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

Abstract

Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have dierent pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound in¯uences on the target cell lines and may explain a number of ®ndings previously attributed to normoxic cell-cell interactions. Thus, we ®rst sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions aect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF1a via pericellular diusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions. Oncogene (2001) 20, 7624 ± 7634.

Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway

“…Upregulation of COX-2 expression in NIH3T3 cells due to the hypoxic condition It has been reported that increasing epithelial cell density results in pericellular hypoxia (Tokuda et al, 2000;Sheta et al, 2001), and hypoxia has been reported to induce COX-2 expression in endothelial cells (Schmedtje et al, 1997;Xu et al, 2000;Wu et al, 2003). Therefore, we next examined whether hypoxia can modulate COX-2 expression in NIH3T3 cells.…”

Hypoxic induction ofCtgfis directly mediated by Hif-1

“…2C). Normoxic stabilization of Hif-1␣ has been reported previously and has been correlated with cell culture confluency (58). Given the nature of epithelial growth displaying high cell-to-cell contact, we feel that this normoxic level of Hif-1␣ is representative of normal epithelial cell cultures.…”

Contribution of HIF-1α in 4E-BP1 Gene Expression

“…Cell density has actually been shown to induce HIF-1a (22), and a computer analysis of human 4E-BP1 gene sequence revealed the presence of a putative HRE located in the À278/þ64 4E-BP1 promoter segment (Fig. 4A) that mediates cell density-dependent activation of 4E-BP1 promoter.…”