Symposium Papers |
Correspondence to: H. Dariush Fahimi, Inst. for Anatomy and Cell Biology II, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
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Summary |
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Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.10.3 µm) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 µm on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H2O2, and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (4050% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H2O2 also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O2) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS. (J Histochem Cytochem 47:11411148, 1999)
Key Words: peroxisome proliferation, signal transduction, oxygen radicals, tyrosine kinase, tubular peroxisomes
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Introduction |
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THE PEROXISOME is a ubiquitous multipurpose cell organelle which is involved in a variety of metabolic processes, including generation and breakdown of reactive oxygen species (
Peroxisomes in confluent cultures of human-derived HepG2 cells are typically small spherical particles (0.20.3 µm) resembling those observed in a variety of other cell types in culture (
Ultraviolet irradiation of cells elicits a complex cellular response comprising the induction of expression of several genes, including those of growth factors (
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Materials and Methods |
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Cell Culture
HepG2 cells (-MEM) containing 2 g/liter sodium bicarbonate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (FCS) (all from Biochrom KG; Berlin, Germany) at 37C in a humidified atmosphere of 5% CO2 and 95% air (normoxic conditions). For experiments, the cells were washed with serum-free DMEM/N1 [Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY); N1 = 0.25% BSA; 6.25 x 10-8 M transferrin; 8.3 x 10-7 M insulin; 3 x 10-8 M selenium; 2 x 10-8 M progesteron; 1 x 10-4 M putrescine (all from Sigma Immunochemicals; Munich, Germany); 50 µg/ml penicillin; 50 µg/ml streptomycin; 100 µg/ml neomycin; according to
Indirect Immunofluorescence
HepG2 cells were processed as described (
UV Irradiation
Four hours after the cells were sown, the DMEM/N1 medium was removed and saved (to be added again to the same cultures after UV exposure) and the dishes (with lids removed) were exposed to 40 J/m2 of UVC (~2 sec) in a UV-Crosslinker, Model 2400 (Stratagene; San Diego, CA) according to
Treatment with NAC, OAG, BIM, and Hydrogen Peroxide
One hour before UV irradiation the cells were pretreated with the antioxidant N-acetyl-cysteine (NAC). NAC (Sigma) was dissolved in DMEM/N1 immediately before use. Some cultures were also treated with 1-oleolyl-2-acetyl-sn-glycerol (OAG; 0.210 µm), an activator of PKC, or with bisindolylmaleimide I (BIM; 0.011 µm), a PKC inhibitor, 1 hr before UV irradiation. Hydrogen peroxide was obtained as a 30% solution (Sigma) and diluted to 200 or 400 µM (final concentration in the culture medium) immediately before use.
Hypoxia/Hyperoxia
For exposure to hypoxia (hyperoxia), HepG2 cells grown to confluence under normoxic conditions (95% air) were seeded in DMEM/N1 or DMEM/N1/10%FCS and immediately transferred into a thermostatized (37C) acrylic plastic chamber (volume about 8 liters) that was flushed with sterile-filtered (filter size 0.22 µm) humidified gas composed of various concentrations of O2 (1.5%, 3%, 40%), 5% CO2, and N2 for 24 hr. After an initial flushing with the appropriate gas at a higher rate for 5 min, the gas flow was kept constant at a rate of about 0.2 liter/min during the incubation. The PO2 and PCO2 in the atmosphere of the acrylic plastic chamber and in the culture medium supernatant was checked with a blood gas analyzer (Model 278; Corning, Corning, NY). After 24 hr the cells and appropriate normoxic controls were fixed and processed for immunofluorescence.
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Results |
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UV Irradiation Induces Tubular Peroxisomes via ROS
As shown previously (
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To determine whether dynamic changes of the peroxisomal compartment and the formation of elongated tubular peroxisomes can also be induced by oxidative stress, HepG2 cells grown at low density under serum-free conditions were exposed to a 40 J/m2 dose of UVC radiation and analyzed by immunofluorescence 24 hr after UV treatment. Control cells were cultured either in serum-free DMEM/N1 medium or in the presence of 10% FCS. As shown in Figure 2, in response to UV irradiation tubular peroxisomes were induced in the absence of FCS to a level comparable to that of 10% FCS (Figure 1B and Figure 2). To test whether ROS are involved in the initiation of UV-induced tubular organelles, we applied NAC, which can be rapidly converted into reduced glutathione (GSH) after uptake by cells, thus serving as an intracellular reducing agent (
Formation of Tubular Peroxisomes in HepG2 Cells Depends on Oxygen Partial Pressure
In another set of experiments, HepG2 cells were seeded in serum-free DMEM/N1 or in DMEM/N1/10% FCS and cultured for 24 hr under normoxic (controls), hypoxic (1.5%, 3% O2), or hyperoxic (40% O2) conditions (Figure 3 and Figure 4). Forty percent of HepG2 cells grown in the presence of 10% FCS in normoxia showed elongated tubular peroxisomes extending over several µm (Figure 3A), whereas hypoxia inhibited their formation in a dose-dependent manner (Figure 4). Almost all peroxisomes under hypoxic conditions were spherical, although FCS was present in the medium (Figure 3B). Furthermore, under hypoxia the cells appeared to be enlarged and more flattened. In contrast, hyperoxia (40% O2) had no significant effect on the formation of tubular peroxisomes. Hyperoxia was neither stimulatory on the formation of tubular peroxisomes in cells grown under serum-free conditions nor inhibitory on cells grown in the presence of 10% FCS (Figure 4). Similar to the UV effect, the tubular structure of mitochondria was not altered with changing oxygen concentrations (not shown).
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Discussion |
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Formation of Tubular Peroxisomes Is Mediated by the Tyrosine Kinase Signal Transduction Pathway
This study has shown that UV irradiation of HepG2 cells under serum-free culture conditions induces the formation of tubular peroxisomes in 4050% of cells. Therefore, the inducing effect of UV appears to be comparable to the combined effects of various proteins and lipids present in FCS (
On the other hand, our previous studies with activators and inhibitors of protein kinase C (PKC) showed no effects on tubulation of peroxisomes (
UV irradiation stimulates the upregulation of a wide range of gene products (B. Other activated genes include those for growth factors and cytokines, such as bFGF,
INF, IL-1, TNF
, and signal intermediaries, such as PKC, c-Ras, and many others (reviewed by
Formation of Tubular Peroxisomes Requires Oxygen
An important observation of the present study was the demonstration of oxygen-dependence of the induction of tubular peroxisomes by FCS (Figure 4). This suggests that the peroxisomal response to various (growth) factors and PUFAs in FCS is also mediated by ROS. A number of studies in recent years have shown that ligand stimulation of nonphagocytic cells results in an increase in intracellular oxygen species. Therefore, the participation of ROS as intermediates of response to growth factors acting through the tyrosine kinase pathway is well documented (
The Peroxisomal Response May Be Central to Cellular Rescue from ROS
We showed recently (
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Acknowledgments |
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Supported by grants from the Deutsche Forschungs-gemeinschaft (DFG) through SFB 352 (project C 7) and SFB 601 (project B 1).
The technical assistance of Gabriele Krämer and Inge Frommer is gratefully acknowledged. The support of Markus Grabenbauer in digital processing of micrographs is appreciated. The activator and inhibitor of PKC (OAG and BIM) were a generous gift from Dr M. Radloff (University of Hamburg, Germany). We are also grateful to Prof Alfred Völkl and Dr Eveline Baumgart for helpful discussions.
Received for publication March 26, 1999; accepted March 30, 1999.
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