Induction of Heme Oxygenase-1 During Endotoxemia Is Downregulated by Transforming Growth Factor-β1

Materials

Salmonella typhosa LPS (Sigma Chemical Co) was dissolved in 0.9% saline and stored at –20°C. Recombinant human IL-1β (Collaborative Biomedical) was stored at –80°C until use. Simian TGF-β1 (gift of Bristol-Myers Squibb Pharmaceutical Research Institute) was stored at 4°C. ZnPP (Porphyrin Products) was dissolved in 0.1N NaOH and neutralized with 0.1N HCl immediately before administration as described.²⁷ The ZnPP was also protected from light during its preparation and use.²⁸

Cell Culture

Rat aortic smooth muscle cells (RASMCs) were harvested from male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) by enzymatic dissociation according to the method of Gunther et al.²⁹ The cells were cultured in DMEM (JRH Biosciences) supplemented with 10% FCS, penicillin (100 U/mL), streptomycin (100 μg/mL), and 25 mmol/L HEPES (pH 7.4) (Sigma). RASMCs were passaged every 4 to 7 days, and experiments were performed on cells 6 to 8 passages from primary culture. After the cells had grown to confluence, they were placed in 2% FCS for 12 hours before the addition of cytokines.

Rat Model of Endotoxemia

To measure tissue levels of HO-1 mRNA and protein, we injected conscious male Sprague-Dawley rats (200 to 250 g) with Salmonella typhosa LPS (4 mg/kg IP) as described.^{10 11} Immediately after LPS administration, the rats received an intraperitoneal injection of vehicle (1% serum albumin) or TGF-β1 (20 μg/kg). The rats were killed 10 hours after LPS administration, and tissue was processed for RNA or protein extraction (see below). As controls for animals receiving LPS, rats were also injected with 0.9% saline. Tissue from control rats was harvested and processed in an identical manner.

Northern Blot Analysis

Total RNA was obtained from rat tissue and cultured RASMCs by guanidinium isothiocyanate extraction and centrifuged through cesium chloride.³⁰ RNA was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters. The filters were hybridized at 68°C for 2 hours with ³²P-labeled rat HO-1 or HO-2 probes^{11 31} in QuikHyb solution (Stratagene). Hybridization with the HO-1 probe revealed a single 1.8-kb transcript, whereas hybridization with the HO-2 probe showed transcripts at 1.3 kb and 1.9 kb, as described elsewhere.^{32 33} The hybridized filters were then washed in 30 mmol/L sodium chloride, 3 mmol/L sodium citrate, and 0.1% SDS solution at 55°C and autoradiographed with Kodak XAR film at –80°C for 8 to 12 hours or stored on phosphor screens for 2 to 4 hours. To correct for differences in RNA loading, the filters were washed in a 50% formamide solution at 80°C and rehybridized with a ³² P-labeled oligonucleotide probe complementary to 18S ribosomal RNA. Images were displayed, and radioactivity was measured on a PhosphorImager running the ImageQuant software (Molecular Dynamics).

Western Blot Analysis

We obtained proteins from rat aortic tissue with an extraction buffer containing 50 mmol/L Tris-HCl, 10% glycerol, 5 mmol/L magnesium acetate, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and 1.0 mmol/L phenylmethylsulfonyl fluoride. Protein from cultured RASMCs was obtained with an extraction buffer containing 13.2 mmol/L Tris-HCl, 5.5% glycerol, 0.44% SDS, and 10% β-mercaptoethanol. An equal amount of soluble protein (50 μg) was fractionated by Tris-glycine-SDS–polyacrylamide gel (12%) electrophoresis. An identical gel was stained with 0.5% Coomassie blue dye to ensure equal loading. Proteins on the gels were then transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membranes were incubated for 60 minutes with a 10% nonfat dry milk blocking solution, washed extensively with PBS-T (1.5 mol/L NaCl and 0.5% Tween, pH 7.2), and then incubated for 2 hours with a polyclonal rabbit antibody (1:1000) to recombinant rat HO-1 protein (SPA-895, Stressgen) or a polyclonal rabbit antibody (1:2000) against purified rat testis HO-2 (OSA-200, Stressgen). The blots were washed several times more with PBS-T and incubated for 60 minutes with a donkey anti-rabbit immunoglobulin antibody linked to horseradish peroxidase (Amersham Life Science). Specific proteins were detected by enhanced chemiluminescence (ECL, Amersham Life Science) and evaluated by densitometric analysis with the NIH Image software (National Institutes of Health). A band for HO-1 protein was visible at the expected size of 32 kDa. We tested the specificity of the HO-1 antibody by preincubating it with its immunogen (SPP-730, Stressgen) and confirming the absence of the 32-kDa band. A band for HO-2 protein was visible at the expected size of 36 kDa.

Transfection and Luciferase Activity

RASMCs were transfected by a DEAE-dextran method as described.⁹ In brief, 500 000 cells were plated onto 100-mm tissue culture dishes and allowed to grow for 48 to 72 hours (until 80% to 90% confluent). Luciferase plasmid DNA (pGL2-basic) (5 μg) containing 4.0 kb of the HO-1 5′-flanking sequence and pOPRSVI-CAT (to correct for differences in transfection efficiency) were added to the cells in a solution containing DEAE-dextran (500 μg/mL). The RASMCs were then shocked with a 5% DMSO solution for 1 minute and allowed to recover in medium containing 10% FCS. Twelve hours after transfection, the cells were placed in 2% FCS. The cells were then stimulated with IL-1β (10 ng/mL) or a combination of IL-1β and TGF-β1 (10 ng/mL) for 24 hours. Cell extracts were prepared by a detergent lysis method (Promega), and luciferase activity was measured in duplicate for all samples by using the Promega luciferase assay system and an EG&G AutoLumat LB953 luminometer. A chloramphenicol acetyl transferase (CAT) assay was performed by a modified 2-phase fluor diffusion method.^{9 34} The ratio of luciferase activity to CAT activity in each sample served as a measure of normalized luciferase activity.

HO Enzyme Activity

RASMC extract was prepared with a homogenization buffer (30 mmol/L Tris, pH 7.5, 0.25 mol/L sucrose, and 0.15 mol/L NaCl) containing Complete protease inhibitor (Boehringer-Mannheim). Cell extract was centrifuged at 10 000g for 15 minutes, and the supernatant fraction was then centrifuged at 100 000g for 1 hour. The microsomal pellet was resuspended in 50 mmol/L potassium phosphate buffer (pH 7.4) containing Complete protease inhibitor. HO enzyme activity was measured by bilirubin generation as described.^{11 35 36} The microsomal supernatant fraction from the liver of a normal rat served as the source of biliverdin reductase. A reaction mixture (430 μL) containing hemin (50 μmol/L), rat liver microsomal supernatant fraction (60 μL), NADPH-generating solution (167 μL), and smooth muscle cell microsomal protein (20 μg) was incubated at 37°C for 20 minutes in the dark. The reactions were stopped by placement on ice, and the mixtures were scanned with a spectrophotometer (Beckman). The amount of bilirubin formed was derived as the difference in optical density between 464 nm and 530 nm (extinction coefficient for bilirubin, 40 nm⁻¹ · cm⁻¹). To confirm that we were assessing HO activity, the assay was also performed in the presence of ZnPP. Increasing doses of ZnPP inhibited the formation of bilirubin, and thus HO enzyme activity, in a dose-dependent manner (data not shown). HO enzyme activity was expressed as picomolar units of bilirubin formed per milligram of protein per hour. Protein concentrations were determined by a dye-binding assay (Bio-Rad). Comparisons of HO enzyme activity between groups were made by factorial ANOVA followed by the Fisher least significant difference test. Statistical significance was accepted at P<0.05.

TGF-β1 Inhibits LPS-Induced Tissue Expression of HO-1 In Vivo

To determine whether TGF-β1 affected HO-1 mRNA levels in an animal model of endotoxemia, we injected rats with vehicle (0.9% saline) or LPS (4 mg/kg). Rats receiving LPS were then injected with vehicle (1% serum albumin) or TGF-β1 (20 μg/kg).¹⁰ LPS promoted a marked induction of HO-1 mRNA in heart and lung tissue (Figure 1⇓). Administration of TGF-β1 suppressed this LPS-induced increase in HO-1 mRNA in both the heart (39% decrease) and the lung (68% decrease). Although heart and lung tissue are highly vascularized, we wanted to see whether TGF-β1 would have a similar effect on HO-1 in blood vessels. Aortic tissue, like heart and lung tissue, showed a marked induction of HO-1 mRNA¹¹ that was inhibited (60% decrease) by TGF-β1 (Figure 1⇓). We also studied the effect of TGF-β1 on HO-2 mRNA levels in rat heart, lung, and aortic tissue. TGF-β1 did not suppress HO-2 mRNA in any of these tissues (data not shown).

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Figure 1.

Effect of TGF-β1 on LPS-induced HO-1 mRNA in vivo. Conscious male Sprague-Dawley rats were injected with vehicle (gray bars), LPS (4 mg/kg, open bars), or LPS (4 mg/kg) followed by TGF-β1 (20 μg/kg, solid bars). The rats were killed 10 hours after LPS administration, and total RNA was extracted from heart, lung, and aortic tissue. Northern blot analysis was performed with 10 μg of total RNA per lane. After electrophoresis, the RNA was transferred to nitrocellulose filters, which were hybridized with a ³²P-labeled rat HO-1 probe. Filters were also hybridized with a ³²P-labeled oligonucleotide probe complementary to 18S ribosomal RNA to control for differences in loading. The signal intensity of each RNA sample hybridized to the HO-1 probe was divided by that of each sample hybridized to the 18S probe. Normalized signal intensities were then plotted as a percentage of LPS signal (mean±SE). Each experiment was performed 3 times.

Protein was also extracted from the heart and lung tissue of rats injected with vehicle, LPS, or LPS plus TGF-β1. The extracted protein was subjected to Western analysis to determine the effect of TGF-β1 on HO-1 protein levels in vivo. Like LPS-induced HO-1 mRNA expression, LPS-induced HO-1 protein expression was reduced by TGF-β1 in both the heart (48% decrease) and the lung (50% decrease) (Figure 2⇓). These 2 experiments indicate that TGF-β1 can suppress LPS-induced HO-1 mRNA and protein in vivo.

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Figure 2.

Effect of TGF-β1 on LPS-induced HO-1 protein expression in vivo. Heart and lung tissue from rats injected with vehicle, LPS, or LPS followed by TGF-β1 (as described for Figure 1⇑) was homogenized, and 50 μg of soluble protein was fractionated by Tris-glycine-SDS–PAGE. HO-1 protein was detected by immunoblotting with a polyclonal rabbit antibody against recombinant rat HO-1. Bar graph shows mean±SE from 3 analyses.

TGF-β1 Downregulates IL-1β–Induced HO-1 mRNA in Vascular Smooth Muscle Cells

Vascular smooth muscle cells play a critical role in the hemodynamic compromise of endotoxemia, and TGF-β1 inhibits HO-1 message in the aortic tissue of rats injected with LPS (Figure 1⇑). To further characterize this phenomenon, we measured the time course of the effect of TGF-β1 on HO-1 mRNA in cultured RASMCs. Because proinflammatory cytokines such as IL-1β are downstream mediators of LPS-associated disease,² we studied the effect of TGF-β1 on IL-1β–induced HO-1 expression. As we have shown elsewhere,¹¹ 24 hours of IL-1β stimulation (10 ng/mL) produced a significant induction of HO-1 mRNA in RASMCs in vitro (Figure 3A⇓, inset). Administration of TGF-β1 either before or after IL-1β stimulation led to a downregulation (49% to 55% decrease) of HO-1 message (Figure 3A⇓). TGF-β1 had its maximal inhibitory effect on HO-1 expression at a dose of 10 ng/mL (Figure 3B⇓); this dose was used in the remaining in vitro experiments.

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Figure 3.

Downregulation of HO-1 mRNA by TGF-β1 in vitro. A, RASMCs were stimulated with IL-1β (10 ng/mL) for 24 hours, and total RNA was extracted from the cells at the end of this period. The cells were also exposed to vehicle (open bars) or TGF-β1 (10 ng/mL, solid bars) either before (30 minutes) or after (2, 4, 8, and 12 hours) the addition of IL-1β. Inset, RASMCs were exposed to a vehicle control (gray bar) or IL-1β (10 ng/mL, open bar) for 24 hours, and total RNA was extracted from the cells. B, RASMCs that had been treated for 30 minutes with increasing doses of TGF-β1 were exposed to IL-1β. Total RNA was extracted after 24 hours of IL-1β stimulation. In all experiments, Northern blot analyses were performed with 10 μg of total RNA per lane as described for Figure 1⇑. HO-1 signal intensities (mean±SE) were normalized against those of 18S and then plotted as percentage of vehicle signal intensity (A), fold increase from control (inset), or percentage of signal intensity at 0 ng/mL TGF-β1 (B). Each experiment was performed 3 times.

To measure the effect of TGF-β1 on HO-1 promoter activity, we transfected an HO-1 promoter construct containing 4 kb of the HO-1 5′-flanking sequence into RASMCs. After transfection, the cells were stimulated with IL-1β or a combination of IL-1β plus TGF-β1 for 24 hours. In the presence of TGF-β1, HO-1 promoter activity decreased by 56% (as measured by luciferase reporter activity, Figure 4A⇓). This inhibitory effect on HO-1 promoter activity was similar to the downregulatory effect of TGF-β1 on HO-1 mRNA (Figure 3A⇑). TGF-β1 did not shorten the half-life of HO-1 mRNA (Figure 4B⇓). Taken together, these data suggest that TGF-β1 downregulates HO-1 mRNA by inhibiting its transcription.

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Figure 4.

Effect of TGF-β1 on HO-1 promoter activity and mRNA half-life. A, Plasmids (pGL2-basic containing 4 kb of the HO-1 5′-flanking sequence and pOPRSVI-CAT [to correct for differences in transfection efficiency]) were cotransfected into RASMCs. The smooth muscle cells were then stimulated with IL-1β (10 ng/mL, open bar) or a combination of IL-1β and TGF-β1 (10 ng/mL each, solid bar) for 24 hours, after which the cell extracts were harvested. Luciferase activity was expressed as a percentage of IL-1β–induced HO-1 activity (mean±SE, n=8 in each group). B, RASMCs were stimulated with IL-1β (10 ng/mL) for 15 hours. Then vehicle (○) or TGF-β1 (10 ng/mL, •) was coincubated with IL-1β for 3 hours. Actinomycin D (10 μg/mL) was administered to the RASMCs after this coincubation period, and total RNA was extracted from the RASMCs at the indicated times. Northern blot analyses were performed with 10 μg of total RNA per lane as described for Figure 1⇑. The signal intensity of each RNA sample hybridized to the HO-1 probe was divided by that hybridized to the 18S probe. The normalized intensity was then plotted as percentage of 0-hour value against time (in log scale). The experiments were performed 3 times.

TGF-β1 Decreases IL-1β–Induced HO-1 Protein in Vascular Smooth Muscle Cells

To determine whether the effect of TGF-β1 on HO-1 message translated into a decrease in HO-1 protein, we performed Western blot analysis on protein extract from RASMCs stimulated for 48 hours with vehicle alone, IL-1β alone, IL-1β plus TGF-β1, or TGF-β1 alone. IL-1β produced an increase in HO-1 protein in cultured vascular smooth muscle cells (Figure 5⇓). Time-course experiments (data not shown) revealed that peak induction of HO-1 protein occurred after 48 hours of IL-1β stimulation. TGF-β1 decreased IL-1β–induced HO-1 protein by 52% (Figure 5⇓), a reduction similar to its effect on IL-1β–induced HO-1 mRNA. TGF-β1 had no effect on basal HO-1 protein expression (Figure 5⇓, vehicle alone compared with TGF-β1 alone).

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Figure 5.

Effect of TGF-β1 on HO-1 protein expression in RASMCs. Cultured RASMCs were stimulated with vehicle alone (gray bar), IL-1β alone (10 ng/mL, open bar), IL-1β plus TGF-β1 (10 ng/mL each, solid bar), or TGF-β1 alone (10 ng/mL, hatched bar) for 48 hours. Protein extracted from the cells (50 μg) was fractionated by Tris-glycine-SDS–PAGE. HO-1 protein was detected by immunoblotting with a polyclonal rabbit antibody against recombinant rat HO-1 protein. Bar graph shows mean±SE from 3 experiments.

TGF-β1 Does Not Downregulate HO-2 in Vascular Smooth Muscle Cells

We also analyzed the HO-2 response to TGF-β1 after IL-1β stimulation in RASMCs to determine whether the effect of TGF-β1 was selective for HO-1. As we have shown elsewhere,¹¹ IL-1β does not significantly increase HO-2 mRNA (Figure 6A⇓, inset). TGF-β1 also had a minimal effect on HO-2 mRNA levels in vascular smooth muscle cells after IL-1β stimulation (Figure 6A⇓), unlike its effect on HO-1 mRNA levels after IL-1β stimulation (Figure 3A⇑). Moreover, TGF-β1 did not decrease HO-2 protein levels (Figure 6B⇓).

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Figure 6.

Effect of TGF-β1 on HO-2 mRNA and protein expression in RASMCs. A, RASMCs were stimulated with IL-1β (10 ng/mL) for 24 hours, and total RNA was extracted from the cells at the end of this period. The cells were also exposed to vehicle (open bars) or TGF-β1 (10 ng/mL, solid bars) either before (30 minutes) or after (2, 4, 8, and 12 hours) the addition of IL-1β. Inset, RASMCs were exposed to a vehicle control (gray bar) or IL-1β (10 ng/mL, open bar) for 24 hours, and total RNA was extracted from the cells. Northern blot analyses were performed with 10 μg of total RNA per lane as described for Figure 1⇑. HO-2 signal intensities were normalized against those of 18S and then plotted as percentage of vehicle signal intensity (A) or fold increase from control (inset). Each experiment was performed 3 times. B, Protein from cultured RASMCs stimulated with vehicle alone (gray bar), IL-1β alone (10 ng/mL, open bar), IL-1β plus TGF-β1 (10 ng/mL each, solid bar), or TGF-β1 alone (10 ng/mL, hatched bar) was fractionated by Tris-glycine-SDS–PAGE. HO-2 protein was detected by immunoblotting with a polyclonal rabbit antibody against purified rat testis HO-2. Bar graph shows mean±SE from 3 experiments.

TGF-β1 Decreases IL-1β–Induced HO Enzyme Activity in Vascular Smooth Muscle Cells

To confirm that the decrease in HO-1 protein led to an overall decrease in enzymatic activity, we measured HO enzyme activity in RASMCs after 48 hours of stimulation with vehicle alone, IL-1β alone, IL-1β plus TGF-β1, or TGF-β1 alone (Figure 7⇓). IL-1β alone produced an increase in HO enzyme activity in vascular smooth muscle cells. The addition of TGF-β1 to cells stimulated with IL-1β resulted in a 77% decrease in HO enzyme activity (Figure 7⇓), bringing the enzymatic activity to a level not different from that in cells treated with vehicle alone. These effects are similar to the effect of TGF-β1 on HO-1 protein levels after IL-1β stimulation (Figure 5⇑). Because TGF-β1 did not suppress HO-2 mRNA or protein expression after IL-1β stimulation, the decrease in IL-1β–induced enzyme activity by TGF-β1 most likely reflects an inhibition of HO-1 enzyme activity.

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Figure 7.

Effect of TGF-β1 on HO enzyme activity in RASMCs. RASMCs were stimulated with vehicle alone (gray bar), IL-1β alone (10 ng/mL, open bar), IL-1β plus TGF-β1 (10 ng/mL each, solid bar), or TGF-β1 alone (10 ng/mL, hatched bar) for 48 hours. HO enzyme activity (mean±SE) was measured from the microsomal pellet of the cell extract. The experiment was performed 3 times. *P<0.05 vs all other groups.