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Am J Physiol Heart Circ Physiol 288: H2647-H2652, 2005. First published January 28, 2005; doi:10.1152/ajpheart.00937.2004
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Extracellular acidosis induces heme oxygenase-1 expression in vascular smooth muscle cells

Helen Christou, Nicole Bailey, Morgan S. Kluger, S. Alex Mitsialis, and Stella Kourembanas

Division of Newborn Medicine, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts

Submitted 9 September 2004 ; accepted in final form 25 January 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Extracellular acidosis (EA) has profound effects on vascular homeostasis, including vascular bed-specific alterations in vascular tone. Regulation of gene expression by EA has been observed in a variety of cells including vascular endothelial cells. Whether EA regulates gene expression in vascular smooth muscle cells (VSMCs) is not known. Heme oxygenase (HO)-1 is expressed in vascular cells, and its expression is regulated by cellular stressors such as heat, radiation, and hypoxia. Increased HO-1 expression in VSMCs leads to increased production of CO and its second messenger cGMP, which are important regulators of vascular tone and paracrine interactions in the vasculature. We examined whether EA regulates the expression of HO-1 in VSMCs. Exposure of VSMCs to acidic medium (pH 6.8) significantly increased HO-1 mRNA and protein compared with exposure to medium of physiological pH (pH 7.4). The acidic induction of HO-1 expression was time dependent and involved both transcriptional activation of the HO-1 gene and enhanced stability of HO-1 mRNA. Nitric oxide did not appear to mediate this response. We conclude that HO-1 is transcriptionally and posttranscriptionally upregulated by EA in VSMCs. This induction is time dependent and reversible. We speculate that EA, as an important tissue and cellular stressor for VSMCs, may elicit changes in gene expression patterns that contribute to the maintenance or disruption of vascular homeostasis.

transcriptional activation; ribonucleic acid stabilization

EXTRACELLULAR ACIDOSIS (EA) is a common clinical state that has profound cardiovascular effects. Although extracellular pH is normally maintained within narrow limits around 7.4, a number of acute and chronic disorders disrupt the acid-base homeostatic mechanisms and lead to systemic acidemia. In addition, local tissue acidosis is found in areas of inflammation, ischemia, and/or hypoxia as well as within solid tumors (36). Systemic acidemia leads to vascular bed-specific alterations in vascular tone and modulation of cardiac contractility. Local tissue acidosis is an important modulator of tumor invasiveness (20) and susceptibility of tumors to radiation, chemotherapy (20), and antiangiogenic therapy (34). The cellular and molecular events mediating the effects of acidosis on vascular homeostasis are incompletely understood. Because acidosis often coexists with hypoxia, the role of acidosis as an independent modulator of vascular cell behavior has, until recently, been overlooked. However, recent studies in vitro and in vivo support the idea that EA has cellular effects independent of hypoxia (2, 9, 21, 27, 32, 35).

Heme oxygenase (HO) and its enzymatic products biliverdin and CO have received increasing recognition as biologically important modulators of cellular interactions in the vasculature. The inducible form of this enzyme, HO-1, is ubiquitously distributed in mammalian tissues, and its expression is regulated by a number of nonheme inducers such as cytokines, heavy metals, hormones and endotoxin (1). In addition, a number of cellular stressors including ultraviolet radiation, hypoxia, hyperoxia, heat shock, and glutathione depletors induce the expression of HO-1 in a variety of cell types (1). Converging lines of investigation in vitro and in vivo suggest that HO-1 induction may represent an adaptive response to these stimuli. In vascular smooth muscle cells, HO-1 is known to be regulated by hypoxia (25), inflammatory (40) and oxidant (29) mediators, nitric oxide (13), and hemodynamic forces (33). The mechanisms of regulation involve predominantly transcriptional activation of the HO-1 gene, whereas nitric oxide has a posttranscriptional effect via stabilization of HO-1 mRNA (13). Smooth muscle cell-derived CO produced by HO-1 activity directly inhibits vascular smooth muscle cell (VSMC) proliferation (24) and has a paracrine effect on endothelial cells whereby endothelin-1, PDGF-B, and VEGF gene expression is altered (19, 23). In vivo experiments have shown that increased HO-1 expression has anti-inflammatory effects in the lung (22, 26), prevents cardiac transplant arteriosclerosis (11), determines cardiac xenograft survival (30), and confers cytoprotection in the setting of cardiac ischemia-reperfusion injury (12). Ongoing in vitro studies are focused on understanding both the effector molecules and the intracellular signaling events involved in these protective effects of HO-1. We hypothesized that EA will induce HO-1 expression in VSMCs, and we examined whether transcriptional and posttranscriptional mechanisms are involved in this regulation.


   METHODS
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 METHODS
RESULTS
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Cell culture and exposure to EA. Primary cultures of rat aortic smooth muscle cells (RASMCs) or rat pulmonary artery smooth muscle cells (RPASMCs) grown in DMEM supplemented with 10% FCS and 2 mM glutamine were passaged every 3–4 days and were used between passages 5 and 9. When they reached ~80% confluence, cells were subjected to 48 h of serum deprivation (0.5% FCS) before exposure to media of different pH values. We used DMEM-F12 (1:1)-0.5% FCS media, pH adjusted to the desired value with 100 mM HEPES buffer. The cells were incubated in a 37°C incubator for different time periods, and they were then harvested for extraction of nuclei, RNA, or protein. Because subtle cellular stimuli such as medium replenishment may lead to altered HO-1 expression, we also did a time course of HO-1 mRNA and protein in medium of physiological pH (7.4).

Northern and Western blot analysis. Northern and Western blot analyses were done by standard methods (22). We used a cDNA probe specific for rat HO-1 (7) for Northern blot analysis and an anti-rat HO-1 antibody (SPA 896, Stressgen) at 1:1,000 dilution for Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used for normalization of the Northern blots. Quantification of signals was done with NIH Image software.

Nuclear run-on assay. Confluent cultures of RASMCs exposed to pH of 6.8 or 7.4 for 18 h were lysed and nuclei were isolated as described by Kavanaugh et al. (17). One hundred microliters of nuclear suspension was incubated with CTP, ATP and GTP at 0.5 mM and 250 µCi of [{alpha}-32P]UTP (3,000 Ci/mmol; New England Nuclear, Boston, MA). In vitro transcription was performed, and the samples were phenol/chloroform extracted, precipitated, and resuspended at equal counts per minute (cpm) per milliliter in hybridization buffer (10–20 x 106 cpm/ml). Hybridization to denatured probes (1 µg) slot-blotted on nitrocellulose filters was performed at 40°C for 4 days in the presence of formamide. cDNA probes for HO-1 and GAPDH were used.

RNA stability. After 48 h of serum deprivation, near-confluent cultures of RASMCs were exposed to medium of pH 6.8 or 7.4 for 18 h. Actinomycin D (5 µg/ml; Calbiochem) was then added to the medium, the cells were harvested at various time points, RNA was isolated, and Northern blot analyses were performed. The half-life of HO-1 mRNA under different conditions was estimated from the time-dependent decay of HO-1 mRNA.

Treatment with N{omega}-nitro-L-arginine. After 48 h of serum deprivation, RASMCs were treated with 2.5 mM N{omega}-nitro-L-arginine (L-NNA) or diluent alone for 1 h before and during exposure to EA (pH 6.8) or medium of pH 7.4 overnight.

Statistical methods. The nonparametric ANOVA (Kruskal-Wallis) test was used to compare median values among the experimental groups, and differences were considered significant if P < 0.05. For the RNA stability graph (see Fig. 5B) our data (average of each time point) were plotted on a semilogarithmic scale (log-lin) and curve fitting to the best exponential curve was done (Deltagraph).



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  		Fig. 5. Effect of extracellular acidosis on HO-1 mRNA stability in RASMCs. A: Northern blot analysis of HO-1 and GAPDH mRNA stability in medium of pH 7.4 or 6.8. RASMCs were cultured in media of the 2 different pH values (6.8 and 7.4) for 18 h after 48 h of serum deprivation. Actinomycin D (5 µg/ml) was added to the medium, and mRNA was collected at 2, 4, 8, and 12 h and analyzed by Northern blot. B: quantitative analysis was performed with NIH Image analysis. Comparison of HO-1 mRNA levels in the cells at each time point was achieved by normalization to GAPDH mRNA. Data represent 6 independent experiments. We have at least 3 values for each time point, and the average of each time point was used to construct the graph. The data were plotted on a semilogarithmic scale (log-lin), and curve fitting to the best exponential curve was performed (Deltagraph). This resulted in correlation coefficients (r) values of –0.80 and –0.82 for pH 6.8 and pH 7.4, respectively.

 

   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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EA induces HO-1 mRNA production in RASMCs and RPASMCs. To determine the potential effect of EA on HO-1 expression, we exposed primary RASMCs to media of various pH values from 7.4 to 6.8 (7.4, 7.2, 7.0 and 6.8) for 18 h. RNA was then isolated, and a Northern blot was performed with a radiolabeled rat HO-1 cDNA as a probe. As shown in Fig. 1C, we found that an extracellular pH of 7.0 is sufficient to induce HO-1 mRNA sixfold above levels at pH 7.4. This induction is also seen at pH 6.8, whereas a milder degree of EA (pH 7.2) does not affect HO-1 mRNA levels in vitro. A similar response was seen in RPASMCs (Fig. 1D). Levels of GAPDH mRNA did not change with acidosis and were used as loading control. A time course of HO-1 mRNA induction by acidosis is shown in Fig. 1, A and B. Figure 1B shows the normalized ratios of HO-1 mRNA at the corresponding time points during incubation in pH 6.8 vs. 7.4. An average fourfold induction is seen in HO-1 mRNA levels after 15 h of exposure to pH 6.8 compared with exposure in medium of pH 7.4 for the same time period. HO-1 mRNA levels increased further by 18 and 24 h of exposure to acidosis and began to decline by 48 h of continued exposure. Of note, cell morphology appeared unchanged after 48 h of exposure to acidosis. To determine whether the decline in HO-1 mRNA could be explained by decreased cell viability after 48 h of exposure to acidosis, cell number was determined by Trypan blue exclusion and was found to be unchanged between conditions of EA and pH 7.4 after 24 and 48 h of continued exposure (data not shown).



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  		Fig. 1. Time course of heme oxygenase (HO)-1 mRNA induction by exposure of rat aortic smooth muscle cells (RASMCs) to acidic medium (pH 6.8) compared with exposure to medium of pH 7.4. A: representative Northern blot depicting HO-1 mRNA levels compared with levels of GAPDH mRNA. B: quantitative analysis of HO-1 mRNA levels after exposure of RASMCs to acidic medium for different time points compared with exposure to medium of pH 7.4 for the corresponding time points (normalized ratios). Data are expressed as means and SE. *P < 0.05, statistically significant difference compared with 1 h. Eight independent experiments are represented, and quantitative analysis was done with NIH Image software and normalized to GAPDH mRNA levels. C and D: Northern blot analysis of HO-1 and GAPDH mRNA levels after exposure of RASMCs (C) or rat pulmonary artery smooth muscle cells (RPASMCs; D) to media of various pH values (6.8, 7.0, 7.2, 7.4) for 18 h. A representative of 3 independent experiments for each cell type and the relative HO-1 mRNA levels compared with GAPDH mRNA levels are shown.

 
Reversibility of HO-1 mRNA induction by EA. We then examined whether the effect of EA on HO-1 mRNA is reversible on return to medium of pH 7.4. We subjected RASMCs to EA (pH 6.8) followed by a period of "recovery" in medium of pH 7.4 and assessed steady-state HO-1 mRNA levels by Northern blot analysis. We found that 8 h of recovery in medium of pH 7.4 was required for HO-1 mRNA levels to return to baseline levels, and this effect persisted at 48 h of recovery (Fig. 2).



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  		Fig. 2. Study of the reversibility of the induction of HO-1 mRNA by acidic pH. A: representative Northern blot depicting relative HO-1 mRNA levels compared with levels of GAPDH mRNA. B: quantitative analysis of HO-1 mRNA levels after exposure of RASMCs to acidic medium and "recovery" in medium of pH 7.4. Cells were incubated in medium of pH 7.4 or acidic medium (pH 6.8) for 18 h. The cells incubated in acidic medium were then returned to medium of pH 7.4, and their content of HO-1 and GAPDH mRNA was measured at 8, 24, and 48 h after the return to medium of pH 7.4 (R8 h, R24 h, and R48 h). Data are expressed as means and SE. *P < 0.05, statistically significant difference compared with 18 h of exposure to medium of pH 7.4. Seven independent experiments are represented, and quantitative analysis was done with NIH Image software and normalized to GAPDH mRNA levels.

 
Induction of HO-1 protein production in RASMCs by EA. To determine whether HO-1 protein levels changed in parallel with levels of HO-1 mRNA, we performed a Western blot analysis on whole cell lysates from RASMCs exposed to EA or medium of pH 7.4. As shown in Fig. 3, HO-1 protein was also induced by exposure to EA in a time-dependent manner. The pattern of induction is similar to that of HO-1 mRNA, with a peak at 18 h and a subsequent decline by 48 h of continued exposure to acidosis. No changes in HO-1 protein levels were seen after exposure to medium of pH 7.4 for 6–48 h.



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  		Fig. 3. Time course of HO-1 protein induction by exposure of RASMCs to acidic medium (pH 6.8). A: representative Western blot of 3 independent experiments. B: quantitative analysis of HO-1 protein levels after exposure of RASMCs to acidic medium for different time points compared with exposure to medium of pH 7.4 for 18 h (N). Data are expressed as means and SE of triplicate experiments. *P < 0.05, statistically significant difference compared with N.

 
Transcriptional regulation of HO-1 expression by acidic pH. To determine whether EA increases HO-1 mRNA in RASMCs via a transcriptional mechanism, we performed three independent nuclear run-on assays. Consistent with Northern blot analysis data, newly transcribed HO-1 mRNA (but not that of GAPDH) in RASMCs increased threefold in cells incubated in acidic medium (pH 6.8) for 18 h compared with cells incubated in medium of pH 7.4 (Fig. 4).



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  		Fig. 4. Transcriptional analysis of HO-1 and GAPDH genes by nuclear run-on assay. One microgram of denatured cDNA for HO-1 (top) or GAPDH (bottom) bound to nitrocellulose membrane was hybridized with {alpha}-32P-labeled run-on transcripts from nuclei isolated from RASMCs cultured under physiological (pH 7.4) or acidic (pH 6.8) conditions for 18 h. A representative of 3 independent experiments is shown.

 
Regulation of HO-1 mRNA stability by acidic pH. To evaluate the effect of acidic pH on HO-1 mRNA stability, we estimated the half-life of HO-1 mRNA in RASMCs incubated in medium of pH 7.4 and in acidic medium (pH 6.8) for 18 h. After exposure to the experimental conditions, cells were treated with actinomycin D to inhibit gene transcription. Total RNA was collected at hourly intervals from 0 to 9 h and at 12 h after addition of actinomycin D, and Northern blot analysis was performed. As shown in Fig. 5, in medium of pH 7.4 HO-1 mRNA decreased gradually, with a half-life of ~3.5 h. In contrast, in acidic conditions (pH 6.8) the rate of decline of HO-1 mRNA was slower, with a half-life of ~6.5 h, indicating that EA increases HO-1 mRNA stability. These data combined with the nuclear run-on studies indicate that EA increases HO-1 mRNA in RASMCs by both transcriptional activation and mRNA stabilization.

Nitric oxide availability does not mediate induction of HO-1 by EA. Because nitric oxide is known to be an inducer of HO-1, we examined whether nitric oxide mediates the induction of HO-1 by acidosis. Treatment of RASMCs with the competitive inhibitor of nitric oxide synthases, L-NNA, did not abrogate the induction of HO-1 mRNA in the setting of EA and did not affect levels of HO-1 mRNA at pH 7.4 (Fig. 6).



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  		Fig. 6. Effect of N{omega}-nitro-L-arginine (L-NNA) on the induction of HO-1 mRNA by extracellular acidosis. A: Northern blot analysis of HO-1 and GAPDH mRNA from RASMCs. RASMCs were cultured in media of 2 different pH values (6.8 and 7.4) for 18 h after 48 h of serum deprivation. One hour before and during the experimental conditions the cells were treated with L-NNA (2.5 mM) or diluent alone. B: quantitative analysis was performed with NIH Image analysis. Comparison of HO-1 mRNA levels in the cells at each condition was achieved by normalization to GAPDH mRNA. Data represent 3 independent experiments. *P < 0.05, statistically significant difference compared with pH 7.4; **P < 0.05, statistically significant difference compared with pH 7.4 + L-NNA.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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We report here that acidic extracellular pH upregulates the expression of HO-1 mRNA and protein in RASMCs. Increased HO-1 mRNA was also detected in RPASMCs exposed to media of acidic pH. Acidic conditions (pH 6.8) led to increased transcription of the HO-1 gene and increased stability of HO-1 mRNA in RASMCs. Thus both transcriptional and posttranscriptional mechanisms were responsible for the increase in HO-1.

To our knowledge, this is the first report of gene regulation by EA in VSMCs. Further studies need to address the acid-sensing and signaling pathways involved as well as the functional implications of this regulation. We found that inhibition of nitric oxide synthesis by L-NNA did not abrogate the induction of HO-1 mRNA by acidosis, suggesting that nitric oxide is not an intermediary molecule of the acidosis response in RASMCs. Acid-sensing ion channels have been identified and are known to mediate the pH responses in a variety of neuronal cells (16, 39). Changes in ion channel physiology and alterations in intracellular calcium concentrations have been described in VSMCs placed in acidic or hypoxic environments (3, 28), but the extent to which they may contribute to regulation of gene expression by acidosis has not been defined. In cardiac myocytes, p38 MAPK-mediated signaling was reported to contribute to hypoxic injury via intracellular acidosis (42). p38 MAPK signaling was also shown to be involved in the regulation of cardiac contractility by acidosis (41). Whether this pathway is involved in VSMC responses to acidosis will need to be examined.

The molecular mechanisms underlying acid regulation of gene expression in other cell types involve both transcriptional activation and mRNA stabilization. Several transcription factors including activating transcription factor-2, NF-{kappa}B, and activator protein-1 are known to mediate the transcriptional regulation of renal phosphoenolpyruvate carboxykinase, macrophage inducible nitric oxide synthase and tumor IL-8 by EA (4, 15, 38). In addition, EA leads to mRNA stabilization of the renal glutaminase (GA) gene, and this effect is mediated by an AU-rich region in the 3'-untranslated region of the mRNA. As shown by Tang and Curthoys (31), this region functions as a pH-response element (8) where {zeta}-crystallin/NADPH:quinone reductase binds with increased affinity during acidosis and confers increased stability of the mRNA.

HO-1 expression is highly regulated by a variety of cellular stressors in VSMCs, but EA has not been previously recognized as an inducing factor. Most known inducing agents of HO-1, including hypoxia, have a transcriptional effect. The cis-acting regulatory elements mediating the transcriptional activation of HO-1 are clustered in two regulatory regions, ~4 kb and ~10 kb upstream of the transcription initiation site of the HO-1 promoter (6). A hypoxia-inducible factor-binding site located at the 10 kb site upstream of the HO-1 promoter (18) was reported to mediate the hypoxic induction of HO-1 in vascular cells. The transcriptional mechanisms mediating the acid induction of HO-1 in RASMCs remain to be defined.

EA is not the only known perturbation in the extracellular environment that has a posttranscriptional effect on HO-1 expression. A posttranscriptional mechanism has also been reported to contribute to HO-1 induction by nitric oxide in RASMCs (13). This was confirmed in primary human fibroblasts and the HeLa human cervical cancer cell line by Bouton and Demple (5). HO-1 mRNA half-life was 2 h in control cells and increased in a dose-dependent manner on induction of endogenous nitric oxide production (to 6–10 h). The molecular mechanisms contributing to HO-1 mRNA stabilization in response to nitric oxide have not been described. We found that HO-1 mRNA half-life increased from ~3.5 h to ~6.5 h on reduction of extracellular pH from 7.4 to 6.8. Further studies are needed to address the molecular basis of both the transcriptional effect of EA on the HO-1 gene and the acid-induced stabilization of HO-1 mRNA.

The relevance of the acidic conditions used in our experiments is supported by in vivo studies. Even though arterial pH values of 7.0 or lower are rarely seen with systemic acidemia, local tissue acidosis of much greater magnitude is known to occur in systemically ill patients (10), in areas of inflammation or ischemia, and within solid tumors. The local tissue pH in human and rodent solid tumors is on average 0.5 units more acidic than normal tissue pH because of a local decrease in oxygen tension and nutrients due to inadequate vasculature (14, 36). Tissue pH as low as 5.8 has been measured in solid tumors (36). In addition, Woo et al. (37) reported tissue pH in the rat between 7.14 and 7.16 under normal conditions followed by a drop to 6.54–6.90 after incision.

In summary, we report that in RASMCs EA induces the expression of HO-1, a gene that catalyzes the production of CO, biliverdin, and iron. This induction is time dependent and reversible and involves transcriptional and posttranscriptional mechanisms. The functional significance of our observation remains to be examined in suitable animal models. Future studies are likely to further define the importance of EA as a contributor to gene regulation in vascular cells and to identify the molecular mechanisms involved.


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H. Christou is supported by National Heart, Lung, and Blood Institute (NHLBI) Grant KO8-HL-03917 and a Charles H. Hood Foundation Child Health Research Grant. S. A. Mitsialis is supported by NHLBI Grant Specialized Center of Research (SCOR) P50-HL-67669, and S. Kourembanas is supported by NHLBI Grants RO1-HL-55454 and SCOR P50-HL-l67669.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Christou, Div. of Newborn Medicine, Children's Hospital, 300 Longwood Ave., Enders 9, Boston, MA 02115 (E-mail: helen.christou{at}childrens.harvard.edu)

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. Section 1734 solely to indicate this fact.


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