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Am J Physiol Heart Circ Physiol 276: H1493-H1501, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 5, H1493-H1501, May 1999

TNF-alpha and IL-1alpha induce heme oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells

Christi M. Terry1, Jennifer A. Clikeman2, John R. Hoidal2,3, and Karleen S. Callahan1,2,3

Departments of 1 Pharmacology and Toxicology and 3 Internal Medicine, University of Utah, Salt Lake City 84132; and 2 Veterans Affairs Medical Center, Salt Lake City, Utah 84148


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heme oxygenase-1 (HO-1), an enzyme important in protection against oxidant stress, is induced in human vascular endothelial cells by the cytokines tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1alpha (IL-1alpha ). However, the signaling mediators that regulate the induction are not known. This study examined the involvement of protein kinase C (PKC), phospholipase A2 (PLA2), calcium, and oxidants in cytokine induction of HO-1. Acute exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated HO-1 mRNA. However, prolonged exposure, which downregulates most PKC isoforms, blocked induction of HO-1 mRNA by IL-1alpha and TNF-alpha . Additionally, the phosphatase inhibitors okadaic acid and calyculin enhanced cytokine induction of HO-1. Mepacrine, a PLA2 inhibitor, prevented HO-1 induction by cytokine, suggesting a role for arachidonate, the product of PLA2 hydrolysis of phospholipids, in HO-1 expression. The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) blocked cytokine induction of HO-1. Paradoxically, the calcium ionophore A-23187 prevented HO-1 induction by cytokine but not by PMA. Finally, the oxidant scavenger N-acetylcysteine inhibited HO-1 induction by cytokines. These results demonstrate that TNF-alpha and IL-1alpha induction of HO-1 requires PKC-mediated phosphorylation and PLA2 activation as well as oxidant generation.

cytokine; phorbol 12-myristate 13-acetate; phosphatase; protein kinase C downregulation; protein kinase C isoforms; tumor necrosis factor-alpha ; interleukin-1alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXIDATIVE STRESS IS implicated in the pathogenesis of many diseases associated with inflammation such as the acute respiratory distress syndrome, pulmonary fibrosis, and asthma and also may contribute to the pathophysiology of atherosclerosis, carcinogenesis, and aging. The enzyme heme oxygenase is often elevated after oxidant exposure and appears to be an important cellular response to stress. Heme oxygenase cleaves the heme molecule, releasing free iron and producing biliverdin, a precursor to bilirubin (54). Heme oxygenase activity makes iron available for sequestration by the storage protein ferritin, which prevents the metal from participating in redox reactions (5). Thus heme oxygenase acts not only in iron conservation but may protect against oxidant damage by removal of reactive heme iron and by producing bilirubin, which has antioxidant properties (51).

Heme oxygenase has three known isoforms that consist of the inducible isoform heme oxygenase-1 (HO-1), the constitutive isoform heme oxygenase-2, and a recently identified isoform, heme oxygenase-3 (35). The expression of HO-1 is highly sensitive to induction by oxidant stress, and much recent work has shown HO-1 activity to be protective against oxidant injury both in vitro and in vivo (1, 2, 12, 14, 33, 36, 40, 42, 44, 59, 64). Thus increased HO-1 activity is an important cellular response to oxidative stress, but the signaling events that are responsible for enhanced HO-1 gene expression are not completely elucidated.

The vascular endothelium, a primary target for oxidant-mediated effects, is also subject to cytokine-mediated changes in cellular function. We have recently shown that the inflammatory cytokines interleukin-1alpha (IL-1alpha ) and tumor necrosis factor-alpha (TNF-alpha ) are effective inducers of HO-1 in cultured human endothelial cells (56). This induction occurs at the transcriptional level and requires protein synthesis, but the involvement of specific signaling mediators in cytokine induction of HO-1 in endothelium is undefined.

TNF-alpha and IL-1alpha utilize the signaling enzyme protein kinase C (PKC) to affect a number of endothelial cell responses (15, 25, 32, 43, 55), whereas others are PKC independent (19, 46). PKC plays an important role in the control of many cell functions including regulation of transcription of a variety of genes (37). Transcriptional activation of genes by phorbol ester, a nonphysiological activator of PKC, occurs via binding of the AP-1 family of transcription factors to the 12-O-tetradecanoylphorbol 13-acetate response element (TRE) (3). We have previously shown that curcumin, an AP-1 inhibitor, blocks cytokine induction of HO-1 mRNA (56). The HO-1 gene contains a TRE within its promoter region (38, 53), and Kurata et al. (30) showed that PKC is required for HO-1 induction by phorbol ester in a mouse leukemia cell line. However, investigations in human cell types have found that PKC-mediated induction of HO-1 is highly variable (4, 6, 26, 38). PKC can regulate the activity of cytosolic phospholipase A2 (PLA2), which hydrolyzes membrane phospholipids releasing arachidonic acid. Arachidonate has previously been shown to participate in induction of HO-1 by ultraviolet light in skin fibroblasts (6). Thus the current study examined whether cytokine induction of HO-1 in endothelial cells is PKC mediated and/or involves PLA2.

In addition to PKC activation, IL-1alpha and TNF-alpha induce various reactive oxygen species in endothelium (22, 34, 39), a characteristic that is shared by many inducers of HO-1 such as ultraviolet light (27) and asbestos (24). Studies by Keyse and Tyrrell (29) show that ultraviolet light and hydrogen peroxide act through hydroxyl radical formation to enhance HO-1 expression. Interestingly, phospholipid peroxidation has been shown to enhance PKC activation of PLA2 in vitro (45). We hypothesize that oxidants may be critical for the induced HO-1 expression by TNF-alpha and IL-1alpha . Thus the current study also investigates whether reactive oxygen species act as intracellular messengers in the stimulation of endothelial HO-1 by these inflammatory cytokines.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Endothelial cells were isolated from human umbilical veins by collagenase detachment and cultured according to the established method described previously (18). Umbilical cords were obtained from Columbia St. Marks Hospital (Salt Lake City, UT). Cells were grown to confluency in 75-cm2 plastic flasks in endothelial cell growth media (EGM) (Clonetics, San Diego, CA). Confluent cells were harvested with trypsin and transferred to 1% gelatin-coated tissue-culture plasticware. The cells were grown to confluency in 60-mm dishes for RNA extraction and Western immunoblot analysis. Confluent, first-passage cells were used for all experiments. During agonist treatment, cultures were observed for any sign of cell injury, and if cells appeared pyknotic or retracted, the cultures were excluded from the experiment.

Cell treatment with agonists and inhibitors. Confluent endothelial cells were incubated at 37°C with 5% CO2-95% air in serumless Neuman Tytell (NT) (GIBCO, Grand Island, NY) for 3-4 h before agonist addition to allow the cells to recover from washing before treatment. Endothelial cells were incubated with either human recombinant IL-1alpha (50 U/ml) (Boehringer Mannheim, Indianapolis, IN), human recombinant TNF-alpha (500 U/ml) (Genzyme, Cambridge, MA), phorbol 12-myristate 13-acetate (PMA; 0.1 or 1.0 µg/ml) (Sigma), or 0.5 µM thymeleatoxin (LC Laboratories, Woburn, MA) for the time periods indicated in RESULTS. In D609 experiments, cells were preincubated with 50-167 µM D609 (Biomol, Plymouth Meeting, PA) for 30 min then exposed to cytokine for 4 h. In other experiments, the PLA2 inhibitor mepacrine (Sigma) at 1 × 10-5 M was added to the preincubation media 30 min before cytokine addition. Cells were then exposed to cytokine in the continued presence of mepacrine for 4 h. To study a role for oxidants in HO-1 expression, endothelial cells were preexposed to 20 mM N-acetylcysteine (NAC) (Sigma) for 1 h before exposure to IL-1alpha or TNF-alpha . Cells were incubated for 4 h and harvested for determination of mRNA levels.

PKC downregulation. To downregulate PKC, endothelial cells were preincubated with 0.1 µg/ml PMA, 0.1 µg/ml 4alpha -PMA (negative control), or 0.2% DMSO vehicle alone (blank) in EGM for 24 h. At the end of 24 h, the EGM was replaced with NT media with the same concentration of agents as in the 24-h pretreatment, and the cells were incubated for another 4 h. The cells were then exposed to IL-1alpha , TNF-alpha , 0.1 µg/ml PMA, or 25 µM sodium arsenite for 4 h and then harvested for RNA analysis. To avoid toxicity, cells were exposed to sodium arsenite for only 30 min, then the cells were washed, and NT medium was replaced for the remaining 3.5-h incubation period.

Phosphatase inhibition. Endothelial cells were treated with TNF-alpha or IL-1alpha in the absence or presence of 1 or 30 nM okadaic acid (Biomol) or 2 nM calyculin (LC Laboratories) for 4 h. Okadaic acid and calyculin were added 15 min before cytokine addition, whereas control cells were treated with DMSO vehicle alone.

Calcium studies. For chelation studies, endothelial cells were treated with IL-1alpha or TNF-alpha in the absence or presence of 4 or 8 mM EGTA, or 10 or 20 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Molecular Probes, Eugene, OR). The EGTA or BAPTA-AM was added 15 min before cytokine addition. After 4 h, the cells were harvested for RNA analysis. Control cells were exposed to DMSO vehicle.

For ionophore studies, endothelial cells were exposed to IL-1alpha , TNF-alpha , or PMA for 4 h in the absence or presence of 0.5 µM A-23187 (Calbiochem, La Jolla, CA) and then harvested for RNA analysis. The A-23187 was added 15 min before cytokine treatment. Control cells were exposed to vehicle (DMSO) alone. In the calpeptin studies, cells were preincubated with calpeptin (30 or 60 µM) and A-23187 for 15 min before treatment with agonist as described above.

Northern blot analysis. RNA was extracted from cells and analyzed by Northern blot technique as previously described (56). Lane loading equivalences were determined by hybridizing the membranes with 32P-labeled cDNA CHO-B probe. CHO-B message expression in human endothelial cells has previously been shown to be unaffected by cytokine treatments (55). The autoradiographic images from the HO-1 labels and the CHO-B labels were scanned with a laser densitometer (Ultrascan XL Enhanced Laser Densitometer, LKB Bromma, Pisctaway, NJ) that converted the densities of the bands to relative absorbance units. The densities of the CHO-B bands were used to correct any variances in lane loading. The normalized HO-1 mRNA values were plotted in graph form by either comparing mRNA induction to control levels or by setting cytokine induction as 100% and reporting the effect of treatments as a percentage of this maximal induction.

Probes. The human HO-1 cDNA probe was made by PCR amplification of a 762-bp fragment of HO-1 as previously described (56). The probe specifically recognizes an mRNA band of ~1.8 kb from cells exposed to sodium arsenite, a characteristic inducer of HO-1.

The CHO-B cDNA probe was a generous gift from Dr. Bruce Marshall of the Division of Pulmonary Medicine, University of Utah School of Medicine, and recognizes an mRNA species of 1.0 kb in size.

Immunoblotting. Analysis of PKC downregulation in cultures treated for 24 h with PMA was done by Western blot analysis of PKC isoforms. After PMA treatment, the cells were washed with ice-cold phosphate-buffered saline and then harvested into ice-cold freshly prepared sucrose extraction buffer [20 mM Tris · HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 µg/ml leupeptin, and 100 µg/ml aprotonin]. The cell lysates were frozen in liquid nitrogen and stored at -70°C until resolved on SDS-PAGE gels. The protein concentrations were determined by using a commercial bicinchoninic acid (BCA) spectrophotometric assay (Pierce, Rockford, IL).

Western blot analysis of PKC isoforms was done by resolving 50 µg of protein on a 10% polyacrylamide gel by denaturing discontinuous gel electrophoresis according to the Laemmli method. After electrophoresis, the proteins were immobilized on a polyvinylidene difluoride membrane (Pall Biodyne, East Hills, NY) using electrophoretic transfer (Bio-Rad Laboratories, Hercules, CA). The PKC isoforms were detected by incubating the immunoblots with primary mouse anti-human PKC-alpha (1:5,000 dilution) or PKC-zeta (1:500 dilution) (Transduction Laboratories, Lexington, KY). Primary antibody binding was detected with an anti-mouse secondary antibody conjugated to horseradish peroxidase (Transduction Laboratories) using a chemiluminescent detection system (Amersham).

PKC activity studies. Confluent endothelial cells were treated with either IL-1alpha , TNF-alpha , or PMA in NT media for 15 min. The cells were quickly harvested into 300 µl of cold PKC assay homogenization buffer (100 mM beta -glycerophosphate, 0.5 M sucrose, 2 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 1 mM PMSF, and 50 µg/ml leupeptin). The harvested cells were frozen immediately in liquid nitrogen and stored at -70°C until assayed.

PKC activity was measured by [gamma -32P]ATP phosphorylation of myelin basic protein-(4---14) substrate peptide. A 10-µl sample of cell lysate was combined with 20 µl of reaction buffer [25 mM MOPS, pH 7.0, 0.5 mM dithiothreitol, 10 mM magnesium acetate, 0.2 mM calcium chloride, and 0.02 mM myelin basic protein-(4---14) peptide] and 20 µl of the cofactors phosphatidylserine (24 µM final concentration) and diacylglycerol (6 µM final concentration). Each treatment was assayed in duplicate. Background reactions to determine nonspecific phosphorylation and 32P binding received the same components except that phosphatidylserine and diacylglycerol were omitted and 6 mM EGTA was added to chelate calcium. Reactions were started by addition of 10 µl of [gamma -32P]ATP (2,000 cpm/pmol) and incubated in a 30°C water bath. A 20-µl aliquot was removed at 10 and 30 min and transferred onto P81 filter paper (Whatman) held in a 96-well dot-blot apparatus (Mini blot I, Schleicher and Schull, Keene, NH). The filter paper was washed in several changes of 75 mM phosphoric acid to remove unincorporated radioactivity, rinsed in 95% ethanol, and dried. The filter paper was then placed in a Flexi-filter holder (Packard), and 30 µl of Microscint scintillation cocktail (Packard) were added to each well and then counted in a Top Count 96-well scintillation counter (Packard Instrument, Meriden, CT). Phosphorylation of the substrate peptide was calculated by subtracting the counts in the baseline reaction from the counts in the complete reaction containing all cofactors. The protein concentration of each sample was determined by BCA assay, and the PKC activity was expressed as nanomoles of substrate protein phosphorylated per microgram of protein per minute.

Statistical analysis. The means ± SE for HO-1 message levels were calculated after normalization of the absorbance units derived from scanning densitometry of the autoradiographic images of Northern blots. A pooled t-test analysis of the data was used for determination of statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently showed that TNF-alpha and IL-1alpha induce HO-1 in endothelial cells with maximal mRNA levels at 4 h and maximal protein levels by 6 h (56). Both cytokines are potent stimulators of HO-1 mRNA, with IL-1alpha causing increases between 5- and 20-fold and TNF-alpha inducing increases between 3- and 10-fold above control levels. Induction by both agents is concentration dependent with 50 U/ml IL-1alpha and 500 U/ml TNF-alpha being the most effective concentrations. These concentrations of IL-1alpha and TNF-alpha were therefore used in the present study examining signaling regulation of HO-1 in cytokine-stimulated endothelial cells.

Although IL-1alpha and TNF-alpha bind distinct receptors, these agents may act through a common signaling event to affect endothelial cell HO-1 induction. If the maximal response seen with the most stimulatory cytokine, IL-1alpha , is not enhanced by TNF-alpha , it would imply that these cytokines share a common, rate-limiting step in HO-1 induction. To examine this, endothelial cells were exposed to IL-1alpha and TNF-alpha together at their respective maximally effective concentrations, and the resulting HO-1 mRNA levels were measured. Figure 1 shows that the combination of TNF-alpha and IL-1alpha causes induction of a HO-1 mRNA level similar to that observed with IL-1alpha alone. This result was consistently observed in five separate experiments, with Fig. 1, inset, displaying a typical autoradiographic result. The inability of TNF-alpha to enhance IL-1alpha induction of HO-1 message is not due to maximal stimulation by IL-1alpha because sodium arsenite causes significantly greater increases of HO-1 mRNA in endothelial cells, i.e., 25- to 50-fold of control (data not shown).


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  		Fig. 1.   HO-1 mRNA induction by interleukin-1alpha (IL-1alpha ) or tumor necrosis factor-alpha (TNF-alpha ) alone or in combination. Cells were exposed to either 50 U/ml IL-1alpha or 500 U/ml TNF-alpha alone or in combination for 4 h. Cells were then harvested for RNA analysis by Northern blotting followed by densitometric quantification and normalization for lane loading differences. HO-1 mRNA induction is expressed as mean ± SE from an value of n >=  5 for each treatment. Inset: a typical autoradiograph of a Northern blot probed with radiolabeled human HO-1-specific cDNA (top) and blot then probed with radiolabeled CHO-B cDNA (bottom) to determine lane loading variation. Cells were treated with vehicle (lane 1), IL-1alpha (lane 2), TNF-alpha (lane 3), or IL-1alpha and TNF-alpha (lane 4) combined as described.

As previous investigations show that PKC plays a regulatory role in HO-1 expression in some other tissues, studies next examined the ability of the PKC activator PMA to induce HO-1 mRNA in endothelial cells. Figure 2 shows that PMA exposure causes increases in HO-1 mRNA by 2 h. This induction appears maximal by 4-6 h and stays elevated for up to 10 h and returns to baseline by 28 h (data not shown). In addition, when thymeleatoxin, an activator of conventional PKC isoforms, is added to cells for 4 h, a fourfold enhancement of HO-1 mRNA is observed (Fig. 2). These results indicate that PKC activation can regulate HO-1 mRNA expression in endothelial cells. Thus additional studies were performed to examine whether induction of HO-1 mRNA by IL-1alpha or TNF-alpha involves PKC.


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  		Fig. 2.   HO-1 mRNA induction by protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (Thym.). Cells were exposed to either 1.0 µg/ml PMA or 0.5 µM thymeleatoxin for times indicated and then harvested for mRNA assessed by Northern blot. HO-1 mRNA induction is expressed as mean ± SE with an n value of 3 or 4 per time point.

Studies were next carried out to determine whether PKC activation occurs as a consequence of exposure of endothelial cells to either IL-1alpha or TNF-alpha . Cells were exposed to TNF-alpha , IL-1alpha , or PMA for 15 min, then total cellular PKC activity was determined via measurement of phosphorylation of a synthetic substrate peptide. As shown in Table 1, PKC activity was significantly increased in cytokine- and PMA-treated cells. IL-1alpha and TNF-alpha induced increases of two- to threefold, whereas PMA exposure resulted in a fivefold stimulation of PKC.

                              
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  		Table 1.   IL-1alpha , TNF-alpha , and PMA increase PKC activity in endothelial cells

Chronic activation of PKC with PMA leads to the proteolytic degradation of the conventional and novel PKC isoforms (8, 21). To examine the involvement of these PKC isoforms in cytokine induction of HO-1 mRNA, endothelial cells were exposed to PMA for 28 h and then to IL-1alpha , TNF-alpha , or PMA for 4 h. As controls, cells were also preexposed to an inactive isomer of PMA, 4alpha -PMA, which does not activate PKC. Figure 3 shows that prolonged PMA exposure causes a significant attenuation of HO-1 mRNA induction by TNF-alpha , IL-1alpha , and PMA. Pretreatment with the inactive 4alpha -PMA isomer has no affect on HO-1 induction by the cytokines or PMA (Fig. 3). HO-1 mRNA levels in 4alpha -PMA-pretreated cells are induced to the same extent as in cells treated with vehicle only (data not shown). The endothelial cells responded to sodium arsenite treatment with maximal increases in HO-1 mRNA after PMA pretreatment (Fig. 3, inset, lane 2). This finding suggests that PKC downregulation does not have a global dampening effect on HO-1 induction.


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  		Fig. 3.   Effect of PMA pretreatment on induction of HO-1 mRNA by cytokines, PMA, or sodium arsenite. Cells were pretreated with either 0.1 µg/ml PMA or 0.1 µg/ml 4alpha -PMA for 28 h then exposed to either 50 U/ml IL-1alpha , 500 U/ml TNF-alpha , or 0.1 µg/ml PMA for 4 h. Cells were then harvested for mRNA and Northern blot analysis performed with induction expressed as mean ± SE from an n value >=  3 for all treatments. Inset: an autoradiograph in which 25 µM sodium arsenite is the stimulus. In this inset, cells were pretreated with 0.1 µg/ml PMA (lanes 1 and 2) or 0.1 µg/ml 4alpha -PMA (lanes 3 and 4) for 28 h followed by exposure to vehicle alone (lanes 1 and 3) or to sodium arsenite (lanes 2 and 4).

Haller et al. (21) reported that prolonged exposure of endothelial cells to PMA resulted in the downregulation of the alpha - and beta  (conventional)-isoforms of PKC, whereas the zeta  (atypical)-isoform is unaffected. To verify this in our cells, Western blot analysis was carried out on endothelial cell protein after prolonged exposure to PMA. Figure 4 shows that endothelial cells exposed to PMA for 24 h have markedly diminished levels of the conventional alpha -isoform of PKC compared with vehicle pretreated cells, whereas the atypical zeta -isoform protein levels remained similar to control cells. These results support the supposition that extended PMA exposure is causing downregulation of protein expression for conventional PKC isoforms.


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  		Fig. 4.   Effect of PMA pretreatment on PKC isoforms alpha  and zeta  protein expression. Cells were treated with either DMSO vehicle (Con.) or 0.1 µg/ml PMA (PMA pretreat.) for 24 h. Cell protein was then harvested and analyzed by Western blotting technique with PKC-alpha - or PKC-zeta -specific antibodies.

The aforementioned results showing that PKC, a protein serine-threonine kinase, is important in regulating HO-1 expression by cytokines suggests that a phosphorylation event is critical. Thus inhibiting dephosphorylation should enhance the HO-1 induction. The specific serine-threonine protein phosphatase inhibitors okadaic acid and calyculin were used to examine the effect of protein phosphatase inhibition on cytokine induction of HO-1 mRNA. Endothelial cells were exposed to cytokine in the absence or presence of 1 or 30 nM okadaic acid or 2.0 nM calyculin. The IC50 value of okadaic acid is 0.5-1.0 nM for protein phosphatase 2A and 60-200 nM for protein phosphatase 1 (17). Thus, at 1 and 30 nM, okadaic acid primarily inhibits protein phosphatase 2A. Calyculin inhibits protein phosphatase 1 and protein phosphatase 2A with similar IC50 values (0.5-2.0 nM). Figure 5 shows that both okadaic acid and calyculin significantly enhance the induction of HO-1 mRNA by IL-1alpha . The induction of HO-1 by IL-1alpha plus 2.0 nM calyculin is not significantly different from the induction observed with IL-1alpha plus 30 nM okadaic acid. Similar results were observed with TNF-alpha (data not shown).


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  		Fig. 5.   Phosphatase inhibitors increase IL-1alpha induction of HO-1 mRNA. Cells were treated with 50 U/ml IL-1alpha for 4 h in absence or presence of indicated concentrations of okadaic acid or calyculin or for 4 h with okadaic acid or calyculin alone. Cells were then harvested for mRNA and Northern blot analysis performed with induction expressed as mean ± SE from an n >=  3 for all treatments. Significant differences were observed among the following treatments: IL-1alpha vs. IL-1alpha  + 1 nM okadaic acid, alpha  = 0.05, P = 0.0147; IL-1alpha vs. IL-1alpha  + 30 nM okadaic acid, alpha  = 0.05, P = 0.0045; IL-1alpha vs. IL-1alpha  + calyculin, alpha  = 0.05, P = 0.0141.

TNF-alpha and IL-1alpha can cause the production of diacylglycerol, which is a direct activator of PKC, via a phosphatidylcholine-specific phospholipase C (PC-PLC) (50). However, when endothelial cells were exposed to the PC-PLC inhibitor D609, in the presence of cytokine, HO-1 levels were actually enhanced, and D609 alone induced significant amounts of HO-1 (data not shown), indicating that PC-PLC does not mediate cytokine induction of HO-1. PLA2 is another lipase that is activated in endothelial cells after TNF-alpha and IL-1alpha exposure (9, 19). PKC phosphorylation of PLA2 enhances its activity (45), and arachidonic acid, the product of PLA2 hydrolysis of phospholipid, can upregulate PKC activity (47). To investigate a possible involvement of PLA2 in cytokine induction of HO-1, cells were incubated with cytokine in the presence or absence of mepacrine, an inhibitor of PLA2. Figure 6 shows that mepacrine completely attenuates TNF-alpha and IL-1alpha induction of HO-1. The results shown in this autoradiograph are representative of those found in three separate experiments.


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  		Fig. 6.   Mepacrine inhibits HO-1 mRNA induction by TNF-alpha or IL-1alpha . Cells were treated with vehicle only, 500 U/ml TNF-alpha , or 50 U/ml IL-1alpha in absence or presence of 1 × 10-5 M mepacrine. An autoradiograph from representative Northern blots (from 3 experiments with n >=  6 per mepacrine treatment) that were probed with a radiolabeled HO-1-specific cDNA probe is shown.

Because maximal activation of the conventional PKC isoforms depends on calcium, studies next examined the effect of manipulation of cellular calcium concentrations on HO-1 induction. Cells were incubated in the presence of the extracellular calcium chelator EGTA then exposed to a cytokine. Cells exposed to IL-1alpha or TNF-alpha plus EGTA show only a small decrease in mRNA levels (i.e., 77 ± 6% of IL-1alpha alone and 75 ± 10% of TNF-alpha alone). Cells exposed to EGTA alone express HO-1 mRNA levels similar to control cells. Thus EGTA has little effect on cytokine induction of HO-1 mRNA. However, when cells are exposed to cytokines in the presence of the membrane-permeable calcium chelator BAPTA-AM, the cytokine induction of HO-1 mRNA is completely blocked as shown in Fig. 7. Because chelation of intracellular calcium prevents HO-1 mRNA induction, the effect of increasing intracellular calcium concentration was examined. For these studies, the calcium ionophore A-23187 was utilized. Figure 8 illustrates that when endothelial cells are exposed to A-23187 in the presence of cytokine, induction of HO-1 mRNA is suppressed by ~90%. A-23187 by itself had no significant effect on HO-1 mRNA levels. Of interest, A-23187 exposure inhibits PMA induction of HO-1 mRNA by only ~30% (Fig. 8). Increases in calcium activate calpain, a protease that can degrade AP-1 (60), a transcription factor that is often involved in HO-1 expression (10-12, 31, 41, 56). To determine if activation of the calcium-activated neutral protease calpain is involved in the inhibitory effect observed with ionophore, cells were exposed to IL-1alpha and A-23187 in the absence or presence of the calpain-specific inhibitor calpeptin. It was found that IL-1alpha induction of HO-1 mRNA is inhibited to the same degree by ionophore in the presence of either 30 or 60 µM calpeptin (data not shown), suggesting that activation of calpain does not contribute to the inhibitory effect of A-23187.


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  		Fig. 7.   BAPTA-AM attenuates cytokine induction of HO-1 mRNA. Cells were treated with 50 U/ml IL-1alpha or 500 U/ml TNF-alpha in absence or presence of 10 µM BAPTA-AM (BAPTA) for 4 h. Cells were then harvested for RNA analysis by Northern blotting followed by densitometric analysis and normalization. Induction of HO-1 mRNA is expressed as mean ± SE with n = 3 for TNF-alpha alone and BAPTA alone treatments and n >=  4 for other treatments.



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  		Fig. 8.   Effect of calcium ionophore A-23187 on cytokine and PMA induction of HO-1 mRNA. Cells were exposed to either 50 U/ml IL-1alpha , 500 U/ml TNF-alpha , or 1.0 µg/ml PMA in absence or presence of 0.5 µM A-23187 or to A-23187 alone for 4 h. Cells were then harvested for mRNA and Northern blot analysis performed with changes expressed as means ± SE from an n value of >=  3 for all treatments.

Reactive oxygen species that, in excess, are detrimental to cell viability can, in subtoxic concentrations, participate in signaling cascades (52, 62). IL-1alpha , TNF-alpha , and PMA all cause the production of reactive oxygen species (22, 34, 39, 48) and may utilize this production to affect gene expression. NAC scavenges free radicals and increases cellular glutathione levels (13). Therefore, this agent was used to investigate the involvement of oxidants in cytokine induction of HO-1 message. When cells are exposed to either cytokine in the presence of NAC, induction of HO-1 mRNA levels is completely prevented (Fig. 9). This result implies that cytokine-mediated induction of HO-1 message levels is dependent on the presence of reactive oxygen species.


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  		Fig. 9.   N-acetylcysteine (NAC) inhibits HO-1 mRNA induction by TNF-alpha or IL-1alpha . Cells were treated with vehicle only, 500 U/ml TNF-alpha , or 50 U/ml IL-1alpha in absence or presence of 25 mM NAC. An autoradiograph of a representative Northern blot (from 3 experiments with n >=  5 per NAC treatment) that was probed with a radiolabeled HO-1-specific cDNA probe is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HO-1 is upregulated in cells by many oxidative stimuli including ultraviolet light (28), hyperoxia (14), and hydrogen peroxide (28). Our recent studies demonstrated that the inflammatory cytokines TNF-alpha and IL-1alpha are also potent inducers of HO-1 in human endothelium (56). Although there are numerous reports of many stimuli that upregulate this key enzyme, and a number of studies have characterized the transcription factors and 5'-DNA sequences required for HO-1 expression, less is known about the intracellular signaling intermediaries that regulate HO-1 expression. The present investigation examined likely signaling mediators that may control cytokine-mediated HO-1 in endothelium.

Although IL-1alpha and TNF-alpha bind to separate specific membrane receptors, these cytokines produce many similar cellular effects and may utilize the same signaling pathways in some instances. The present results suggest that HO-1 induction occurs via a common mediator because exposure of the cells to both cytokines did not cause a synergistic response (Fig. 1). Our results and studies by others show that IL-1alpha and TNF-alpha can activate endothelial PKC (15); therefore, several experiments focused on this enzyme as playing a critical role in regulating HO-1 message levels. The data obtained indicate that both TNF-alpha and IL-1alpha increase HO-1 mRNA via a PKC-mediated pathway because prolonged exposure to PMA, which downregulates endogenous levels of conventional and novel PKC isoforms, significantly attenuated cytokine-induced HO-1 message levels (Fig. 3).

Activation of PC-PLC by TNF has been shown to elevate levels of diacylglycerol, an endogenous PKC activator (50). However, in this study, D609, an inhibitor of PC-PLC, did not prevent cytokine induction of HO-1. Another lipase, PLA2, hydrolyzes membrane phospholipids to yield arachidonic acid, a biologically active fatty acid that is also metabolized to a variety of other vasoactive compounds. PLA2 has been shown to be a substrate for PKC in vitro, and PKC phosphorylation of PLA2 enhances its activity (45). Additionally, TNF-alpha (9) and IL-1alpha (19) increase endothelial production of arachidonic acid, and arachidonate has PKC activating properties (47). Therefore, to examine an involvement of PLA2 in HO-1 expression by cytokines, the PLA2 inhibitor mepacrine was used. Mepacrine completely inhibited HO-1 induction by both TNF-alpha and IL-1alpha . Although our study shows that PLA2 activity is important in cytokine induction of HO-1, where PLA2 acts in the signaling cascade was not determined. IL-1alpha and TNF-alpha may utilize arachidonic acid generation to activate PKC and subsequently upregulate HO-1 expression, or cytokine activation of PKC may stimulate arachidonate production that then either directly or indirectly affects HO-1 expression.

In addition to activating PKC, both IL-1alpha and TNF-alpha can inactivate protein phosphatases (20) that could contribute to induced gene expression by these cytokines. When endothelial cells were exposed to either of the serine-threonine protein phosphatase 1 and 2A inhibitors, calyculin or okadaic acid, cytokine-induced HO-1 expression was greatly enhanced (Fig. 5). These observations support the involvement of a serine-threonine protein phosphorylation event in cytokine regulation of HO-1 as well as suggest that cytokine inactivation of phosphatases may also contribute to upregulated HO-1 expression. Because the phosphatase inhibitors caused a marked enhancement of cytokine-induced HO-1 expression, it suggests that the basal phosphatase activity regulating HO-1 expression is relatively high compared with protein kinase activity. Both of the phosphatase inhibitors alone caused a small increase of HO-1 message as well (~80% by okadaic acid and 300% by calyculin; Fig. 5), which also supports the conjecture that basal protein kinase activity is relatively low in endothelial cells.

Inducers of HO-1 often share the characteristics of producing oxidants and/or depleting reduced glutathione (28, 57, 58). Because the involvement of oxidant generation on HO-1 induction by TNF-alpha or IL-1alpha has previously not been examined, we studied the effect of the antioxidant NAC on cytokine induction of HO-1. NAC scavenges free radicals and increases cellular glutathione levels. We found that NAC completely inhibits IL-1alpha and TNF-alpha induction of HO-1. IL-1alpha and TNF-alpha can increase production of various reactive oxygen species in endothelium (22, 34, 39) via induction of enzymes such as xanthine oxidase (22), cyclooxygenase (49), and NADPH oxidoreductase (61) as well as through other sources (25, 39, 43). It is not known whether the generation of reactive oxygen species, the resultant decrease in reduced glutathione, the elevation in oxidized glutathione, or a combination of these is responsible for the activation of the HO-1 gene by cytokines. It is noteworthy that TNF has recently been shown to deplete endothelial-glutathione levels via a PKC-dependent generation of superoxide and nitric oxide (43). Additionally, Yee et al. (63) found that nitric oxide induction of HO-1 in endothelial cells was accompanied by oxidation of glutathione. However, others (16, 36) have found that chemical nitric oxide donors can induce HO-1 in endothelial cells at concentrations that do not significantly deplete reduced glutathione. Furthermore, Yet et al. (64) reported that IL-1beta induction of HO-1 in vascular smooth muscle cells was not significantly diminished by an inhibitor of nitric oxide synthase. Thus oxidant production is often involved in HO-1 upregulation, but more detailed studies are required to determine the relative roles of glutathione and various oxidant species in cytokine signaling of the HO-1 gene in endothelial cells.

Although BAPTA-AM, an intracellular calcium chelator, completely abrogates HO-1 mRNA induction by cytokine, enhancement of calcium levels with A-23187 also prevents HO-1 expression. We expected BAPTA-AM to attenuate HO-1 induction by inhibiting delivery of calcium to PKC and the ionophore to enhance HO-1 induction through increased PKC activation. The observed paradoxical effect of calcium changes is not understood, but others have reported that ionophore caused a decrease in endogenous HO-1 mRNA in rat vascular smooth muscle cells (23). It is possible that the rapid increase in intracellular calcium caused by ionophore may activate enzymes or gene expression(s) that have a negative affect on HO-1 induction by cytokine. For instance, a recent report showed that calcium-mobilizing agents inhibited TNF-mediated stimulation of nitric oxide synthase activity in mouse endothelial cells (7). Of note, the ionophore effect on cytokine induction of HO-1 may be occurring upstream of PKC activation, since the PMA induction of HO-1 message was only partially inhibited by A-23187 exposure (Fig. 8).

In summary, the results of this study show that PKC and PLA2 participate in HO-1 upregulation and that changes in intracellular calcium and inhibition of protein phosphatase activity can alter the induction of HO-1 expression by both IL-1alpha and TNF-alpha . In addition, the generation of oxidant species by these inflammatory mediators appears to contribute significantly to HO-1 upregulation.


    ACKNOWLEDGEMENTS

We thank Dr. Donald K. Blumenthal for donation of the myelin basic protein substrate peptide and for helpful comments during the manuscript preparation. In addition, we thank the labor and delivery nursing staff of Columbia St. Mark's Hospital in Salt Lake City, UT, for providing the umbilical cords used in this study.


    FOOTNOTES

This study was supported in part by Department of Veterans Affairs Medical Research Funds (to K. S. Callahan and J. R. Hoidal), by the Western Institute for Biomedical Research (to C. M. Terry and K. S. Callahan), and by a predoctoral fellowship from the American Foundation for Pharmaceutical Education (to C. M. Terry).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. S. Callahan, Univ. of Utah, Pulmonary Division, 50 N. Medical Dr., Salt Lake City, UT 84132.

Received 8 June 1998; accepted in final form 11 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, N. G., Y. Lavrovsky, M. L. Schwartzman, R. A. Stoltz, R. D. Levere, M. E. Gerritsen, S. Shibahara, and A. Kappas. Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity. Proc. Natl. Acad. Sci. USA 92: 6798-6802, 1995[Abstract/Free Full Text].

2.   Agarwal, A., J. Balla, J. Alam, A. J. Croatt, and K. A. Nath. Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat. Kidney Int. 48: 1298-1307, 1995[Medline].

3.   Angel, P., M. Imagaua, R. Chiv, B. Stein, R. J. Imbra, H. J. Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49: 729-739, 1987[Medline].

4.   Applegate, L. A., P. Luscher, and R. M. Tyrrell. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 51: 974-978, 1991[Abstract/Free Full Text].

5.   Balla, G., H. S. Jacob, J. Balla, M. Rosenberg, K. Nath, F. Apple, J. W. Eaton, and G. M. Vercellotti. Ferritin: a cytoprotective antioxidant strategem of endothelium. J. Biol. Chem. 267: 18148-18153, 1992[Abstract/Free Full Text].

6.   Basu-Modak, S., P. Luscher, and R. M. Tyrrell. Lipid metabolite involvement in the activation of the human heme oxygenase-1 gene. Free Radical Biol. Med. 18: 721-730, 1996.

7.   Bereta, M., J. Bereta, I. Georgoff, F. D. Coffman, S. Cohen, and M. C. Cohen. Methylxanthines and calcium-mobilizing agents inhibit the expression of cytokine-inducible nitric oxide synthase and vascular cell adhesion molecule-1 in murine microvascular endothelial cells. Exp. Cell Res. 212: 230-242, 1994[Medline].

8.   Borner, C., S. N. Guadagno, D. Fabbro, and I. B. Weinstein. Expression of four protein kinase C isoforms in rat fibroblasts: distinct subcellular distribution and regulation by calcium and phorbol esters. J. Biol. Chem. 267: 12892-12899, 1992[Abstract/Free Full Text].

9.   Cabre, F., D. Tost, N. Suesa, M. Gutierrez, P. Udedo, D. Mauleon, and G. Carganico. Synthesis and release of platelet-activating factor and eicosanoids in human endothelial cells induced by different agonists. Agents Actions 38: 212-219, 1993[Medline].

10.   Camhi, S. L., J. Alam, L. Otterbein, S. L. Sylvester, and A. M. K. Choi. Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am. J. Respir. Cell. Mol. Biol. 13: 387-398, 1995[Abstract].

11.   Camhi, S. L., J. Alam, G. W. Wiegand, B. Y. Chin, and A. M. Choi. Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1. Am. J. Respir. Cell. Mol. Biol. 18: 226-234, 1998[Abstract/Free Full Text].

12.   Choi, A. M., and J. Alam. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am. J. Respir. Cell. Mol. Biol. 15: 9-19, 1996[Abstract].

13.   Deneke, S. M., and B. L. Fanburg. Regulation of cellular glutathione. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L163-L173, 1989[Abstract/Free Full Text].

14.   Dennery, P. A., K. J. Sridhar, C. S. Lee, H. E. Wong, V. Shokoohi, P. A. Rodgers, and D. R. Spitz. Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J. Biol. Chem. 272: 14937-14942, 1997[Abstract/Free Full Text].

15.   Ferro, T. J., D. M. Parker, L. M. Commins, P. G. Phillips, and A. Johnson. Tumor necrosis factor-alpha activates pulmonary artery endothelial protein kinase C. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L9-L14, 1993.

16.   Foresti, R., J. E. Clark, C. J. Green, and R. Motterlini. Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells: involvement of superoxide and peroxynitrite anions. J. Biol. Chem. 272: 18411-18417, 1997[Abstract/Free Full Text].

17.   Fujiki, H., and M. Suganuma. Tumor promotion by inhibitors of protein phosphatases 1 and 2A: the okadaic acid class of compounds. Adv. Cancer Res. 61: 143-193, 1993[Medline].

18.   Garcia, J. G. N., R. G. Painter, J. W. Fenton, E. D. English, and K. S. Callahan. Thrombin-induced human endothelial cell PGI2 biosynthesis: role of guanine nucleotide-regulatory proteins in stimulus/coupling responses. J. Cell. Physiol. 142: 186-193, 1990[Medline].

19.   Garcia, J. G. N., J. E. Stasek, C. Bahler, and V. Natarajan. Interleukin-1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction. J. Lab. Clin. Med. 120: 929-940, 1992[Medline].

20.   Guy, G. R., J. Cairns, S. B. Ng, and Y. H. Tan. Inactivation of a redox-sensitive protein phosphatase during the early events of tumor necrosis factor/interleukin-1 signal transduction. J. Biol. Chem. 268: 2141-2148, 1993[Abstract/Free Full Text].

21.   Haller, H., W. Ziegler, C. Lindschau, and F. Luft. Endothelial cell tyrosine kinase receptor and G-protein coupled receptor activation involves distinct protein kinase C isoforms. Arterioscler. Thromb. Vasc. Biol. 16: 678-686, 1996[Abstract/Free Full Text].

22.   Ishii, Y., C. A. Partridge, P. J. Del Vecchio, and A. B. Malik. Tumor necrosis factor-alpha -mediated decrease in glutathione increases the sensitivity of pulmonary vascular endothelial cells to H2O2. J. Clin. Invest. 89: 794-802, 1992.

23.   Ishizaka, N., and K. K. Griendling. Heme oxygenase-1 is regulated by angiotensin II in rat vascular smooth muscle cells. Hypertension 29: 790-795, 1997[Abstract/Free Full Text].

24.   Janssen, Y. M. W., J. P. Marsh, M. P. Absher, E. Gabrielson, P. J. A. Borm, K. Driscoll, and B. T. Mossman. Oxidant stress responses in human pleural mesothelial cells exposed to asbestos. Am. J. Respir. Crit. Care Med. 149: 795-802, 1994[Abstract].

25.   Johnson, A., D. T. Phelps, and T. J. Ferro. Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L318-L325, 1994[Abstract/Free Full Text].

26.   Kantengwa, S., and B. S. Polla. Flavonoids, but not protein kinase C inhibitors, prevent stress protein synthesis during erythrophagocytosis. Biochem. Biophys. Res. Commun. 180: 308-314, 1991[Medline].

27.   Keyse, S., L. Applegate, Y. Tromvoukis, and R. M. Tyrell. Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts. Mol. Cell. Biol. 10: 4967-4969, 1990[Abstract/Free Full Text].

28.   Keyse, S. M., and R. M. Tyrrell. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. USA 86: 99-103, 1989[Abstract/Free Full Text].

29.   Keyse, S. M., and R. M. Tyrrell. Induction of the heme oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365 nm) radiation: evidence for the involvement of the hydroxyl radical. Carcinogenesis 11: 787-791, 1989[Abstract/Free Full Text].

30.   Kurata, S., M. Matsumoto, and H. Nakajima. Transcriptional control of the heme oxygenase gene in mouse M1 cells during their TPA-induced differentiation into macrophages. J. Cell. Biochem. 62: 314-324, 1996[Medline].

31.   Lee, P. J., J. Alam, S. L. Sylvester, N. Inamdar, L. Otterbein, and A. M. Choi. Regulation of heme oxygenase-1 expression in vitro and in vivo in hyperoxic lung injury. Am. J. Respir. Cell. Mol. Biol. 14: 556-568, 1996[Abstract].

32.   Magnuson, D. K., R. V. Maier, and T. H. Pohlman. Protein kinase C: a potential pathway of endothelial cell activation by endotoxin, tumor necrosis factor and interleukin-1. Surgery 106: 216-222, 1989[Medline].

33.   Maines, M. D., R. D. Mayer, J. F. Ewing, and W. K. McCoubrey, Jr. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32. J. Pharmacol. Exp. Ther. 264: 457-462, 1993[Abstract/Free Full Text].

34.   Matsubara, T., and M. Ziff. Increased superoxide anion release from human endothelial cells in response to cytokines. J. Immunol. 137: 3295-3298, 1986[Abstract].

35.   McCoubrey, W. K. J., T. J. Huang, and M. D. Maines. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur. J. Biochem. 247: 725-732, 1997[Medline].

36.   Motterlini, R., R. Foresti, M. Intaglietta, and R. M. Winslow. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H107-H114, 1996[Abstract/Free Full Text].

37.   Mufson, R. A. The role of serine/threonine phosphorylation in hematopoietic cytokine receptor signal transduction. FASEB J. 11: 37-44, 1997[Abstract].

38.   Muraosa, Y., and S. Shibahara. Identification of a cis-regulatory element and putative transacting factor responsible for 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of heme oxygenase expression in mylemonocytic cell lines. Mol. Cell. Biol. 13: 7881-7891, 1993[Abstract/Free Full Text].

39.   Murphy, H. S., J. A. Shayman, G. O. Till, M. Mahrougui, C. B. Owens, U. S. Ryan, and P. A. Ward. Superoxide responses of endothelial cells to C5a and TNF-alpha : divergent signal transduction pathways. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L51-L59, 1992[Abstract/Free Full Text].

40.   Nath, K. A., G. Balla, G. M. Vercellotti, J. Balla, H. S. Jacob, M. D. Levitt, and M. E. Rosenberg. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J. Clin. Invest. 90: 267-270, 1992.

41.   Oguro, T., M. Hayashi, S. Numazawa, K. Asakawa, and T. Yoshida. Heme oxygenase-1 gene expression by a glutathione depeletor, phorone, mediated through AP-1 activation in rats. Biochem. Biophys. Res. Commun. 221: 259-265, 1996[Medline].

42.   Otterbein, L., S. L. Sylvester, and A. M. K. Choi. Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxygenase-1. Am. J. Respir. Cell. Mol. Biol. 13: 595-601, 1995[Abstract].

43.   Phelps, D. T., T. J. Ferro, P. J. Higgins, R. Shankar, D. M. Parker, and A. J. Johnson. TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L551-L559, 1995[Abstract/Free Full Text].

44.   Poss, K. D., and S. Tonegawa. Reduced stress defence in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. USA 94: 10925-10930, 1997[Abstract/Free Full Text].

45.   Rashba-Step, J., A. Tatoyan, R. Duncan, D. Ann, T. R. Pushpa-Rehka, and A. Sevanian. Phospholipid peroxidation induces cytosolic phospholipase A2 activity: membrane effects versus enzyme phosphorylation. Arch. Biochem. Biophys. 343: 44-54, 1997[Medline].

46.   Ritchie, A. J., D. R. Johnson, B. M. Ewenstein, and J. S. Pober. Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation: studies with phorbol myristate acetate and staurosporine. J. Immunol. 146: 3056-3062, 1991[Abstract].

47.   Routtenberg, A., and K. Murakami. Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2+. FEBS Lett. 192: 189-193, 1985[Medline].

48.   Schreck, R., P. Rieber, and P. A. Baeuerle. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 10: 2247-2258, 1991[Medline].

49.   Schuger, L., J. Varani, R. M. Marks, S. L. Kunkel, K. J. Johnson, and P. A. Ward. Cytotoxicity of tumor necrosis factor-alpha for human umbilical vein endothelial cells. Lab. Invest. 61: 62-68, 1989[Medline].

50.   Schutze, S., D. Berkovic, O. Tomsing, C. Unger, and M. Kronke. Tumor necrosis factor induces rapid production of 1',2'diacylglycerol by a phosphatidylcholine-specific phospholipase C. J. Exp. Med. 174: 975-988, 1991[Abstract/Free Full Text].

51.   Stocker, R., Y. Yamamoto, A. F. McDonagh, A. N. Glazer, and B. N. Ames. Bilirubin is an antioxidant of possible physiological importance. Science 235: 1043-1046, 1987[Abstract/Free Full Text].

52.   Suzuki, Y. J., H. J. Forman, and A. Sevanian. Oxidants as stimulators of signal transduction. Free Radical Biol. Med. 22: 269-285, 1997[Medline].

53.   Takeda, K., S. Ishiazwa, M. Sato, T. Yoshida, and S. Shibahara. Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene. J. Biol. Chem. 1994: 22858-22867, 1994.

54.   Tenhunen, R., H. S. Marver, and R. Schmid. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. USA 61: 748-755, 1968[Free Full Text].

55.   Terry, C. M., and K. S. Callahan. Protein kinase C regulates cytokine-induced tissue factor transcription and procoagulant activity in human endothelial cells. J. Lab. Clin. Med. 127: 81-93, 1995.

56.   Terry, C. M., J. A. Clikeman, J. R. Hoidal, and K. S. Callahan. Effect of tumor necrosis factor alpha  and interleukin-1alpha on heme oxygenase-1 expression in human endothelial cells. Am. J. Physiol. 274 (Heart Circ. Physiol. 43): H883-H891, 1998[Abstract/Free Full Text].

57.   Tomaro, M. L., J. Frydman, and R. B. Frydman. Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylkhydrazine, and diamide: evidence for oxidative stress involvement. Arch. Biochem. Biophys. 286: 610-617, 1991[Medline].

58.   Tyrrell, R. M., L. A. Applegate, and Y. Tromvoukis. The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activation by a variety of agents including oxidants. Carcinogenesis 14: 761-765, 1993[Abstract/Free Full Text].

59.   Vile, G. F., S. Basu-Modak, C. Waltner, and R. M. Tyrrell. Heme oxygenase-1 mediates an adaptive response to oxidative stress in human skin fibroblasts. Proc. Natl. Acad. Sci. USA 91: 2607-2610, 1994[Abstract/Free Full Text].

60.   Watt, J., and P. L. Molloy. Specific cleavage of transcription factors by the thiol protease, m-calpain. Nucleic Acids Res. 21: 5092-5100, 1993[Abstract/Free Full Text].

61.   Weber, C., W. Erl, A. Pietsch, M. Strobel, H. W. Ziegler-Heitbrock, and P. C. Weber. Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. Arterioscler. Thromb. 14: 1665-1673, 1994[Abstract/Free Full Text].

62.   Wolin, M. S. Reactive oxygen species and vascular signal transduction mechanisms. Microcirculation 3: 1-17, 1996[Medline].

63.   Yee, E. L., B. R. Pitt, R. R. Billiar, and Y.-M. Kim. Effect of nitric oxide on heme metabolism in pulmonary endothelial cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L512-L518, 1996[Abstract/Free Full Text].

64.   Yet, S.-F., A. Pellacani, C. Patterson, L. Tan, S. C. Folta, L. Foster, W.-S. Lee, C.-M. Hsieh, and M. A. Perrella. Induction of heme oxygenase-1 expression in vascular smooth muscle cells: a link to endotoxic shock. J. Biol. Chem. 272: 4295-4301, 1997[Abstract/Free Full Text].

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