Francoise Bono

Hypoxia-inducible factor-2alpha (HIF-2alpha) is involved in the apoptotic response to hypoglycemia but not to hypoxia

Deprivation of oxygen (hypoxia) and/or glucose (hypoglycemia) represents a serious stress that affects cellular survival. The hypoxia-inducible transcription factor-1α (HIF-1α), which has been implicated in the cellular response to hypoxia (Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551–578), mediates apoptosis during hypoxia (Halterman, M. W., Miller, C. C., and Federoff, H. J. (1999) J. Neurosci. 19, 6818–6824 and Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998)Nature 394, 485–490), but the function of its homologue HIF-2α remains unknown. Therefore, the role of HIF-2α in cellular survival was studied by targeted inactivation of theHIF-2α gene (HIF-2α−/−) in murine embryonic stem (ES) cells. In contrast to HIF-1α deficiency, loss of HIF-2α did not protect ES cells against apoptosis during hypoxia. Both HIF-1α−/− and HIF-2α−/− ES cells were, however, resistant to apoptosis in response to hypoglycemia. When co-cultured with wild type ES cells, HIF-2α−/− ES cells became rapidly and progressively enriched in hypoglycemia but not in hypoxia. Thus, HIF-1α and HIF-2α may have distinct roles in responses to environmental stress, and despite its name, HIF-2α may be more important in the survival response to environmental variables other than the level of oxygen.

Influence of Heparin Mimetics on Assembly of the FGF-FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.