(Received for publication, June 19, 1995; and in revised form, August 7, 1995)
From the
The hypoxia-associated proteins (HAPs) are five cell-associated
stress proteins (M 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
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.
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) ()offer an
excellent example of adaptation to extreme hypoxia. For example,
pulmonary arterial EC normally reside in a pO
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. (
)
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 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.
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 - and
-subunits of human enolase. The 12-amino acid
sequence (B) was 75 and 58% identical to the
- and
-subunits, respectively. Significant identity was also found with
the 44-kDa enolase
-subunit.
Figure 2:
Identification of HAP47 as non-neuronal
enolase (). Normoxic BAEC were maintained in 21% O
(C) or exposed to 0% O
for 18 h (H). H446 cells (S), a human small-cell lung cancer
line coexpressing both
- 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
- 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 and is maximally up-regulated
after 18-24 h(1) . Western analysis of whole cell lysates
from BAEC grown in 21% O
and then exposed to 0% O
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
(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 (C) or exposed to 0% O
for 18 h (H). After hypoxic exposure, cells were placed in 21% O
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.
Figure 4:
Time course of NNE mRNA and protein levels
during hypoxia. Panel A, normoxic BAEC were either maintained
in 21% O or exposed to 0% O
for various lengths
of time, as indicated. In certain experiments, after 48 h in 0%
O
, EC were returned to 21% O
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
-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
exposure. Total mRNA was isolated and analyzed as
described previously. Alternatively, BAEC were exposed to 0% O
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 or 21% O
(C). Parallel
cultures of BAEC and BPAEC maintained in either 3% O
or 21%
O
were subsequently exposed to 0% O
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
. Panel C, non-neuronal enolase mRNA level of each cell type and
condition is compared with normoxic BPAEC maintained in 21% O
(21% BPAEC-C).
We have previously described the HAPs, a set of
cell-associated stress proteins (M 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
, 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 -,
-, and
-subunits(13) . Non-neuronal enolase, a homodimer of
47-kDa
-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
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
-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 is below this level.
This degree of hypoxia is more extreme than in our system in which the pO
of medium is 20-25 mm Hg after exposure
to 0% O
(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.