Cell density mediated pericellular hypoxia leads to induction of HIF-1 alpha via nitric oxide and Ras/MAP kinase mediated signaling pathways

Environmental signals in the cellular milieu such as hypoxia, growth factor s, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environme nt to the nucleus. Several groups have evaluated gene expression or signali ng pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assume d that cells grown at various densities in standard in vitro incubator cond itions do not have different pericellular oxygen levels. However, pericellu lar hypoxia can be induced by increasing cell density, which can exert prof ound influences on the target cell lines and may explain a number of findin gs previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normo xic culture conditions results in pericellular hypoxia in prostate cancer c ells. Second, we sought to evaluate whether such interactions affect transc ription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HR E in response to cell density induced pericellular hypoxia in routine normo xic culture conditions. Our results indicate that paracrine cell interactio ns can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We als o make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracri ne factor secreted by densely cultured cells. These results suggest that pa racrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a po ssible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.