Abstract
The hypoxia-associated proteins (HAPs) are five cell-associated stress proteins (M(r) 34, 36, 39, 47, and 57) up-regulated 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.
Highlights
Tolerance to acute hypoxia and adaptation to chronic hypoxia are crucial to the survival of cells and the organisms they comprise
In studying the endothelial cells (EC) response to hypoxia, we have previously described the hypoxia associated proteins (HAPs), a unique set of five cell-associated stress proteins (Mr 34, 36, 39, 47, and 57) up-regulated in a time and oxygen concentration-dependent manner in EC exposed to hypoxia [1,2,3]
The hypoxic upregulation of HAPs in EC occurs in lieu of the stereotypic hypoxic induction of heat-shock and glucose-regulated proteins seen in more hypoxia-sensitive cells and correlates with the ability of EC to tolerate hypoxia
Summary
Materials—Bovine serum albumin was obtained from Hyclone Corp (Logan, CT). Protein quantification was performed spectrophotometrically with a kit from Bio-Rad. A rabbit derived polyclonal antibody directed against the ␣-subunit of NNE with cross-reactivity against the ␥-subunit of neuron-specific enolase (NSE) was obtained from Biogenesis (Franklin, MN). A murine monoclonal antibody raised against human NSE and specific for the enolase ␥-subunit was obtained from Dako Corp. Experiments were performed using 85–90% confluent EC monolayers of passage number 3–10. Control and hypoxic conditions for an individual experiment were performed in parallel on identical cell lines of identical passage number. As a positive control for enolase, H446 cells, a human small-cell lung cancer line (American Type Culture Collection; ATCC, Rockville, MD) were used.
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