Hypoxia CREB phosphorylation

Figure 6 Effect of hypoxia on the phosphorylation of Akt. PC12 cells were pretreated with either wortmannin (100 nM), a specific inhibitor of PI3-Kinase, or vehicle (0.01% DMSO) for 1 hr prior to exposure to either normoxia (21% O2) or hypoxia (5% O2) for 6 hr. Whole-cell lysates were then immunoblotted for either phospo-Akt (a), total Akt (b), phospho-GSK3 (c), phospho-CREB (d), or EPASl (e). Note that CREB phosphorylation and EPASl phosphorylation and accumulation persist in the presence of wortmannin.

Figure 7 Effect of hypoxia on CREB phosphorylation. PC12 cells were exposed to either normoxia or hypoxia (5% O2) for the indicated time. Whole-cell lysates were immunoblotted with an antibody to Ser phospho-CREB (a) or total (phosphorylation-state-independent) CREB (b).

Figure 8 CREB phosphorylation by hypoxia does not require Ca, PCK, RSK-2, MAPK, or p38. Cells were pretreated with various drugs or vehicle (0.1% dimethyl sulfoxide) as indicated. Cells were then exposed to either normoxia (C, 21% O2) or hypoxia (H, 5% O2) for 6 hr, and whole-cell lysates were immunoblotted with an antibody specific for Ser phospho-CREB. (a) Cells were preincubated for 40 min in serum-fi ee medium in the presence of Ca and vehicle (—) or in serum-iree medium formulated without Ca and supplemented with 1 mM EGTA-I-100 pM BAPTA-AM (-h). The medium was then replaced (minus drug or vehicle) and cells were exposed to either normoxia or hypoxia, (b) Cells were pretreated for 40 min in serum-free medium with either vehicle (—) or 20 pM chelerythrine chloride, an inhibitor of PKC (CHL -P), and exposed to either normoxia or hypoxia, (c) Cells were pretreated for 40 min in serum-fi ee medium with either vehicle (—) or 0.3 pM Ro 31-8220, an inhibitor of RSK and p70 S6 kinase (-P), and exposed to either normoxia or hypoxia, (d) Cells were pretreated for 40 min in serum-lree medium with either vehicle (—) or 50 pM PD098059, an inhibitor of MEKl and the downstream MAPKS (-P), and exposed to either normoxia or hypoxia, (e) Cells were pretreated for 1 hr in serum-fiee medium with either vehicle (—) or 10 nM rapamycin, an inhibitor of p70 S6 kinase (-P), and exposed to either normoxia or hypoxia, (f) Cells were pretreated for 1 hr in serum-fiee medium with either vehicle (—) or 20 pM SB203580, an inhibitor of p38a kinase and MAPKAP kinase (-P), and then exposed to either normoxia or hypoxia. In all of these experiments, hypoxia did not alter the total levels of CREB.

A catecholaminergic cell line, rat pheochromo-cytoma (PC 12) cells, that has been proven to be a useful system to study hypoxia-regulated gene expression, at 5% O2 rapidly induced a persistent phosphorylation of cyclic AMP response element binding protein (CREB) on Ser (Beitner-JOHNSON and Millhorn 1998), an element that is required for CREB-mediated transciptional activation (Gonzalez and Montminy 1989, Bonni et al. 1995). PC 12 cell cultures exhibited a loss of cells and increase in intracellular oxidative stress when exposed to ethanol for 24 h (Li et al. 2001). Catalase (EC 1.11.1.6) can attenuate this ethanol-induced cell loss and oxidative stress. 

Hypoxia induced a robust Ser phosphorylation of CREB (Fig. 7) that was greater than that prodnced by two prototypical stimuli used to activate CREB,... 

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