Non-neuronal enolase is an endothelial hypoxic stress protein

Abstract

The hypoxia-associated proteins (HAPs) are five cell-associated stress proteins (M(r) 34, 36, 39, 47, and 57) upregulated in cultured vascular endothelial cells (EC) exposed to hypoxia. While hypoxic exposure of other cell types induces heat shock and glucose-regulated proteins, EC preferentially up-regulate HAPs. In order to identify the 47-kDa HAP, protein from hypoxic bovine EC lysates was isolated, digested with trypsin, and sequenced. Significant identity was found with enolase, a glycolytic enzyme. Western analyses confirmed that non-neuronal enolase (NNE) is up-regulated in hypoxic EC. Western analysis of subcellular fractions localized NNE primarily to the cytoplasm and confirmed that it was up-regulated 2.3-fold by hypoxia. Interestingly, NNE also appeared in the nuclear fraction of EC but was unchanged by hypoxia. Northern analyses revealed that NNE mRNA hypoxic up- regulation began at 1-2 h, peaked at 18 h, persisted for 48 h, and returned to base line after return to 21% O2 for 24 h. Hypoxia maximally up-regulated NNE mRNA levels 3.4-fold. While hypoxic up-regulation of NNE may have a protective effect by augmenting anaerobic metabolism, we speculate that enolase may contribute to EC hypoxia tolerance through one or more of its nonglycolytic functions.