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MS 9158 Received 15 January 1999; accepted after revision 10 March 1999.
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ABSTRACT |
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(HIF-1). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0·05).
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INTRODUCTION |
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Sufficient O2 delivery is essential for energy metabolism of tissues. The regulation of O2 supply involves acute changes in ventilation and perfusion, and the more delayed genetic adaptation of erythropoiesis and angiogenesis (Gleadle & Ratcliffe, 1998). Red blood cell production is under the control of the glycoprotein hormone erythropoietin (EPO) which is synthesized in the kidneys and the liver in response to hypoxia (Jelkmann & Metzen, 1996). Angiogenesis is physiologically important in the prenatal period and childhood, but also occurs in wounds and solid tumors due to the local lack of O2. Here, the most specific mitogen appears to be vascular endothelial growth factor (VEGF) which is produced at low PO2 in normal and malignant tissues (Ferrara & Davis-Smyth, 1997). As a result of alternative exon splicing, VEGF may exist as different molecular species, with the 165 amino acid component being the predominant, secreted form.
Both augmented gene transcription and post-transcriptional processes account for the increase in EPO and VEGF production rates at low PO2 (Goldberg & Schneider, 1994). EPO and VEGF synthesis share some regulatory mechanisms including the participation of a haem protein as the O2 sensor and cis-acting DNA elements that are responsive to hypoxia-inducible factor-1 (HIF-1; Wang & Semenza, 1996; Wenger & Gassmann, 1997; Gleadle & Ratcliffe, 1998). In addition, there are similarities in the signal transduction pathways with several other proteins involved in the control of the O2 supply and energy metabolism of the tissue (Maxwell et al. 1993). Calcium ions may influence the production of secreted proteins at different steps, including gene transcription, mRNA stabilization, glycosylation and secretion (Morgan & Curran, 1986; Wodnar-Filipowicz & Moroni; 1990; Oda, 1992; Kimball & Jefferson, 1992; Kuznetsov et al. 1993). However, the role of Ca2+ in the control of hypoxia-induced EPO and VEGF gene expression and protein secretion is incompletely understood. Low extracellular Ca2+ has been shown to increase EPO production in a human renal carcinoma cell line (Nagakura et al. 1987). The calcium ionophore A23187 has been reported not to induce EPO production by human hepatoma (Hep3B) cells (Goldberg et al. 1988). However, treatment with the intracellular Ca2+ chelator BAPTA-AM reduced VEGF mRNA levels in cultures of adenovirus-transformed human fetal renal (HEK 293) cells (Mukhopadhyay & Akbarali, 1996).
In the present work the role of Ca2+ was studied in hypoxic Hep3B cells. This human hepatoma cell line is a very useful model because it expresses both the EPO and the VEGF gene in a PO2-dependent manner (Goldberg et al. 1987; Goldberg & Schneider, 1994) and, thus, enables one to compare directly responses of the two growth factors. Compounds used for the study included modulators of the extracellular Ca2+ concentration (CaCl2, EGTA), a calcium ionophore (ionomycin), membrane permeant Ca2+ chelators (BAPTA-AM, EGTA-AM) and an endoplasmic reticulum Ca2+-ATPase inhibitor (thapsigargin). The results suggest that manipulation of the Ca2+ flux and the intracellular concentration of free Ca2+ have little influence on VEGF and EPO synthesis. VEGF secretion tended to be inversely correlated with the cytosolic Ca2+ concentration, while EPO production was inhibited by increases and decreases of intracellular calcium levels.
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METHODS |
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Reagents
BAPTA-AM and ionomycin were purchased from Calbiochem (Bad Soden, Germany); EGTA was obtained from Serva (Heidelberg, Germany); and EGTA-AM and thapsigargin were from MoBiTec (Göttingen, Germany). A cytotoxicity assay based on the capacity of the cells to reduce tetrazolium salt to formazan was used as reported previously (Wolff & Jelkmann, 1993) to ensure that the compounds used did not affect cell viability.
Cell culture
Human hepatoma cells of the line Hep3B (ATCC HB 8064) were grown in RPMI 1640 medium (Gibco BRL) supplemented with 10 % fetal bovine serum and 2 mM glutamine. Cultures were maintained in 75 cm2 flasks (Falcon, Becton Dickinson, Heidelberg, Germany) and plated onto 24-well dishes (Nunc, Wiesbaden-Biebrich, Germany) for all experiments except when cells were scheduled for Western blot analysis (see below). After 6-7 days cell cultures were confluent. Medium was exchanged 24 h prior to all incubations. In the 24-well dishes experiments were started by addition of 1 ml culture medium with or without the desired reagents. Under these conditions pericellular PO2 reached a value of less than 2 mmHg within 2 h and then remained constant, i.e. cells were hypoxic (Metzen et al. 1995). All experiments were carried out on at least four separate cultures. After the incubation, medium was collected for EPO and VEGF determination.
Measurement of intracellular free calcium concentration ([Ca2+]i)
Cells were cultured in 24-well plates (Nunc) as above. Six to seven days after subcultivation cells were incubated with Hepes-buffered Tyrode salt solution (pH 7·4, Gibco BRL) containing the fluorescent calcium indicator fura2-AM (5 µM, MoBiTec). After an incubation period of 60 min at 37°C the cells were washed to remove extracellular fura2-AM. Measurement of [Ca2+]i was started 20 min later using a Deltascan 4000 system (Photon Technology International, Wedel, Germany) linked to an inverted phase-contrast microscope (× 20 objective, Olympus, Hamburg, Germany). During measurements, the temperature was kept constant by perfusion of the space between the culture wells with water heated to 37°C. Each field of vision contained approximately 150 cells which were alternately illuminated at 340 and 380 nm. Fura2 emissions (510 nm) were amplified by a photomultiplier and recorded twice per second. At the end of each experiment fura2 fluorescence was quenched by the addition of MnCl2 (8 mM) to determine background fluorescence. After subtraction of background fluorescence, the 340 nm/380 nm ratio was computed and transformed into intracellular free calcium concentration. A calibration curve was obtained by recording fluorescence of 10 standards that contained defined concentrations of Ca2+ and EGTA.
Assay of EPO and VEGF
EPO concentration in cell culture media was measured by radioimmunoassay (RIA) as described elsewhere (Wolff & Jelkmann, 1993). In brief the assay system included 125I-labelled recombinant human EPO (high specific activity; Amersham Buchler, Braunschweig, Germany), rabbit anti-EPO serum and human urinary EPO as the standard, which was calibrated by bioassay against International Reference Preparation B (Annable et al. 1972). Free EPO and antibody-bound EPO were separated by precipitation with polyethlene glycol 6000 (Merck, Darmstadt, Germany). Performance parameters were as follows: lower detection limit, 5 U ml-1; intra-assay variance < 6 %; and interassay variance < 12 % in the range 20-100 U ml-1.
In the same samples VEGF was determined by enzyme-linked immunosorbent assay (ELISA; Quantikine, R & D Systems, Minneapolis, USA). Antibodies were directed against human recombinant VEGF. The immobilized antibody was monoclonal, while the secondary horseradish peroxidase-conjugated antibody was polyclonal. The intra- and interassay coefficients of variance were 5 and 7·5 %, respectively. The lower detection limit was 9 pg ml-1.
Northern blot analysis
At the end of the experiments Hep3B cells were washed with ice-cold phosphate-buffered saline (PBS, pH 7·4) and lysed with guanidinium thiocyanate (4 M in PBS with 0·1 M 
-mercaptoethanol). Total RNA was isolated by the acidic phenol-chloroform method (Chomczynski & Sacchi, 1987); RNA concentration was calculated from the absorption at 260 nm. Total RNA (15 µg per lane) was separated by electrophoresis in denaturing gels containing 1·1 % (w/w) agarose in Mops buffer (40 mM Mops, 10 mM sodium acetate, 1 mM EDTA, pH 7·0) with 0·7 M formaldehyde. The RNA was transferred onto nylon membranes (Nytran plus, Schleicher & Schuell, Dassel, Germany) by vacuum blotting (Pharmacia vacuum blotter) with 20 × SSPE buffer, then cross-linked to the filters by exposure to UV light and baked for 2 h at 80°C. Prehybridization was in 5 × SSC with 45 % formamide, 5 × Denhardt's solution, 0·1 % SDS and 100 µg ml-1 sonicated, denatured salmon sperm DNA for 3 h at 42°C. Hybridizations were performed in fresh solution of identical composition supplemented with the radioactive probe (0·5 × 106 to 2 × 106 c.p.m. ml-1) for 2 days at 42°C. After hybridization filters were washed twice for 15 min in 2 × SSC-0·1 % SDS at 50°C, and subsequently two times in 0·2 × SSC-0·1 % SDS at 50°C. Filters were sealed in plastic bags and analysed in a Fuji BAS 2000 phosphoimager (Fuji, Germany).
Hybridization probes were generated by PCR from published sequences for human EPO and VEGF and were labelled using a commercially available DNA labelling kit (MBI Fermentas, Vilnius, Lithuania).
Western blot analysis
HIF-1 content of the cell cultures was determined by immunoblotting. For these experiments cells were cultured in 177 cm2 dishes (Sarstedt, Nümbrecht, Germany) using 15 ml cell culture medium for an incubation period of 4 h. This time course is desirable because it has been shown for Hep3B cells that HIF-1 levels are maximal after 4 h of hypoxia and decline thereafter (Wiesener et al. 1998). To induce hypoxia rapidly cells were removed from a normoxic atmosphere (20 % O2, 5 % CO2, balance N2) and placed in an incubator that contained 3 % O2, 5 % CO2, balance N2.
Whole-cell protein extraction and immunoblotting were done as described previously by Wiesener et al. (1998). In brief, cultures were washed with ice-cold PBS and detached by scraping. Cells were lysed in extraction buffer (7 M urea, 10 % glycerol, 10 mM Tris-HCI pH 6·8, 1 % sodium dodecyl sulfate (SDS), 5 mM dithiothreitol, 0·5 mM phenylmethyl sulfonyl fluoride (PMSF), with 1 mg l-1 each aprotinin, pepstatin and leupeptin). Protein concentration was determined using the biuret and Folin phenol reagents (Sigma). Samples were run on SDS-polyacrylamide (7·5 %) gels and transferred to nitrocellulose membranes (Amersham) electrophoretically (Trans-Blot SD, BioRad, München, Germany). Equal loading and transfer were verified by staining with 2 % Ponceau S. Membranes were blocked overnight with PBS-5 % fat-free skimmed milk and then incubated with a monoclonal mouse antibody raised against human HIF-1 (Transduction Laboratories, Lexington, USA) diluted to 0·5 µg ml-1, for 2 h at room temperature. For detection a horseradish peroxidase-linked anti-mouse IgG antibody (Santa Cruz, Heidelberg, Germany; 1 : 2000, 1 h incubation at room temperature) and enhanced chemiluminescence (ECL) substrate (Amersham) were used.
Statistical analysis
The data are presented as means ± standard deviation (S.D.). Student's t test for unpaired data was used to determine significant differences between two group means. Dunnett's post hoc test was applied to compare a control mean with several treatment means. P < 0·05 was considered statistically significant.
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RESULTS |
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Variation of extracellular Ca2+ concentration
In all experiments RPMI 1640 medium was used, which contains 0·5 mM Ca2+ according to the manufacturer's declaration. [Ca2+]o was decreased by application of 0·5 mM EGTA or increased by addition of CaCl2 to yield a final concentration of 1 mM. Higher doses of Ca2+ reduced cellular viability. None of the manipulations of [Ca2+]o resulted in a significant change in the 24 h rate of production of EPO or VEGF (data not shown).
Cytosolic free Ca2+ concentration
The membrane permeant versions of BAPTA and EGTA are widely used to chelate cytosolic Ca2+ and thus to decrease [Ca2+]i (e.g. Ju & Allen, 1998). In the presence of extracellular calcium BAPTA-AM enhanced EPO synthesis in a dose-dependent manner (control, 21·3 ± 3·5 mU ml-1; 1 µM BAPTA, 27·1 ± 6·0 mU ml-1, P < 0·05; 5 µM BAPTA, 33·4 ± 2·6 mU ml-1, P < 0·01). However, VEGF production was not affected (Fig. 1A). RNA analysis did not reveal different levels of EPO or VEGF mRNA in the cells (Fig. 2). It is questionable, however, whether the effect of BAPTA-AM on EPO secretion was caused by a decrease of [Ca2+]i because BAPTA has been shown to bind not only Ca2+ but also Fe2+ (Britigan et al. 1998). Furthermore, chelation of cytosolic calcium was possibly compensated by an increased inward calcium leak. We therefore tested the effects of EGTA-AM in the absence of extracellular calcium. Incubation with a combination of 0·5 mM EGTA to chelate extracellular Ca2+ and 10 µM EGTA-AM induced a remarkable accumulation of HIF-1 (Fig. 4). VEGF secretion was consistently and significantly enhanced (Fig. 1B; control, 3·1 ± 0·6 ng ml-1; 5 µM EGTA-AM, 4·0 ± 0·5 ng ml-1; 10 µM EGTA-AM, 4·2 ± 0·4 ng ml-1). However, there was a dose-dependent reduction in EPO secretion (control, 24·1 ± 1·2 mU ml-1; 5 µM EGTA-AM, 16·2 ± 1·1 mU ml-1; 10 µM EGTA-AM, 14·1 ± 1·4 mU ml-1).
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(C) on EPO and VEGF production in hypoxic Hep3B cells
EPO ( | ||
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Fifteen micrograms of total RNA were loaded per lane and hybridized with an EPO or VEGF cDNA probe. Equal loading was confirmed by ethidium bromide staining of 28S and 18S rRNA. | ||
The calcium ionophore ionomycin is known to elevate [Ca2+]i by triggering a rapid release of Ca2+ from intracellular stores (Smith et al. 1989). In our experiments 1 µM ionomycin led to an immediate increase of [Ca2+]i that was sustained for at least 10 min (Fig. 3B). Ionomycin significantly decreased EPO synthesis (100·2 ± 25·0 vs. 67·2 ± 14·9 mU ml-1, P < 0·05) and also tended to decrease VEGF production (1·9 ± 0·1 vs. 1·7 ± 0·2 ng ml-1); this effect was not statistically significant, however. Ionomycin did not affect cellular HIF-1 content (Fig. 4).
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A, the effect of 10 nM thapsigargin on [Ca2+]i. B, superimposed Ca2+ traces from 3 different cultures. * Effect of 1 µM ionomycin without pretreatment. | ||
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expression in Hep3B cellsNormoxic control was incubated for 4 h in an atmosphere of 20 % O2, 5 % CO2, balance N2. Hypoxia was induced by placing the cultures in an atmosphere of 3 % O2, 5 % CO2, balance N2 for 4 or 24 h as indicated. Protein extraction and Western blotting were done as described in the Methods section. Reactive proteins were detected using the ECL system (Amersham). | ||
Store depletion
The tumor-promoting agent thapsigargin inhibits endoplasmic reticulum Ca2+-ATPase (Kijima et al. 1991). Thus it depletes intracellular calcium stores and frequently elicits a marked elevation of [Ca2+]i (Gericke et al. 1993). In Hep3B cells, however, 10 nM thapsigargin induced only a small increase of [Ca2+]i within 10 min after application (Fig. 3A). After an incubation period of 24 h the calcium content of intracellular stores was considerably reduced (Fig. 3B) while cytosolic Ca2+ concentration was unaffected. Thapsigargin (10 nM) inhibited EPO production (control, 38·2 ± 4·7 mU ml-1; 5 nM thapsigargin, 41·0 ± 7·9 mU ml-1, n.s.; 10 nM thapsigargin, 23·1 ± 6·6 mU ml-1, P < 0·01). At 5 nM the drug significantly enhanced VEGF synthesis (4·6 ± 1·2 vs. 6·4 ± 1·3 ng ml-1, P < 0·05). Interestingly, this effect was not seen after application of 10 nM thapsigargin (Fig. 1C) although this dosage has been shown to be non-cytotoxic. Differences in EPO or VEGF mRNA content of the cells were hardly detectable on Northern blots (Fig. 2). The effects of thapsigargin on EPO and VEGF secretion seem to be independent of HIF-1 activation because HIF-1 levels remained unchanged (Fig. 4).
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DISCUSSION |
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Calcium ions are important in the control of the activity of various intracellular enzymes and may influence the rate of secretion of proteohormones. We have studied whether alterations of the extra- and intracellular Ca2+ concentration have a significant impact on the production of EPO and VEGF in hypoxic Hep3B cells. This cell line, as well as another human hepatoma cell line, HepG2, have proved to be very useful models to study PO2-dependent EPO and VEGF gene expression (Bunn & Poyton, 1996). The cellular responses to hypoxia are largely mediated by a transcription factor termed hypoxia-inducible factor-1 (HIF-1). Therefore we also investigated whether manipulations of cellular calcium induce an accumulation of HIF-1. We found that only chelation of cytosolic calcium in the absence of extracellular Ca2+ elevated cellular HIF-1 levels while treatment with the calcium ionophore ionomycin or store depletion with thapsigargin failed to have a gross effect on HIF-1. Interestingly, there was a good correlation between cellular HIF-1 levels and VEGF gene expression. Conversely, EPO production was decreased by chelation of cytosolic Ca2+, by the addition of ionomycin, and by depletion of intracellular storage sites. Thus, our experiments provide further evidence that the synthesis of these two hypoxia-inducible proteins is controlled by overlapping, yet distinct signalling mechanisms.
While this study was in progress, Bae et al. (1998) performed a differential display analysis of normoxic and hypoxic HepG2 cells. Interestingly, the oscillin gene was among the differentially expressed genes induced with hypoxia. Oscillin mediates Ca2+ release from intracellular stores (Galione et al. 1997). When we blocked Ca2+ uptake into the endoplasmic reticulum, thus depleting this intracellular calcium store, we obtained different effects on the two hypoxia-induced genes VEGF and EPO. Figure 3B shows that our depletion manoeuvre was effective. Thus, one might interpret the inhibition of EPO synthesis to be a result of the depletion of intracellular Ca2+ stores. This would be in line with results reported by Mukhopadhyay & Akbarali (1996) who found that the hypoxia-induced VEGF production is inhibited by caffeine in HEK293 cells. However, we did not find a significant decrease in VEGF mRNA or protein production in Hep3B cells with 10 nM thapsigargin. The moderate increase in VEGF secretion caused by 5 nM thapsigargin remains poorly understood.
Unexpectedly and in contrast to what Mukhopadhyay & Akbarali (1996) have reported with respect to VEGF mRNA levels, BAPTA-AM did not lower hypoxia-induced VEGF or EPO gene expression. In fact, BAPTA-AM at 1 and 5 µM significantly enhanced EPO secretion into the culture medium of Hep3B cells. Two explanations for this discrepancy have to be considered. First, we used only 5 µM BAPTA-AM since Hep3B cells show clear signs of reduced viability with higher concentrations, whereas Mukhopadhyay & Akbarali (1996) pretreated their cells with 20 µM BAPTA-AM. Second, BAPTA-AM has been found to have several pharmacological effects (Parekh & Penner, 1995) that may be more or less pronounced in different cell types. In particular BAPTA-AM has recently been described as a potent iron chelator (Britigan et al. 1998). This may be relevant in view of the fact that a haem-containing protein is involved in the regulation of hypoxia-inducible gene expression (Bunn & Poyton, 1996). Interestingly, VEGF gene expression was found to be relatively unaffected by BAPTA-AM. To avoid these hazards and to exclude the possibility that cytosolic calcium chelation is balanced by an increased inward leak, we repeated the experiments with another membrane permeant Ca2+-chelating agent, EGTA-AM, in the absence of extracellular calcium ions. This treatment led to an apparent accumulation of HIF-1 in parallel with an increase in VEGF secretion. Surprisingly, EPO production was dose-dependently and significantly reduced. These data may indicate that elevated HIF-1 levels are not sufficient to drive hypoxia-induced EPO gene expression.
In general, however, the effect of all substances was moderate, even when the effects reached statistical significance. In Hep3B cells hypoxia is able to elicit a 50- to 100-fold stimulation of EPO mRNA levels compared with normoxia (Fandrey & Bunn, 1993). In our experiments alterations of VEGF or EPO mRNA levels were hardly detectable. The inhibition of EPO protein secretion exerted by 10 µM thapsigargin was the only effect that was confirmed by lowered EPO mRNA levels.
In conclusion, we believe that intracellular Ca2+ concentration does not play a major role in the control of hypoxia-induced gene expression. Other divalent cations that interfere with redox-sensitive proteins like Fe2+ (Fandrey et al. 1997) or Ni2+ and Co2+ (Porwol et al. 1998) appear to be of much greater importance with respect to oxygen-dependent gene expression.
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E. Metzen: Institute of Physiology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
Email: metzen{at}physio.mu-luebeck.de
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) and VEGF (
Effect of 1 µM ionomycin in Ca2+-free buffer, i.e. after 2 min of incubation with excess EGTA. The increase of [Ca2+]i was caused solely by release of Ca2+ from intracellular stores. 
Effect of 1 µM ionomycin in Ca2+-free buffer after a 24 h incubation with 10 µM thapsigargin in the presence of Ca2+.