown each HSP90 isoform in bcl-2 overexpressing cells, we found that HSP90b, but not HSP90a, is required for HIF-1a protein stabilization by bcl-2. Moreover, in agreement with these data, we found that only HSP90b binds HIF-1a protein in bcl-2 overexpressing cells exposed to hypoxia. These results are in a good accordance with very recent data demonstrating an association between b isoform of HSP90 and bcl-2 protein in response to VEGF in leukemia cells [39] or to CpG oligodeox- ynucleotide in macrophages [40]. All together, these results confirm that HSP90b is an important regulator of HIF-1a stability and indicate that this molecular chaperone may be one of the mediators of bcl-2 pro-angiogenic function. A recent report demonstrated that RACK1 protein promotes ubiquitination of HIF-1a induced by the HSP90 inhibitor 17-AAG and its subsequent VHL-independent proteasomal degradation compet- ing with HSP90 for binding to PAS domain of HIF-1a [2]. Notwithstanding, when exposing melanoma cells to the HSP90 inhibitor 17-AAG, we observed that bcl-2 overexpression counteracts both HIF-1a protein degradation induced by 17- AAG, and the reduction of interaction between HIF-1a and HSP90 induced by the inhibitor. Besides, we did not observe any difference in the HIF-1a binding to RACK1 after forced expression of bcl-2 under hypoxia even after 17-AAG exposure (data not shown), suggesting that bcl-2 does not regulates RACK1/Elongin-C dependent HIF-1a degradation pathways. So far we cannot exclude that other molecular players, such as HSP70, JNK1 and the COMMD1 proteins [41–43], may be modulated by bcl-2 and play a role in the stabilization process of HIF-1a protein mediated by bcl-2. In conclusion, our study establishes a molecular link and highlights the possibility that bcl-2 is a new HIF-1a-binding protein whose multivalent interactions are required for the stabilization of HIF-1a, and that nuclear localization of bcl-2 may have an important role in protecting HIF-1a from ubiquitination and proteasomal degradation that commences in the nucleus. Materials and Methods Cell cultures, hypoxia exposure, transfections and viral infection Human melanoma cell lines were cultured in complete RPMI medium (Invitrogen, Carlsbad, CA). JR1, JR8, M14, PLF2 [44], and ASM-SC, bcl-2 overexpressing clones (Bcl2/5 and Bcl2/37) and a control clone (puro) derived from the M14 line after stable transfection, bcl-2 overexpressing (JR8/Bcl-2 and PLF2/Bcl-2) and control (JR8/puro, PLF2/puro) cells derived from the JR8 and PLF2 line after stable transfection were used. ASM-SC was cloned by limiting dilution from A375.S2 melanoma cell line (ATCC, Manassas, VA). For hypoxia exposure, culture dishes were placed in a hypoxia chamber allowing the formation of a hypoxic environment of 5% CO2, 95% N2. Unless stated otherwise, these hypoxic levels (1% of oxygen concentration, 24 h) was used in all experiments. For experiments under low or high cell density conditions, 100 cells/mm2 or 700 cells/mm2 were respectively plated and 24 h later cells were harvested and subjected to different assays. The cells were stably or transiently transfected with the expression vector encoding the human wild type bcl-2. Transfec- PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11772