©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Non-neuronal Enolase Is an Endothelial Hypoxic Stress Protein (*)

(Received for publication, June 19, 1995; and in revised form, August 7, 1995)

Robert M. Aaronson Krista K. Graven Marisa Tucci Robert J. McDonald Harrison W. Farber (§)

From the Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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% O(2) 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.


INTRODUCTION

Tolerance to acute hypoxia and adaptation to chronic hypoxia are crucial to the survival of cells and the organisms they comprise. Among mammalian cells, vascular endothelial cells (EC) (^1)offer an excellent example of adaptation to extreme hypoxia. For example, pulmonary arterial EC normally reside in a pO(2) of 35-40 mm Hg and may be exposed to lower oxygen concentrations in disease states. Yet, we and others have shown that bovine and human EC in culture are extremely tolerant to decreases in ambient oxygen tension(1, 2, 3, 4, 5, 6, 7, 8) . We have speculated that this tolerance may be, in part, acquired. For instance, although exposure to 0% oxygen for 4-5 days is lethal to bovine pulmonary artery and aortic EC (BPAEC and BAEC, respectively) not acclimatized to hypoxia, BPAEC and BAEC grown in 3% oxygen and then exposed to 0% oxygen survive, proliferate, and maintain characteristic EC appearance for more than 6 days. (^2)

In studying the EC response to hypoxia, we have previously described the hypoxia associated proteins (HAPs), a unique set of five cell-associated stress proteins (M(r) 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 up-regulation 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. As part of our efforts to elucidate the function of the HAPs in physiologic and hypoxic conditions, we have previously identified the 36-kDa HAP as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase(3) . In the current study, we report the isolation and identification of HAP47 as non-neuronal enolase (NNE) and examine its hypoxic regulation.


EXPERIMENTAL PROCEDURES

Materials

Bovine serum albumin was obtained from Hyclone Corp (Logan, CT). All other tissue culture materials were purchased from Life Technologies, Inc. Oxygen gas mixtures were purchased from Wesco (Billerica, MA). Protein quantification was performed spectrophotometrically with a kit from Bio-Rad. A rabbit derived polyclonal antibody directed against the alpha-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. (Carpinteria, CA). Secondary antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA). Renaissance Western blot chemiluminescence reagent was obtained from DuPont NEN. TriReagent-LS was obtained from Molecular Research Center (Cincinnati, OH), random primer labeling was performed using a kit from Promega (Madison, WI), and QuikHyb was obtained from Stratagene (La Jolla, CA). Poly(vinylidene difluoride) membranes were purchased from Applied Biosystems (Foster City, CA), nitrocellulose membranes (0.2-µm pore size) were purchased from Schleicher & Schuell, and nylon membranes (Hybond-N+) were purchased from Amersham Corp.

Cell Culture

Bovine EC were isolated from freshly excised calf pulmonary arteries and aortas as described previously(1, 2, 3, 4, 5, 6) . Cultures were maintained from isolation at 37 °C in a humidified incubator in 21% O(2), 5% CO(2), balance N(2) (21% O(2), normoxia) or in a humidified sealed chamber (Billups-Rothenburg, Del Mar, CA) gassed with 3% O(2), 5% CO(2), balance N(2) (3% O(2), chronic hypoxia)(1, 3, 4, 5, 6) . The partial pressures of oxygen in the media under these conditions are 140 and 40 mm Hg, respectively(1, 4) . Endothelial cell purity was confirmed by typical cobblestone appearance, factor VIII immunofluorescence, and uptake of fluorescent acetylated low density lipoprotein(1, 6) . 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.

Protein Induction and Labeling

Hypoxia associated proteins were induced using EC (3 times 10^6 cells) maintained chronically in 21% O(2) or 3% O(2) and then exposed to 0% O(2), 5% CO(2), balance N(2) (O% O(2)) for various times in a humidified sealed chamber(1, 2, 3) . The partial pressure of oxygen in the medium under this condition is approximately 20-25 mm Hg (1, 4) . After exposure to 0% O(2) cells were allowed to recover in 21% O(2) for 3 h. In other experiments, EC proteins were radiolabeled with 75 µCi/ml [S]methionine in methionine-free modified Eagle's medium supplemented with 10% bovine calf serum and 1 mM sodium pyruvate for the last 1 h of the recovery period.

Protein Isolation

Endothelial cell and H446 monolayers were washed three times with phosphate-buffered saline (PBS). Whole cell lysates were prepared by solubilization in Laemmli buffer supplemented with aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride, boiled for 2 min, and frozen at -20 °C until electrophoresis. In some experiments, EC subcellular fractionation was performed as described previously(3) . Subcellular fractions were diluted in 5 times sample buffer (3.8 g of Tris, pH 6.8, 50% glycerol v/v, 5 g of SDS, 5% beta-mercaptoethanol v/v in 100 ml) and boiled for 2 min prior to electrophoresis.

Gel Electrophoresis, Autoradiography, and Protein Sequencing

Whole cell lysates and subcellular fractions were analyzed using 10% SDS-PAGE(1, 2, 3) . For HAP47 sequencing, a maximal quantity of unlabeled protein was loaded in all but two lanes; these two lanes were loaded with equal radioactive counts of [S]methionine-labeled protein from normoxic and hypoxic conditions in order to identify the desired band. The radioactive lanes were excised, dried, and exposed to Kodak XAR-5 film (Eastman Kodak Co.) for 1-2 days. The remaining gel containing unlabeled protein was stored in electrophoresis buffer until it was compared with the autoradiograph. Protein bands were visualized with 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and destained with 50% methanol, 10% acetic acid. The band corresponding to HAP47 was excised from eight lanes and combined into a single lane on a second SDS-PAGE. Following electrophoresis of the second gel, protein was electroblotted onto a poly(vinylidene difluoride) membrane and amino-terminal sequencing was performed(3) . Initial sequencing with an Applied Biosystems 470A gas phase sequencer equipped with a 120 PTH analyzer was unsuccessful due to amino-terminal blockage. Thus, tryptic digestion, HPLC separation, and protein sequencing were performed by the Harvard Microchemistry Facility (Cambridge, MA)(9, 10) . The two oligopeptide sequences obtained were compared with the ProSwiss and Medline data bases for significant identity.

Western Analysis

Using equal amounts of protein from whole cell lysates and/or subcellular fractions of EC and H446 per lane, 10% SDS-PAGE was performed. Protein was then electrophoretically transferred in buffer (20% methanol (v/v), 25 mM Tris, 192 mM glycine, pH 8.3) to nitrocellulose for 2500 mAmp-h. The nitrocellulose membrane was soaked for 1 h in PBST-M (phosphate-buffered saline, 0.1% Tween-20 (v/v), 5% non-fat powdered milk (w/v)) to block nonspecific binding. The nitrocellulose membrane was then washed with PBST (same buffer as above without milk) twice for 30 s, once for 15 min, and then twice for 5 min. The polyclonal antibody directed against enolase alpha- and -subunits was diluted in PBST according to the manufacturer's instructions and incubated with the nitrocellulose membrane for 1 h. The membrane was washed as before with PBST-M, incubated for 1 h in secondary antibody diluted in PBST-M and washed as before with PBST. Immunodetection was performed with Renaissance chemiluminescence reagent and DuPont Reflection film according to the manufacturers' instructions. Protein levels were assessed by densitometry using a Molecular Dynamics computing densitometer. In some experiments, antibody was removed from the nitrocellulose membrane by incubating with stripping buffer (100 mM beta-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) for 30 min at 50 °C and washing twice with PBST-M for 10 min at room temperature; immunodetection was repeated to ensure antibody removal. Western blot analysis using a monoclonal antibody specific for the -subunit of enolase was then performed using the same procedure.

RNA Isolation and Northern Analysis

Total RNA was isolated from cell monolayers with TriReagent-LS according to the manufacturer's instructions. Endothelial cells grown long term in 21% O(2) were exposed to 0% O(2) for various times and then placed in 21% O(2) for 1 h. Parallel cultures of control cells were maintained at 21% O(2). Fifteen µg of total RNA were separated electrophoretically through a 1% agarose-formaldehyde gel(11) . After ethidium bromide staining, RNA was transferred to a nylon membrane and UV cross-linked (Stratalinker, Stratagene, La Jolla, CA). Prehybridization was performed with QuikHyb solution at 68 °C for 1 h. The full-length 1.7-kilobase pair human NNE cDNA (ATCC) was labeled with [alpha-P]CTP by the random primer method(12) . Herring sperm DNA (50 µg/ml QuikHyb) and the NNE probe (1 times 10^6 cpm/ml QuikHyb) were added, and hybridization was carried out at 68 °C for 3 h. The blot was washed with SSC (1 times = 3 M NaCl, 0.3 M sodium citrate) and SDS as follows: 2 times SSC, 0.1% SDS at 68 °C twice for 20 min at room temperature, then once with 0.2 times SSC, 0.1% SDS for 20 min at 68 °C, and exposed to Kodak XAR-5 film for 1-4 days. To document equal loading, the nylon membranes were stripped with boiling 0.8% SDS and reprobed with either a human beta-actin cDNA probe (ATCC) labeled as above or a 28 S ribosomal RNA oligonucleotide (kind gift of Dr. Alan Fine, Boston University Medical School) end-labeled with [-P]ATP. Hybridization signals were quantitated by densitometry and normalized to either actin or 28 S ribosomal levels.

Data

Each experiment was performed three to six times; results were reproducible and qualitatively similar independent of passage number or primary cell line used. Results presented are representative examples of each experiment.


RESULTS

Protein Sequencing

Tryptic digestion and HPLC separation produced two peptide fragments: one containing 22 amino acids and a second containing 12 amino acids (Fig. 1). Significant identity was found with the glycolytic enzyme enolase. In mammalian species, three genetically distinct subunits, alpha, beta and (M(r) 47, 44, and 47, respectively) form three dimeric enolase isoforms: NSE ( and alpha), NNE (alphaalpha), and muscle-specific enolase (MSE, betabeta). While bovine enolase has not been sequenced, enolase protein and DNA sequences are known in human, mouse, and rat. The 22-amino acid sequence (excluding a 1-amino acid gap) was 100% identical with each human enolase subunit. The 12-amino acid sequence was 75, 54.5, and 58.3% homologous with the human alpha-, beta-, and -subunits, respectively.


Figure 1: Amino acid sequence of HAP47. Isolated HAP47 was internally digested with trypsin, separated by HPLC, and amino-terminally sequenced as described under ``Experimental Procedures.'' The 22-amino acid sequence (A) was 100% identical (excluding a 1-amino acid gap) to the 47-kDa alpha- and -subunits of human enolase. The 12-amino acid sequence (B) was 75 and 58% identical to the alpha- and -subunits, respectively. Significant identity was also found with the 44-kDa enolase beta-subunit.



Western Analysis and Subcellular Fractionation

Since MSE is specific for muscle and composed of lower M(r) subunits than HAP47, it was not investigated further. However, both NSE and NNE contain 47-kDa subunits and share significant identity with the HAP47 amino acid sequence. To determine whether one or both of these isoforms is HAP47, Western blot analyses with enolase antibodies were performed. Because a monoclonal antibody specific for the enolase alpha-subunit is not commercially available, the protein-containing nitrocellulose membranes were hybridized with a polyclonal antibody directed against the alpha-subunit but known to cross-react with the -subunit. The membrane was then washed and rehybridized with a monoclonal antibody specific for the isomer. Lysates from H446, a human small-cell lung cancer line which coexpresses NSE and NNE, served as a positive control (Fig. 2). These studies showed that NSE is not present in either normoxic or hypoxic EC and that HAP47 is NNE.


Figure 2: Identification of HAP47 as non-neuronal enolase (alphaalpha). Normoxic BAEC were maintained in 21% O(2) (C) or exposed to 0% O(2) for 18 h (H). H446 cells (S), a human small-cell lung cancer line coexpressing both alpha- and -subunits, served as a positive control. Whole cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analyses were performed with a polyclonal antibody directed against both the alpha- and -subunits (A) and a monoclonal antibody specific for the -subunit (B). Protein levels were assessed using chemiluminescence, autoradiography, and densitometry, as described under ``Experimental Procedures.''



Prior studies have demonstrated that HAP47 up-regulation is first detected after 4 h of exposure to 0% O(2) and is maximally up-regulated after 18-24 h(1) . Western analysis of whole cell lysates from BAEC grown in 21% O(2) and then exposed to 0% O(2) for 18 h confirmed NNE protein up-regulation by hypoxia, with a 2-fold increase in protein level (Fig. 2A). Analysis of cytosolic fractions from these cells revealed a 2.3-fold increase in protein levels after 18 h 0% O(2) (Fig. 3). Non-neuronal enolase was also consistently found in the nuclear fractions of EC. However, in contrast to cytosolic NNE, the level of NNE present in the nuclear fraction did not change with hypoxia (Fig. 3).


Figure 3: Subcellular localization of NNE protein during hypoxia. Normoxic BAEC were maintained in 21% O(2) (C) or exposed to 0% O(2) for 18 h (H). After hypoxic exposure, cells were placed in 21% O(2) for 2 h, subcellular fractionation was performed, and protein was separated by SDS-PAGE, as described under ``Experimental Procedures.'' Western blotting was performed with a polyclonal antibody directed against NNE. Densitometric protein levels are expressed as percent of control.



Northern Analysis

A time course analysis of BAEC grown in 21% O(2) and exposed to 0% O(2) showed that NNE mRNA levels began to rise after 1-2 h of hypoxia, reached a peak increase of 2.7-fold at 18 h, and remained elevated at 48 h. After return of the cells to 21% O(2) for 24 h, NNE mRNA levels returned to base line (Fig. 4A). These findings correlate with and slightly precede the time course of HAP47 protein levels (Fig. 4B). Non-neuronal enolase mRNA hypoxic regulation was examined and compared in BAEC and BPAEC grown in either 21% or 3% O(2). Bovine aortic EC and BPAEC grown in 21% O(2) up-regulated NNE mRNA 2.7- and 3.4-fold, respectively, when exposed to 0% O(2) for 18 h (Fig. 5A). Bovine aortic EC and BPAEC grown in 3% O(2) had base-line NNE mRNA levels 2-4-fold higher, respectively, than their 21% O(2) counterparts (Fig. 5B) and increased NNE mRNA 2.3-fold in hypoxia (Fig. 5C). Comparing NNE mRNA levels for the different EC conditions to the lowest level of NNE mRNA expression (BPAEC maintained in 21% O(2)), 3% BAEC, and BPAEC up-regulated NNE mRNA to the greatest extent, approximately 10-fold (Fig. 5B).


Figure 4: Time course of NNE mRNA and protein levels during hypoxia. Panel A, normoxic BAEC were either maintained in 21% O(2) or exposed to 0% O(2) for various lengths of time, as indicated. In certain experiments, after 48 h in 0% O(2), EC were returned to 21% O(2) for 24 h (REOX). Total RNA was isolated using TriReagent, separated on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to a human full-length NNE cDNA probe (middle), as described under ``Experimental Procedures.'' Densitometry values were normalized to beta-actin (bottom) and expressed as percent of control (top). Panel B, time course of NNE mRNA and HAP47 protein levels over increasing time periods of 0% O(2) exposure. Total mRNA was isolated and analyzed as described previously. Alternatively, BAEC were exposed to 0% O(2) for varying time periods, total protein was isolated and separated on a 10% SDS-PAGE, and [S]methionine-labeled HAP47 levels were assessed by densitometry, as described under ``Experimental Procedures.''




Figure 5: Endothelial cell NNE mRNA levels during hypoxia. Panel A, control BAEC and BPAEC were maintained in either 3% O(2) or 21% O(2) (C). Parallel cultures of BAEC and BPAEC maintained in either 3% O(2) or 21% O(2) were subsequently exposed to 0% O(2) for 18 h (H). Total RNA was isolated, transferred to a nylon membrane, and hybridized to a human full-length NNE cDNA probe (top) and a 28 S ribosomal RNA oligonucleotide probe (bottom), as described under ``Experimental Procedures.'' Densitometric values for NNE mRNA levels were normalized to 28 S ribosomal RNA levels and expressed as a percent of control levels. Panel B, control level of NNE mRNA for each cell type and base-line condition is compared with NNE mRNA level after exposure to 0% O(2). Panel C, non-neuronal enolase mRNA level of each cell type and condition is compared with normoxic BPAEC maintained in 21% O(2) (21% BPAEC-C).




DISCUSSION

We have previously described the HAPs, a set of cell-associated stress proteins (M(r) 34, 36, 39, 47, and 57) up-regulated by hypoxia in cultured EC (1, 2, 3) and have identified HAP36 as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase(3) . In the present study, we have identified HAP47 as NNE by amino acid sequencing of two peptide fragments and Western analysis and have demonstrated that NNE mRNA up-regulation by hypoxia begins at 1-2 h, is maximal at 18 h, persists for at least 48 h, returns to base line after 24 h in 21% O(2), and corresponds to the time-course of protein induction.

Enolase catalyzes the interconversion of 2-phosphoglycerate to phosphoenolpyruvate. Higher vertebrates possess three enolase genes, encoding alpha-, beta-, and -subunits(13) . Non-neuronal enolase, a homodimer of 47-kDa alpha-subunits, is the major isoform during embryogenesis and persists in nearly every adult tissue(14, 15) . The -subunit, also 47 kDa, is found in the and alpha dimers known collectively as NSE(16, 17) . Neuron-specific enolase is expressed mainly in normal and neoplastic cells of neuronal and neuroendocrine origin(18, 19) , but has been found in diverse mammalian cell types(20, 21) . Neuron-specific enolase has not been found in EC (21) nor has it previously been studied during exposure to hypoxia. The beta-subunit forms the homodimeric MSE. It is of a different molecular mass than HAP47 (44 kDa) and is found only in heart and skeletal muscle.

How might hypoxia-induced up-regulation of enolase augment hypoxia tolerance in EC? Of interest, hypoxia-sensitive cells, such as renal tubular epithelial cells, type II pneumocytes, fibroblasts, and smooth muscle cells, do not up-regulate a protein corresponding to enolase when subjected to hypoxic stress(2) . In hypoxia-sensitive cell types, decreased oxygen leads to energy insufficiency, followed by increased membrane permeability, loss of ion gradients, and, ultimately, irreversible cellular damage(22) . In order to obviate these effects, invertebrate cells, which are better able to tolerate hypoxia, employ various protective strategies. These include: (i) increased stores of fermentable fuel, (ii) increased tolerance of metabolic acidosis, (iii) improved handling of intracellular calcium, (iv) metabolic rate depression (metabolic arrest), (v) alterations in cellular membranes to decrease the energy requirements for vital ion pumps (channel arrest), and (vi) ATP regeneration by stores of phosphocreatine(22) . However, the ability of mammalian cells to employ these strategies appears limited, and, when exposed to hypoxia, most increase glucose utilization via increased rates of glycolysis (Pasteur effect).

Endothelial cells appear to respond differently to hypoxia than most mammalian cells. They maintain ATP and GTP levels during extended periods of hypoxia(6, 7, 8) ; however, the cellular mechanisms responsible for maintenance of these high energy phosphates are unknown. Metabolic arrest (6) and augmented phosphocreatine utilization (23) may occur in hypoxic EC and could be, in part, responsible for EC hypoxia tolerance. Thus, NNE up-regulation might contribute to hypoxia tolerance by augmenting anaerobic energy production and glycolytic competence in EC. Indeed, hypoxic BAEC up-regulate glucose transporter mRNA and protein levels and increase glucose utilization(23) ; yet, the Pasteur effect appears to be less significant in EC than in other cell types. Specifically, neither decreased oxygen consumption nor increased lactate production have been demonstrated in EC until ambient oxygen tension falls below 3 mm Hg(24) , implying that glycolysis does not increase until ambient pO(2) is below this level. This degree of hypoxia is more extreme than in our system in which the pO(2) of medium is 20-25 mm Hg after exposure to 0% O(2)(1, 4) . Moreover, HAPs are not up-regulated in EC by either glucose deprivation or inhibition of oxidative phosphorylation(2) . Thus, while increased glycolytic metabolism likely contributes to the EC response to extreme hypoxia, it may not fully explain the acquired hypoxia tolerance demonstrated in our system. Perhaps, up-regulation of enolase contributes to hypoxia tolerance through nonglycolytic mechanisms.

We and others have speculated that stress responses actively participate in adaptation to cellular hypoxia. Heat shock proteins and GRPs presumably contribute to the acquisition of tolerance to cellular stresses by aiding in protein translocation and refolding after stress (25, 26) . They are constitutively expressed in most cell types and are induced by various cellular stresses, including hypoxia(27, 28, 29) . Hypoxia-associated protein up-regulation is similar to the traditional stress response in that HAP up-regulation occurs in a time and oxygen concentration-dependent manner and occurs despite a concurrent decrease in total cellular protein. However, HAPs are distinct from HSPs and GRPs and appear to be stress- and EC-specific. In addition, while EC up-regulate HSPs and GRPs when exposed to a variety of stimuli, including heat shock, arsenite, and glucose deprivation, they do not up-regulate these traditional stress proteins when exposed to hypoxia. Thus, HAP up-regulation represents an unusual, and perhaps novel, response to hypoxia in EC(1, 2, 3) .

Like glyceraldehyde-3-phosphate dehydrogenase (HAP36) and other glycolytic enzymes, enolase may have important cellular functions separate from its catalytic function. For instance, enolase binds to F-actin and tubulin (30, 31) and has been localized to the centrosome in HeLa cells (32, 33) and to cell-surface membranes in brain tumor cells(34) . It is a lens crystallin in several vertebrates(35) , undergoes axonal transport in guinea pigs(36) , is a plasminogen receptor in a monocytoid cell line(37, 38) , and binds in vitro to single-stranded DNA in Saccharomyces cerevisiae(39) . Enolase may also play an isoform-specific role in the stress response of S. cerevisiae(40) .

Mammalian enolases may also play isoform-specific roles in enhanced cell survival. For instance, while both NSE and NNE are present in cultured rat neocortical neurons, only NSE appears to promote survival (41) . Similarly, NNE may serve a specific function in promoting EC survival in hypoxia. In this regard, a recent report suggests an association between enolase and c-myc, a proto-oncogene that plays a major role in the control of cell proliferation(42, 43) . This report (43) has identified a 35-40 kDa protein, MBP-1, that binds to and may negatively regulate the P2 promoter of c-myc in HeLa cells. Of interest, the two oligopeptides obtained in the sequencing of HAP47 were 100% homologous with MBP-1. Overall, MBP-1 is 79.8 and 68% homologous with the 215-amino acid carboxyl terminus of human NSE and NNE, respectively. While this homology may represent evolutionary colineage and not a functional similarity between these proteins, it is an intriguing possibility that enolase acts as a transcriptional regulator of c-myc.

In summary, we have identified the glycolytic enzyme NNE as HAP47, one of a distinct family of EC proteins up-regulated specifically by hypoxia. We speculate that enolase contributes directly to the hypoxia tolerance demonstrated by EC and does so, in part, through nonglycolytic mechanisms.


FOOTNOTES

*
This work was supported in part by a Research Training Fellowship (to R. M. A.) and a Career Investigator Award (to H. W. F.) from the American Lung Association, and by National Institutes of Health NHBLI Grants HL-45537 (to H. W. F.) and HL-03125 (to K. K. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Boston University School of Medicine, 80 East Concord St., R-3, Boston, MA 02118. Tel.: 617-638-4860; Fax: 617-536-8093.

(^1)
The abbreviations used are: EC, endothelial cell(s); BAEC, bovine aortic endothelial cell(s); BPAEC, bovine pulmonary artery endothelial cell(s); GRP, glucose-regulated protein; HAP, hypoxia-associated protein; HSP, heat shock protein; MSE, muscle-specific enolase; NNE, non-neuronal enolase; NSE, neuron-specific enolase; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

(^2)
K. K. Graven and H. W. Farber, unpublished observation.


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