(Received for publication, July 25, 1995)
From the
Transcriptional regulation of gene expression by hypoxia is an
important, but yet only marginally characterized mechanism by which
organisms adapt to low oxygen concentrations. The human hepatoma cell
line HepG2 is a widely used model for studying hypoxic induction of the
hematopoietic growth factor erythropoietin. In an attempt to identify
additional genes expressed in HepG2 cells during hypoxia, we
differentially screened a cDNA library derived from hypoxic (1%
O) HepG2 cells using probes isolated from either normoxic
(21% O
) or hypoxic cells. Two genes were identified, one
encoding aldolase, a member of the glycolytic enzymes, and the other
encoding
-antitrypsin which belongs to the family of
the acute phase (AP) responsive proteins. Whereas hypoxic induction of
glycolytic enzymes is well established, oxygen-dependent regulation of
AP genes has not been reported so far. AP proteins are liver-derived
plasma proteins whose production during inflammation is either
up-regulated (positive AP reactants) or down-regulated (negative AP
reactants). In the present study, we demonstrate that on the mRNA level
hypoxic stimulation of HepG2 cells led to (i) an induction of the
positive AP reactants
-antitrypsin,
-antichymotrypsin, complement C3, haptoglobin, and
-acid glycoprotein; (ii) a down-regulation of the
negative AP reactant albumin; (iii) an up-regulation of the negative AP
reactant transferrin; and (iv) unchanged levels of the positive AP
reactants
- and
-fibrinogen as well as hemopexin.
Cycloheximide inhibited hypoxic up-regulation of AP mRNAs demonstrating
that de novo protein synthesis is required for hypoxic
induction. Nuclear run-on assays indicate that the hypoxic increase in
AP mRNAs is mainly due to transcriptional regulation. The hypoxic
response was compared to AP stimulation by interleukin 6. The results
suggest that the adaptive response to hypoxia overlaps with, but is not
identical with, the AP response mediated by interleukin 6.
Many insights into the mechanisms of oxygen-regulated gene
expression have been provided by the study of hypoxia-induced
erythropoietin (EPO) ()gene
expression(1, 2, 3) . The glycoprotein
hormone EPO is the predominant stimulator of erythropoiesis in bone
marrow(4, 5) . EPO is mainly produced in fetal liver
and adult kidney and, to some extent, also in adult liver. Following
exposure to hypoxia caused by high altitude or anemia, for example, EPO
levels in the blood increase 500- to 2000-fold (6) . So far,
the human hepatoma cell lines HepG2 and Hep3B are the only permanent
cell culture models available to study oxygen-regulated EPO
production(7) . When cultured under hypoxic conditions (1% versus 21% O
), both cell lines show an increased
EPO secretion which is mainly transcriptionally regulated (8, 9) .
Other genes have been found to be induced by hypoxia in many different tissues as well (reviewed in (10) ). The wide variety of these genes can be divided roughly into three classes. The first class includes molecules that are favorable for the adaption of the whole organism to general hypoxia, such as EPO which elevates the oxygen transport capacity of the blood. The second class comprises local acting factors that ensure the survival of tissues exposed to local hypoxia due to high oxygen consumption, reduced blood supply, or injury, for example. One example is vascular endothelial growth factor (VEGF), a potent angiogenic factor leading to increased vascularization of the affected tissue. Hypoxic induction of VEGF has been found in many different tissues and tumors(11) , as well as in hepatoma cells(12) . The third class consists of intracellular factors involved in the adaption of the cell to hypoxia, such as ubiquitously expressed glycolytic enzymes which provide ATP through anaerobic glycolysis (13) or transcription factors of the Jun and Fos family which are induced by low oxygen in cardiac myocytes and hepatoma cells(12, 14) . Apart from the assumption that the oxygen sensor might be a hemeprotein(15) , neither the nature of this molecule nor the mechanisms leading to enhanced gene expression have so far been characterized clearly(16) .
The HepG2 and
Hep3B cell lines are not only used extensively to study the regulation
of EPO gene expression, but represent also a common model system for
investigating proinflammatory, cytokine-dependent expression of acute
phase (AP) proteins. The AP response is a protective physiological
reaction of the organism to disturbances of its homeostasis due to
inflammation caused by tissue injury, infection, or neoplastic growth
(reviewed in (17, 18, 19) ). Characteristics
of an AP response after local injury include the release of cytokines (e.g. IL-1, IL-6, IL-11, tumor necrosis factor , leukemia
inhibitory factor, and oncostatin M) which in turn induce a systemic
reaction manifested by, for example, fever, elevated secretion of
glucocorticoids, and changes in the concentration of a specific set of
plasma proteins, termed AP proteins, which are mainly produced in the
liver. These AP proteins are either up-regulated (positive AP
reactants) or down-regulated (negative AP reactants) during the AP
response. Protease inhibitors, blood coagulation factors, transport
proteins, and complement components are examples of positive AP
reactants which are commonly up-regulated 2- to 10-fold on both the
mRNA and protein levels. Typical negative AP reactants include albumin
and transferrin. IL-6 has been shown to be the major mediator of the AP
response in both hepatoma cell lines in vitro and in rats in vivo, but IL-1, tumor necrosis factor
, leukemia
inhibitory factor, and other cytokines are also capable of partially
mediating the AP response. The spectrum of AP proteins induced in
hepatoma cells, however, varies qualitatively and quantitatively
between the different cytokines studied.
Unexpectedly, differential screening of a cDNA library derived from hypoxic HepG2 cells identified hypoxic up-regulation of an AP protein family member, encouraging us to analyze the response of other AP proteins to hypoxia in HepG2 cells.
The first clone was identical with fructose-1,6-bisphosphate aldolase A, starting 18 base pairs upstream of the AUG translation initiation codon(27) . Northern blot hybridizations (Fig. 1) revealed a time-independent 2- to 3-fold accumulation of aldolase mRNA during 24 to 72 h of hypoxia in HepG2 cells (see below). This result was consistent with nuclear run-off experiments in skeletal muscle cells, where aldolase transcription rates have been reported to be induced 2- to 5-fold by low oxygenation(13) . During hypoxia, when anaerobic glycolysis is the major source of ATP, the induction of glycolytic activity ensures constant energy supply to the cell(28) . The cloning of a hypoxia-inducible glycolytic enzyme, however, confirmed the accuracy of our differential screening approach.
Figure 1:
Northern blot analysis of
hypoxia-induced HepG2 cells. HepG2 cells were induced by exposure to 1%
O for 24 h to 72 h at an initial cell density of 1
10
/cm
. Normoxic (21% O
) control
experiments were performed in parallel for each time point. Equal
amounts (10 µg) of total RNA were loaded. The signal obtained with
a 28 S rRNA probe was used to control for equal loading and blotting
efficiency.
The second clone was identified
as -antitrypsin, beginning 11 bp upstream of the
translational start site(29, 30) . Its mRNA was found
to be induced in a time-dependent manner in HepG2 cells (Fig. 1). Since
-antitrypsin belongs to the
group of plasma proteins induced in hepatocytes during the AP
response(17) , we tested if other members of this family are
induced by hypoxia as well.
Figure 2: EPO protein levels in the supernatant of HepG2 cultures. HepG2 cells were induced by hypoxia as described in Fig. 1. EPO concentrations were determined by a radioimmunoassay using recombinant human EPO. Results are expressed as nanounits of EPO per cell.
Northern blot analysis was then
performed using hybridization probes for EPO and VEGF, as well as
positive AP reactants (-antitrypsin,
-antichymotrypsin, complement C3, haptoglobin,
-acid glycoprotein, hemopexin, and
- and
-fibrinogen) and negative AP reactants (albumin and transferrin).
The signals were quantitated by PhosphorImager analysis and corrected
for differences in loading and blotting by hybridization to a 28 S
ribosomal probe as exemplified in Fig. 1.
-Actin, another
commonly used normalization probe, was found to be reproducibly induced
by a factor of approximately 1.5 ( Fig. 1and 3A). A
weak transcriptional activation of
-actin by hypoxia has already
been reported in rat skeletal muscle cells(13) . Hence,
-actin is inappropriate for normalization of these experiments.
Hypoxic response of HepG2 cells was verified by analyzing the induction
rates for EPO and VEGF. Under the stated experimental conditions, EPO
and VEGF mRNAs were induced 3.5- to 7-fold and 4.5- to 11-fold,
respectively, after 24 h to 72 h of hypoxia (Fig. 3A). Fig. 3B revealed a similar 3- to 7-fold time-dependent
induction of AP protein-encoding mRNAs
(
-antichymotrypsin, complement C3,
-antitrypsin, and haptoglobin) after 48 h to 72 h of
hypoxic incubation. The negative AP reactant albumin was down-regulated
by hypoxia, as it is during the in vivo AP response.
Surprisingly, although fibrinogens are among the most prominent
positive AP reactants induced by IL-6 in HepG2 cells(17) , mRNA
levels of the coordinately expressed
- and
-fibrinogen genes (32, 33) were not significantly affected by hypoxia.
The positive AP reactants
-acid glycoprotein and
hemopexin were also only marginally regulated by oxygen. Moreover,
transferrin, which is down-regulated by IL-6 and tumor necrosis factor
in HepG2 cells in vitro and also during the AP response in vivo(17) , was induced by hypoxia up to 4.5-fold.
Hypoxic exposure for up to 24 h at an 8-fold higher initial cell
density did not significantly induce mRNA levels of AP proteins or of
EPO or VEGF (not shown), in accordance with the previously reported
cell density dependence of EPO expression in HepG2 cells(7) .
Time course and extent of hypoxic induction of AP mRNAs was similar to
the mRNAs encoding EPO and VEGF. A maximum was reached at 72 h of
incubation, whereas hypoxic induction of aldolase mRNA remained
relatively constant over the time points examined (Fig. 3A). In summary, these results suggest a common
mechanism for hypoxic induction of EPO, VEGF, and AP mRNAs and possibly
a distinct mechanism for induction of the glycolytic enzyme aldolase.
Figure 3: Time-dependent induction of steady-state mRNA levels following hypoxic treatment of HepG2 cells. Northern blots were quantified by PhosphorImager analysis and normalized by hybridization to a 28 S rRNA probe. Results are shown as percentage of the corresponding normoxic control. A, hypoxic induction of non-AP genes. B, hypoxic induction of AP genes.
Figure 4: IL-6 induction of AP genes in HepG2 cells. The cells were induced with 20 ng/ml IL-6 in the presence of 1 µM dexamethasone (DXM). Steady-state mRNA levels were determined as described in Fig. 3.
A comparison of hypoxic and IL-6/dexamethasone treatment of HepG2 cells in vitro opposed to the AP response in vivo is shown in Table 1. The results suggest that most of the liver AP genes can also be induced by IL-6/dexamethasone in HepG2 cells. However, Table 1revealed a hypoxia-specific AP mRNA pattern in HepG2 cells which was overlapping but not identical with the cytokine-induced pattern observed in vitro and in vivo. This pattern also did not correspond to the two classes of AP proteins, proposed by Baumann and Gauldie (34) based on their hormone requirement.
Figure 5:
Transcriptional regulation of
hypoxia-inducible AP gene expression. Northern blot analysis and
nuclear run-on assays were performed using parallel HepG2 cultures
induced at 1% O for 48 h. Signal intensities were corrected
for vector background, normalized to the signal obtained with a
ribosomal protein L28 cDNA probe, and expressed as percentage of the
normoxic control. The mean values of two independent experiments are
shown. Run-on signals for VEGF were less than 2-fold above background
level and hence not included in this figure. n.d., not
detectable.
Figure 6:
Effect of cycloheximide on hypoxic AP gene
induction. HepG2 cells were induced for 48 h at 1% O with
or without 20 µg/ml cycloheximide, and total RNA was analyzed by
Northern blotting as described in Fig. 3.
Both,
-antitrypsin and
-antichymotrypsin
inhibit proteases (neutrophil-derived elastase and cathepsin G,
respectively) that otherwise could lead to proteolytic degradation of
lung tissue and emphysema, thereby affecting general oxygen supply to
the organism(38, 39) . It is tempting to speculate
that induction of these protease inhibitors might contribute to
protecting the lung from tissue damage caused by neutrophils which are
known to invade the lung of hypoxic mice(40) .
Since iron is an essential component for hemoglobin synthesis during red cell formation in bone marrow, hypoxic up-regulation of the iron transport protein transferrin might support EPO-induced erythropoiesis by enhanced iron supply. Indeed, increased transferrin serum levels in mice (41) and rats (42) that were exposed to 50% atmospheric pressure for 1 to 3 days have been reported. Likewise, hypoxic induction of the hemoglobin-binding transport protein haptoglobin might sustain enhanced erythropoiesis by preventing the loss of heme iron from the kidney. Besides many other functions, complement C3 is involved in erythrocyte degradation, thereby maintaining constant erythrocyte lifespan (reviewed in (43) and (44) ). Complement C3 induction might be required to keep the balance between plasma complement C3 concentrations and decay accelerating factor under conditions of enhanced erythropoiesis. Decay accelerating factor is present on the surface of erythrocytes and protects them from complement-mediated cell lysis.
Cellular stress could be another common inducer of AP proteins and EPO under hypoxic conditions. Heat shock is a well-established activator of stress-responsive genes and the question arose whether heat shock and hypoxia are two different stimuli resulting in the expression of the same genes. Several lines of evidence indicate that this is not the case. Despite reports that anoxia (59) and anoxia followed by reoxygenation (60) induce heat-shock proteins in mammalian cell lines, oxygen tensions similar to those used in our experiments failed to induce the major heat-shock proteins(3) . On the other hand, IL-6 does not induce the heat-shock protein Hsp70 in Hep3B cells(61) . Furthermore, neither EPO (3, 15) nor AP proteins (60) are induced by heat shock in hepatoma cells. In this context, it is noteworthy that although the oxygen tension in our experiments (7 mm Hg) is 21-fold reduced compared to ambient air (140 mm Hg), it represents only about a 3.5-fold reduction compared to the average oxygen tension measured in vivo at the liver surface (approximately 25 mm Hg, (62) ). This suggests that the hypoxic conditions used in our experiments represent a rather mild physiological stimulant compared to the pathological conditions of anoxia or heat shock, both of which result in heat-shock protein expression.
In summary, inflammation-independent hypoxic induction of AP protein gene expression in hepatic and probably also extrahepatic tissues could shed new light on the mechanisms by which organisms adapt to hypoxia.