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Published ahead of print on November 29, 2007, doi:10.1165/rcmb.2007-0260OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 483-490, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0260OC

Acrolein Induces Heme Oxygenase-1 through PKC-{delta} and PI3K in Human Bronchial Epithelial Cells

Hongqiao Zhang1 and Henry Jay Forman1

1 School of Natural Science, University of California at Merced, Merced, California

Correspondence and requests for reprints should be addressed to Henry Jay Forman, School of Natural Sciences, University of California, Merced, P.O. Box 2039, Merced, CA 95340. E-mail: hjforman{at}gmail.com


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Heme oxygenase-1 (HO-1) catalyzes the rate limiting reaction of heme metabolism and plays critical roles in resistance to oxidative stress and other cellular functions. It is well known that HO-1 is induced in response to various stresses; however, the signaling pathways involved remain incompletely elucidated. Acrolein is an {alpha},β-unsaturated aldehyde present in cigarette smoke and also a product of lipid peroxidation. In this investigation we studied HO-1 induction in response to acrolein and determined the signaling pathways involved in human bronchial epithelial cells (HBE1 cells). We demonstrated that acrolein significantly increased the HO-1 mRNA content and promoter activity. Acrolein-mediated HO-1 induction was significantly attenuated by pan–protein kinase C (PKC) inhibitors RO318220, staurosporine, and PKC-{delta} selective inhibitor rottlerin and PKC-{delta} small interfering RNA. The HO-1 induction was also decreased by phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin. No significant effects on HO-1 induction were observed with the pretreatment of mitogen-activated protein kinase pathway inhibitors PD98059 (ERK), SB203580 (p38MAPK) and JNKi, and conventional and atypical PKC inhibitors. Furthermore, Nrf2 silencing significantly attenuated the HO-1 induction by acrolein. Inhibition of PKC-{delta} significantly decreased acrolein-mediated Nrf2 nuclear translocation, though inhibition of PI3K had no effect. Taken together, our results indicate that acrolein up-regulates HO-1 expression through both PKC-{delta} and PI3K pathways in HBE1 cells; PKC-{delta} appears to regulate HO-1 induction via modulating Nrf2 nuclear translocation, while PI3K may work through targeting on downstream signaling molecules other than Nrf2.

Key Words: acrolein • heme oxygenase 1 • PKC-{delta} • Akt



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Heme oxygenase-1 (HO-1) induction is crucial in oxidative resistance. Understanding signaling for HO-1 induction by acrolein, a common toxicant, will help develop new strategies for the prevention of diseases associated with oxidative stress.

 
Heme oxygenase (HO) is the rate-limiting enzyme that catalyzes the degradation of heme into iron, biliverdin, and carbon monoxide. By doing so, HO is not only involved in heme metabolism and erythrocyte turnover, it also plays cytoprotective roles against oxidative injuries and inflammation. Among the three isoenzymes of HO identified so far, HO-1 is the only isoform that can be induced in response to stimuli such as oxidants, hypoxia, heavy metals, and inflammatory molecules (1). Accumulating evidence from both in vivo and in vitro experiments suggest that HO-1 plays critical roles in cellular functions such as adaptation to stress, cellular proliferation, and apoptosis (2). Recently HO-1 has also been implicated in various pathologies, including neurodegenerative diseases (3, 4), cardiovascular diseases (5), and pulmonary diseases (6, 7).

HO-1 induction in response to environmental stress has been being extensively studied. The induction occurs mainly at the transcriptional level. Several DNA cis-elements present in the promoter (5'-flanking) region of HO-1 gene, including AP-1 binding sites, electrophile response element (EpRE), stress response element (StRE), cAMP response element, and NF-{kappa}B binding sites, have been associated with the induction of HO-1 gene (1). Among them, EpRE and its binding protein Nrf2 have been studied extensively. Nrf2 is a basic leucine zipper protein (bZIP) belonging to the cap ‘n’ collar (CNC) family of transcription factors and is a well-established player in EpRE signaling. Under basal conditions, Nrf2 is retained in the cytosol through association with Kelch-like ECH-associated protein 1 (Keap1). Upon exposure to oxidative stimuli, Nrf2 is dissociated from Keap1 and translocated into the nucleus, where it forms heterodimers with other bZIP-CNC factors such as small Maf proteins and binds to EpRE to regulate gene transcription (for recent reviews see Refs. 8–10). Recent studies have shown that Nrf2/EpRE activation is essential for HO-1 induction in response to stimuli, especially those linked with oxidative/electrophile stress (11–19).

Several signaling pathways, including ERK, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3K), have been implicated in HO-1 induction (1). It remains unclear how these signaling kinases work. Recent reports suggest that these pathways may increase HO-1 transcription through regulating Nrf2/EpRE activity (20, 21). Indeed, the phosphorylation of Nrf2 is critical for Nrf2-Keap1 dissociation (22, 23) and Nrf2 nuclear exportation (24), and in some research models, inhibition of the above signaling pathways led to the decrease of Nrf2 nuclear translocation (25).

Acrolein is a highly reactive {alpha},β-unsaturated aldehyde present in cigarette smoke, vehicle exhaust, and overheated cooking oil or food (26); it also occurs as a lipid peroxidation product (27) and a metabolic by-product of allyl alcohol, allylamine, spermine, spermidine, and the antineoplastic drug cyclophosphamide (26). Acrolein concentration is increased in various oxidative stress–implicated diseases such as arteriosclerosis (28) and Alzheimer's disease (29). At higher concentrations, acrolein is very toxic and causes injuries by conjugating to proteins and nucleic acids, and disrupting normal cellular function (26). Recently several studies suggest that at subtoxic concentrations, acrolein could activate a variety of signaling pathways and regulate the expression of cytoprotective genes (30). In this study we investigated the HO-1 induction by acrolein and determined the signaling pathways involved in human bronchial epithelial cells with the aim of further understanding the regulatory mechanism of HO-1 induction.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Chemicals and Reagents
Unless otherwise noted, all chemicals were from Sigma (St. Louis, MO). Antibodies and small interfering RNAs (siRNAs) were from Santa Cruz (Santa Cruz, CA). Protein kinase inhibitors were bought from Calbiochem (La Jolla, CA). TRIzol Reagent was from Life Technologies (Grand Island, NY). DNA-free reagent was from Ambion (Austin, TX). TaqMan Reverse Transcription Reagent and SYBR Green PCR Master Mix were from Applied Biosystems (Foster City, CA). Luciferase activity assay kit was from Promega (Madison, WI). FuGENE 6 transfection reagent was from Roche (Indianapolis, IN). M-PER mammalian protein extraction reagent and NE-PER nuclear extraction reagents were from Pierce (Rockford, IL). All chemicals used were at least analytical grade.

Cell Culture and Treatment
A human bronchial epithelial cell line (HBE1 cell) was cultured in collagen-coated dishes in 5% CO2 at 37°C as described by Harper and coworkers (31). Cells were treated when 90% confluent. Acrolein was freshly diluted to appropriate concentrations with 1x PBS before use.

HO-1 mRNA Assay
The content of HO-1 mRNA was determined with real-time RT-PCR method by following the protocol described before (32). Briefly, RNA samples were treated with DNA-free reagent and reverse transcribed by using the TaqMan reverse transcription system; real-time PCRs were then performed with a Cepheid 1.2 real-time PCR machine (Cepheid, Sunnyvale, CA). GAPDH was used as internal control. The primers are as follows. HO-1: sense 5'-TCTCTTGGCTGGCTTCCTTAC-3', antisense 5'-GGCTTTTGGAGGTTTGAGACA-3'; GAPDH: sense 5'-TGGGTGTGAACCATGAGAAG-3, antisense 5'-CCATCACGACACAGTTTCC-3.

Western Blotting
Western blotting was performed as described previously (33). Briefly, protein was extracted and 15 µg protein was heated for 15 minutes at 95°C in a 2x loading buffer containing SDS (Tris base, pH 6.5, glycerol, DTT, and pyronin Y), electrophoresed on a 4 to 20% Tris-glycine acrylamide gel (Invitrogen, Carlsbad, CA), and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membrane was blocked with 5% fat-free milk and then incubated overnight at 4°C with primary antibody in 5% milk in Tris-buffered saline (TBS). After being washed with TBS containing 0.05% Tween 20 (TTBS), the membrane was incubated with secondary antibody at room temperature for 2 hours. After TTBS washing, the membrane was treated with an enhanced chemiluminescence reagent mixture (ECL Plus; Amersham, Arlington Heights, IL) for 5 minutes. The target bands were imaged on a Kodak Image Station 2000R (Kodak, Rochester, New York).

Plasmid and Transient Transfection
The human HO-1 promoter-driven luciferase plasmid, which was constructed by inserting 4.9 kb of the 5'-flanking region of human HO-1 gene into pGL3 basic vector (34), was kindly provided by Dr. Norbert Leitinger at University of Virginia. Cells were seeded in 12-well collagen-coated plates and transfected at 70 to 80% confluence by using FuGENE 6 transfection Regent. β-galactosidase plasmid was cotransfected as an internal control. After transfection for 24 hours, medium was replaced and cells were treated with/without acrolein for 24 hours. After treatment, cells were collected and lysed with M-PER. The luciferase and β-galactosidase activity assay were performed as described before (32).

siRNA Transient Transfection
Transfection of siRNA was performed using FuGENE 6 transfection reagent by following the procedure provided with the transfection reagent and the protocol described earlier (35). HBE1 cells at approximately 60 to 80% confluence were transfected with 50 nM (PKC-{delta}, Nrf2, or control) siRNA (Santa Cruz Biotechnology). Cell medium was replaced at a certain time after transfection, and cells were treated with/without acrolein as indicated in the figures before being collected for various assays.

Cytotoxicity Assay
Toxicity of acrolein was determined with the MTT assay. Briefly, cells at 70 to 80% confluence were treated with various doses of acrolein for 24 or 48 hours, and MTT was added to medium. Cells were incubated for 2 hours, and then MTT absorbance was read at a wavelength of 570 nm. Survival was reported as the percentage of living cells compared with vehicle group.

Statistical Analysis
The comparative {Delta}{Delta}CT method was used for the relative mRNA quantitation as described previously (25). All data were expressed as the mean ± SE. Sigma Stat software (SPSS, Inc., Chicago, IL) was used for statistical analysis, and statistical significance was accepted when P < 0.05. The Tukey test was used for comparison of mRNA level and luciferase activity.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Acrolein Induces HO-1 Transcription in HBE1 Cells
Acrolein can be lethal, but at subtoxic doses it is a potent signaling agent that can affect various biochemical processes in cells. Acrolein at an initial concentration of 5 to 20 µM was used in the current study. Acrolein is volatile and in our system had a half-life of approximately 5 minutes in media without cells at room temperature. So the actual exposure to acrolein to which the cells were subjected was far less than a continuous exposure to 5 to 20 µM. In the range of doses used, acrolein did not affect the cell survival (Figure 1A) or cause any visible morphologic change of HBE1 cells (data not shown). To investigate HO-1 induction by acrolein, we measured the mRNA content of HO-1 with the real-time PCR method after exposure to acrolein. After exposure for 12 hours, HO-1 mRNA was increased by 2.5-fold by 5 µM acrolein compared with nontreatment; and more markedly, the HO-1 mRNA was increased by approximately 32-fold with 20 µM acrolein treatment (Figure 1B). These data suggest that acrolein increased HO-1 mRNA in a dose-dependent manner. The HO-1 mRNA increase upon acrolein exposure was also time dependent: the increase became significant beginning at 6 hours and remained so at 24 hours, but the maximal increase occurred at 12 hours of acrolein exposure (data not shown).


Figure 1
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  		Figure 1. Acrolein up-regulates heme oxygenase-1 (HO-1) gene expression. (A) In vitro cytotoxicity assay of acrolein (MTT assay). HBE1 cells were pretreated with various concentrations of acrolein for indicated time and MTT assay was then performed as described in MATERIALS AND METHODS (n = 3). (B) Dose-dependent increase of HO-1 mRNA content by acrolein. HBE1 cells were treated with different doses of acrolein for 12 hours and the mRNA level of HO-1 was determined with real time-PCR method. (C) Effect of acrolein on HO-1 promoter activity. HBE1 cells were transiently transfected with hHO-1-Luc reporter for 15 hours and then treated with acrolein. Twenty-four hours later, cells were collected and luciferase activity was measured. The figure shows the value of induction fold of luciferase activity after acrolein treatment (n = 3). *P < 0.05 compared with vehicle control.

 
To determine whether acrolein increases HO-1 expression at the transcription level, the effect of acrolein on HO-1 promoter activity was investigated with a reporter assay. The activation of HO-1 promoter was studied by using a luciferase reporter that was driven by a 4.9-kb proximal region of the human HO-1 promoter (34). As shown in Figure 1C, the HO-1 promoter activity was increased by acrolein in a dose-dependent manner, and it was increased as high as 5.2-fold by 20 µM acrolein. Taken together, data in Figures 1B and 1C reveal that acrolein up-regulated HO-1 expression at the transcription level. Since the induction of HO-1 was more significant with 20 µM acrolein treatment, 20 µM acrolein was used in the following experiments.

MAPK Pathways Are Not Involved in HO-1 Induction by Acrolein
The three major MAPK pathways (ERK1/2, p38MAPK, and JNK) have been suggested to be involved in the regulation of HO-1 by a variety of agents. Using specific inhibitors against these pathways, we investigated the involvement of MAPKs in acrolein-mediated HO-1 induction in HBE1 cells. At concentrations that effectively inhibit targeted pathways (32), PD98059, SB203580, and JNK inhibitor I (specific inhibitors of ERK1/2, p38MAPK, and JNK pathways, respectively) had no significant effect on HO-1 mRNA level that was increased by acrolein (Figure 2A), suggesting that ERK, JNK, and p38MAPK pathways were not involved in HO-1 induction by acrolein.


Figure 2
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  		Figure 2. Signaling pathways involved in HO-1 induction by acrolein. HBE1 cells were pretreated with inhibitors as indicated for 1 hour before being treated with 20 µM acrolein. Cells were collected 12 hours later and the mRNA level of HO-1 was determined. (A) Effect of mitogen-activated protein kinase (MAPK) inhibitors. (B) Effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) inhibitors. (C) Effect of PKC isozyme inhibitors. (D) Effect of PKC and PI3K inhibitors on HO-1 promoter activation. HBE1 cells were transfected with hHO-1-Luc reporter for 15 hours and cells were pretreated with indicated inhibitors for 1 hour before being treated with/without 20 µM acrolein for 24 hours. Then cells were collected and luciferase activity was measured (n = 3). *P < 0.05 compared with vehicle control, #P < 0.05 compared with acrolein treatment.

 
Both PKC and PI3K Pathways Are Involved in HO-1 Induction by Acrolein
We then investigated the involvement of PKC and PI3K pathways in acrolein-mediated HO-1 induction. As shown in Figure 2B, HO-1 induction was inhibited by pan-PKC inhibitor RO318220 (2 µM) by 70% and staurosporin (0.2 µM) by 65%, respectively. Meanwhile, PI3K inhibitors LY294002 (50 µM) and wortmannin (0.5 µM) also partially inhibited the HO-1 induction (by ~ 50% and 58%, respectively) (Figure 2B). Considering the partial inhibitory effects of PKC and PI3K inhibitors, we examined whether the use of both simultaneously would block the HO-1 induction more effectively. Pretreatment with 2 µM RO318220 and 50 µM of LY294002 in combination abrogated the increase of HO-1 mRNA content (Figure 2B), suggesting that PKC and PI3K act through separate pathways to regulate HO-1 expression.

The involvement of PKC and PI3K signaling pathways in HO-1 induction was further studied with the HO-1 reporter assay. Consistent with their effects on mRNA level, pan-PKC inhibitor RO318220 (2 µM), and PI3K inhibitors LY294002 (50 µM) and wortmannin (0.5 µM), also significantly decreased the activation of HO-1 promoter by acrolein (Figure 2D). In addition, combined pretreatment with both RO318220 and LY294002 completely blocked the activation of HO-1 promoter. Taken together, these data suggest that both PKC and PI3K signaling pathways are involved in the acrolein-mediated HO-1 induction. These data also suggests that PKC and PI3K trigger HO-induction through separate downstream molecules.

PKCs are a family of kinases that include three subfamilies of isozymes: classical or conventional PKC (cPKC: {alpha}, β, {gamma}), novel PKC (nPKC: {delta}, {varepsilon}, {theta}, and {eta}), and atypical PKC (aPKC: {zeta} and {iota}/{lambda}). To investigate which PKC isozymes are responsible for acrolein-mediated HO-1 induction, we also determined the effects of relatively specific inhibitors against PKC isozymes on HO-1 mRNA induction. cPKC inhibitors RO31–8425 and RO32–0432, and a pseudosubstrate peptide inhibitor of the aPKC, did not decrease HO-1 mRNA induction. In contrast, rottlerin, a PKC-{delta} inhibitor, increased the basal level of HO-1 mRNA, but significantly attenuated acrolein-mediated HO-1 mRNA induction (37-fold versus 4.5-fold) (Figure 2C). As shown in Figure 2D, rottlerin did not affect the basal activity of HO-1 reporter driven by a 4.9-kb base pair of DNA sequence from the upstream of 5'-untranslated region of human HO-1 gene, but it decreased acrolein-mediated promoter activity by 58%, suggesting that PKC-{delta} is responsible for acrolein-increased HO-1 mRNA.

To avoid any nonspecific effect of rottlerin and further confirm the involvement of PKC-{delta} in HO-1 induction, effect of PKC-{delta} siRNA on acrolein-mediated HO-1 induction was investigated. PKC-{delta} protein was decreased by 56% with the treatment of siRNA for 25 hours (Figure 3A). Meanwhile, PKC-{delta} siRNA decreased HO-1 mRNA induction (Figure 3B) and HO-1 promoter activation (Figure 3C) by approximately 25% and 33%, respectively, indicating that PKC-{delta} is involved in acrolein-mediated HO-1 induction.


Figure 3
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  		Figure 3. PKC-{delta} small interfering RNA (siRNA) decreased HO-1 induction by acrolein. (A) Silencing of PKC-{delta} protein by siRNA treatment. HBE1 cells were transfected with 50 nM of control- or PKC-{delta} siRNA for 12 or 24 hours before being collected for PKC-{delta} protein assay with Western blot. C, control siRNA; P, PKC-{delta} siRNA. (B) Effect of PKC-{delta} siRNA on acrolein-induced HO-1 mRNA. Cells were pretreated with PKC-{delta} siRNA for 24 hours and treated with 20 mM acrolein for 12 hours, then HO-1 mRNA was analyzed with real-time RT-PCR (n = 7). (C) PKC-{delta} siRNA decreased HO-1 promoter activation by acrolein. Cells were transiently transfected with siRNA and hHO-1 reporter for 24 hours before being treated with acrolein for another 24 hours. Then cells were collected and luciferase activity was measured (n = 4). *P < 0.05 compared with vehicle control, #P < 0.05 compared with acrolein treatment.

 
Nrf2/EpRE Is Involved in HO-1 Induction by Acrolein
Many transcription factors including AP-1, NF-{kappa}B, and Nrf2 have been implicated in HO-1 induction by other agents. To test the hypothesis that Nrf2 is involved in acrolein-induced HO-1 expression, we investigated the effect of Nrf2 silencing on HO-1 induction. Nrf2 siRNA (50 nM) markedly decreased Nrf2 protein levels in both the cytosol and nucleus, and a more significant effect was observed in nuclear Nrf2 with acrolein treatment (Figure 4A). Nrf2 siRNA significantly attenuated acrolein-increased HO-1 mRNA (Figure 4B) and promoter activity (Figure 4C). These data suggest that Nrf2 is a crucial transcription factor involved in HO-1 induction by acrolein.


Figure 4
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  		Figure 4. Nrf2 is involved in HO-1 induction by acrolein. (A) Silencing effects of Nrf2 siRNA on Nrf2 protein. Cells were transiently transfected with siRNA for 24 hours and then treated with 20 µM acrolein for 1 hour before Nrf2 protein in both cytosol and nucleus was examined with Western blot. C, control siRNA; N, Nrf2 siRNA. (B) Silencing Nrf2 decreased acrolein-mediated increase in HO-1 mRNA level. HBE1 cells were transiently transfected with 50 nM SiRNA of Nrf2 for 24 hours before being treated with/without acrolein for 12 hours. Then cells were collected and mRNA was determined with aforementioned method. (C) Nrf2 SiRNA abrogated HO-1 promoter activation by acrolein. HBE1 cells were co-transfected with hHO-1-Luc reporter and 50 nM of siRNAs as indicated. Fifteen hours later, medium was replaced and cells were treated with/without 20 µM acrolein for 24 hours. Then cells were collected and luciferase activity was measured (n = 3). *P < 0.05 compared with vehicle control, #P < 0.05 compared with acrolein treatment.

 
Effects of PKC-{delta} and PI3K Inhibitors on Nrf2 Nuclear Accumulation
Based on data above that suggested PKC-{delta}, PI3K, and Nrf2 were involved in HO-1 induction, and that PKC was reported to be involved in Nrf2 activation, we examined whether acrolein activates Nrf2 via PKC-{delta} and PI3K pathways. Nrf2 is located in the cytoplasm at basal conditions, and upon stimulation it translocates into the nucleus to play its role as transcription factor. The nuclear accumulation of Nrf2 is usually considered a marker of Nrf2 activation in response to stressors. As shown in Figure 5, 1 hour of treatment with 20 µM acrolein increased nuclear Nrf2 significantly (~ 2.5-fold) compared with nontreatment in HBE1 cells. Pretreatment with PKC-{delta} inhibitor (rottlerin) for 1 hour abrogated acrolein-caused Nrf2 nuclear translocation, while PI3K inhibitor (LY294002) had no effect on Nrf2 activation at concentrations that inhibited HO-1 induction, suggesting that PKC-{delta} is but PI3K is not involved in the activation of Nrf2.


Figure 5
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  		Figure 5. Effect of PKC-{delta} and PI3K inhibitors on Nrf2 nuclear accumulation. HBE1 cells were pretreated with inhibitors for 1 hour before being treated with acrolein for another 1 hour. Cells were then collected and nucleus was extracted. Nrf2 and Lamin B1 in the nucleus were determined. Lamin B1 was used as the internal control. PO: 10 µM rottlerin; LY: 50 µM LY294002.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
HO-1 induction has been observed in numerous types of cells and upon exposure to diverse agents. This inducible feature of HO-1 makes it a critical component of cellular resistance mechanism against internal and external oxidative stresses such as inflammation and cigarette smoking. In this study we demonstrate that acrolein, an {alpha},β-unsaturated aldehyde generated during oxidative stress and present in cigarette smoke, is a potent inducer of HO-1 in human bronchial epithelial cells (Figures 1B and 1C). This finding is consistent with and confirms previous reports that acrolein could induce HO-1 in bovine artery endothelial cells (36) and human alveolar macrophages (37). This suggests that acrolein-mediated HO-1 induction may play important roles in the adaptive response to environmental insults such as cigarette smoke. Other oxidants and electrophiles generated during oxidative stress or present in cigarette smoke, such as H2O2, NO, and 4-hydroxynonenal, can also up-regulate HO-1 expression (19, 38, 39), but it remains unclear if any synergistic effect exists among these inducers.

Many signaling pathways, including PKC, PI3K, and MAPK pathways (ERK, JNK, and p38MAPK), have been implicated in HO-1 induction (1). The acrolein-mediated HO-1 induction was attenuated by approximately 65% and 50% with inhibition of PKC and PI3K, respectively; and the induction was completely blocked with the inhibition of both pathways (Figures 2B and 2D). This indicates that both PKC and PI3K pathways are required for the induction of HO-1 by acrolein. Other groups also observed a similar phenomenon, namely that combined action of several pathways are needed for HO-1 induction. For instance, McNally and colleagues (40) reported that PKC or p38MAPK inhibitor alone abrogated only about 50% of curcumin-mediated HO-1 induction, and that both PKC and p38MAPK pathways were required for the full induction. In another study, individual inhibition of PKC or MAPK decreased less than 50% of HO-1 induction by oxLDL, whereas the combined inhibition of both pathways completely blocked the induction (15).

PKC is a family of at least 11 isozymes, and our data strongly suggest that PKC-{delta} is the PKC isozyme responsible for acrolein-induced HO-1 expression (Figures 2C, 2D, and 3). The effect of the PKC-{delta} inhibitor rottlerin on HO-1 induction appeared to be more potent compared with that of PKC-{delta} siRNA. The former decreased the HO-1 mRNA induction by 87% and promoter activity by 60%, respectively, while the latter decreased the HO-1 promoter activation by 33% and mRNA by 25%. Nonetheless, the apparently lesser potency of PKC-{delta} siRNA was likely due to the lower efficiency of siRNA in deceasing PKC-{delta} protein than rottlerin was in decreasing activity, as the maximal decrease of PKC-{delta} protein by its siRNA was only 60%. Taking this observation into consideration, the data from the application of rottlerin and siRNA were consistent. Together, the data clearly show that PKC-{delta} is partially involved in acrolein-mediated HO-1 induction in HBE1 cells.

In contrast with the effect on acrolein-induced HO-1, results on the effect of PKC-{delta} inhibition on basal HO-1 expression revealed other aspects of the regulation of this gene. Although rottlerin increased basal expression of HO-1 mRNA (Figure 2C) at the transcriptional level (data not shown), it has no effect on the basal activity of the reporter driven by 4.9-kb base pair of DNA sequence from the upstream of the 5'-untranslated region of human HO-1 promoter, suggesting that rottlerin may work through activating cis-elements further upstream of the DNA sequence used in the reporter construct. One could also speculate equally that rottlerin increased HO-1 expression by inhibiting signaling for regulation of a suppressor element. The current evidence, however, is circumstantial, and the role of other elements is actually beyond the scope of the current study.

The involvement of PKC-{delta} in HO-1 induction was also previously observed in monocytes by curcumin (41) and endothelial cells by piceatannol (42). There are also reports about the involvement of cPKC (43) and aPKC (44, 45) in HO-1 induction. In the current study, however, the cPKC inhibitors RO31–8425 and RO32–0432, and the aPKC pseudosubstrate inhibitor (peptide inhibitor against PKC-{zeta}), had no effect on HO-1 induction, nor did they have an effect on nuclear translocation of Nrf2 (data not shown), indicating that these PKC isozymes are not involved in acrolein-mediated HO-1 induction. The underlying reason that different PKC isoforms are involved in different cells is not entirely clear. Some studies found that the expression profile of PKC isozymes is tissue specific and the cellular spatial distribution of them is cell type specific (46, 47). This may cause the activation of different PKC isozyme profiles in response to HO-1 inducers in different cell systems. Another possible explanation is that the difference in PKC isozymes involved may be related to inducers. For example, Rushworth and coworkers reported that in human monocytes, LPS induced HO-1 through cPKC (43), while curcumin acted via PKC-{delta} (41). Further studies are necessary to elucidate whether different PKC isozymes are involved in HO-1 induction in different cells because of their presence or absence, or because the pathways taken to activation of the transcription factors, Nrf2 and its partner(s) in EpRE activation, differ due to the absence or presence of other members of their signaling pathways.

In contrast to some studies, we did not find the involvement of MAPKs (ERK1/2, p38MAPK, and JNK) in HO-1 induction by acrolein (Figure 2A). In a previous study, Wu and colleagues suggested that in acrolein-caused HO-1 induction in bovine artery endothelial cells, PKC and PI3K were not involved, but that JNK was involved (36). However, in that study the inhibitor SP600125 was used, although it is clearly nonspecific for JNK and inhibits at least 13 other kinases in the same concentration range (48). Regardless of this, studies have shown that signaling pathway involved in HO-1 induction is cell dependent, and this may explain the inconsistency between the two studies (BEAC versus HBE1).

Sequencing of the human HO-1 promoter has revealed putative regulatory cis-elements of AP-1, NF-{kappa}B, C/EBP, SP-1, and multiple EpREs (also called ARE, MRE, or StRE in some publications) that appear likely to be regulated by oxidants and electrophiles like acrolein. It is becoming clear that EpRE plays a critical role in HO-1 regulation and inducers may increase HO-1 gene expression by affecting the proteins binding to EpRE. Consistent with this, we found that knockout of Nrf2, the most established EpRE-binding protein, significantly attenuated acrolein-mediated HO-1 induction (Figures 4B and 4C), indicating the involvement of Nrf2/EpRE signaling in the induction. At least two nonexclusive mechanisms of Nrf2 activation, Keap1 modification and Nrf2 phosphorylation, have been proposed. The former proposes that modification of the active thiol groups on Keap1 disrupts the Nrf2-Keap1 complex and leads to Nrf2 dissociation (49–52). The latter mechanism suggests that phosphorylation of Nrf2 at Ser40 is necessary for its release from Keap1 (23). Based on its electrophilic character, acrolein may dissociate Nrf2 from Keap1 through a mechanism similar to that of other electrophiles such as HNE and 15-PGJ2, that is, by conjugating with the thiol groups on the active site of Keap1 (52). Current data that PKC-{delta} inhibitor rottlerin abrogated acrolein-increased Nrf2 nuclear accumulation suggest that PKC-mediated phosphorylation is also involved in Nrf2 dissociation/activation (Figure 5). On the other hand, although inhibition of PI3K decreased HO-1 induction, it had no significant effect on Nrf2 nuclear translocation, suggesting that PI3K regulated HO-1 induction through signaling molecules other than Nrf2. Some components of the EpRE transcription machinery such as the Nrf2 partners and associated proteins could be potential targets of PI3K pathway, though these proteins may not be directly phosphorylated by PI3K. For instance, CBP, a nuclear protein that binds Nrf2 transactivation domain and increases Nrf2 activity (53, 54), and C/EBP responsive element binding protein (CREB), the coactivator of CBP (55), could be regulated by phosphorylation (56–61) and thus a potential target of the PI3K pathway. Instead of targeting EpRE/Nrf2 machinery, PI3K may also regulate acrolein-mediated HO-1 induction through cis-elements other than EpRE. Rojo and coworkers reported that PI3K was involved in HO-1 induction through modulating the binding of SP-1 to a cis-element between –146 and –58 in the proximal region of human HO-1 promoter (45). In another study, Chung and colleagues found that PI3K was involved in the activation of Ets-2 and it's binding to an Ets-2 binding site at –93 of the HO-1 promoter, which site was critical for LPS-mediated HO-1 induction (62). Indeed, the activation of the HO-1 reporter, which was driven by a 4.9-kb base pair of proximal DNA sequence from the 5'-untranslated region of human HO-1, was partially inhibited by PI3K inhibitor LY294002 and wortmannin (Figure 2D). Furthermore, the inhibitory effect of Nrf2 siRNA on HO-1 induction was incomplete (Figures 4B and 4C), although Nrf2 nuclear translocation was decreased to basal level by siRNA silencing, suggesting that other cis-elements than EpRE or transcription factors other than Nrf2 are potential targets of PI3K involved in the HO-1 induction with acrolein. In other words, PI3K may regulate HO-1 induction through signaling events other than Nrf2, and we are in the process of investigating these downstream signaling molecules activated by acrolein.

HO-1 induction is a crucial mechanism of resistance against oxidative stress, and understanding the signaling pathways involved in HO-1 induction will help develop new strategies for the prevention and treatment of diseases associated with oxidative stress. We found that acrolein, a lipid peroxidation product, markedly induced HO-1 gene expression in human bronchial epithelial cells. We also found that both PKC-{delta} and PI3K signaling pathways were involved in acrolein-mediated HO-1 induction. In addition, our results suggest that PKC-{delta} regulates HO-1 induction through Nrf2, whereas PI3K may work through signaling events other than Nrf2. These data provide evidence that acrolein is a potent inducer of cytoprotective genes such as HO-1 in response to oxidative insults and that multiple signaling pathways are involved in HO-1 induction.


   Footnotes
 
This work was supported by grant 14RT-0059 from the California Tobacco Related Diseases Research Program.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0260OC on November 29, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 9, 2007

Accepted in final form October 19, 2007


   References
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 Abstract
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 MATERIALS AND METHODS
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 DISCUSSION
 References
 

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