Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 98: 732-738, 2005. First published October 8, 2004; doi:10.1152/japplphysiol.00821.2004
8750-7587/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
98/2/732    most recent
00821.2004v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Banks, M. F.
Right arrow Articles by Stenmark, K. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Banks, M. F.
Right arrow Articles by Stenmark, K. R.

HIGHLIGHTED TOPICS
Pulmonary Circulation and Hypoxia

Egr-1 antisense oligonucleotides inhibit hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts

Mark F. Banks, Evgenia V. Gerasimovskaya, Doug A. Tucker, Maria G. Frid, Todd C. Carpenter, and Kurt R. Stenmark

Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, Denver, Colorado

Submitted 2 August 2004 ; accepted in final form 30 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In most mammalian species, chronic exposure to hypoxia leads to pulmonary hypertension and vascular remodeling. The adventitial fibroblast, because of its ability to proliferate in response to hypoxia, is thought to be a critical cell in the remodeling process. However, the transcription factors driving hypoxia-induced fibroblast proliferation have yet to be elucidated. The early growth response-1 (Egr-1) transcription factor has been shown to be upregulated by hypoxia in pulmonary artery adventitial fibroblasts. We therefore hypothesized that Egr-1 is directly involved in hypoxia-induced adventitial fibroblast proliferation. Immunohistochemical analysis of in vivo lung tissue from animals exposed to chronic hypoxia revealed increased expression of Egr-1 in the pulmonary artery fibroblasts vs. expression shown in normoxic controls. In fibroblasts cultured from chronically hypoxic animals, exposure to 1% oxygen upregulated Egr-1 protein and cell proliferation. To evaluate the role of Egr-1 in hypoxia-induced proliferation, we employed an Egr-1 antisense strategy. Addition of antisense Egr-1 oligonucleotides, but not sense oligonucleotides, attenuated the hypoxia-induced upregulation of Egr-1 protein and reduced hypoxia-induced DNA synthesis by 50%. Cell proliferation was also significantly inhibited by the addition of antisense Egr-1 oligonucleotides but not the sense oligonucleotides. In addition, hypoxia-induced upregulations of cyclin D and epidermal growth factor receptor were attenuated by Egr-1 antisense oligonucleotides. We conclude that Egr-1 protein expression is very sensitive to upregulation by hypoxia in pulmonary artery adventitial fibroblasts and that it plays an important role in the autonomous growth phenotype induced by hypoxia in these cells.

early growth response-1; epidermal growth factor receptor; cyclin D; pulmonary hypertension; vascular remodeling

CHRONIC EXPOSURE TO HYPOXIA leads to pulmonary hypertension and vascular remodeling in most mammalian species (30). Extensive fibroproliferative changes have been noted within the adventitia of pulmonary arteries in response to chronic hypoxia (32). In fact, the adventitial compartment demonstrates the earliest and most dramatic changes in response to low oxygen tension (28). Adventitial fibroblasts are thought to be a critical cell type in vascular remodeling (5, 6, 29, 31). Animal studies have demonstrated early and sustained proliferation of fibroblasts in response to hypoxic conditions (2, 7). This response has been shown to exceed that seen in smooth muscle or the endothelium under identical partial pressures of oxygen (8). Although fibroblasts clearly play an important role in hypoxia-induced vascular remodeling, the transcription factors driving this hypoxia-induced proliferation have yet to be fully characterized.

The expression of numerous transcription factors is increased under hypoxic conditions. These factors include activator protein-1, hypoxia-inducible factor-1, high mobility group-1, CCAAT-enhancer binding protein, NF-{kappa}B, and early growth response-1 (Egr-1) (26). Previous studies have demonstrated that Egr-1 is a hypoxia-inducible transcription factor that upregulates the expression of numerous pathophysiologically relevant genes in the vasculature (16). Indeed, Egr-1 upregulates a host of downstream targets that have a role in the cellular response to stressors such as hypoxia (11, 16). Among vascular cell types, Egr-1 expression has been demonstrated in endothelial cells, smooth muscle cells, fibroblasts, and macrophages (11, 13). Stern and colleagues (38) eloquently demonstrated a critical role for Egr-1 in the maladaptive pulmonary vascular response to ischemia-reperfusion. These authors found a reduction in ICAM, VEGF, tissue factor, monocyte chemoattractant protein-1, and plasminogen-activator inhibitor-1 mRNA in the lungs of Egr-1 knockout mice exposed to ischemia-reperfusion compared with wild-type controls. Furthermore, the Egr-1 knockout animals had improved oxygenation, reduced lung inflammation, and improved survival compared with wild-type animals. An earlier study by the same authors (39) showed a unique contribution of Egr-1 to hypoxic pulmonary vascular pathophysiology. Egr-1 mRNA was expressed in murine lung at levels of hypoxia ranging from 6 to 10% O2. This mRNA expression exhibited a biphasic time course, with an Egr-1 mRNA peak observed as early as 30 min and then again after 24–48 h of hypoxia. More recently, Egr-1 was shown to be regulated by a protein kinase C{beta}-dependent pathway and by activation of the downstream MAPKs, ERK-1/2 and JNK (12). Although these studies illustrate the potentially significant role of Egr-1 in hypoxic pulmonary vascular remodeling, none ascribes a specific role for Egr-1 in the regulation of smooth muscle or fibroblast proliferation under hypoxic conditions.

Our laboratory (13) has previously shown that cultured pulmonary artery fibroblasts increase Egr-1 mRNA, Egr-1 protein expression, and Egr-1 DNA binding activity in response to hypoxia. Furthermore, our laboratory (5) also demonstrated that hypoxia induced an autonomous growth phenotype in these cells via the activation of a G-protein-coupled receptor and the downstream MAPK ERK-1/2. It remained for us to demonstrate whether the direct inhibition of Egr-1 would attenuate hypoxic proliferation of adventitial fibroblasts. As such, we investigated whether an oligonucleotide antisense strategy would inhibit Egr-1 protein production, inhibit fibroblast proliferation, and inhibit specific downstream regulators of cell division.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture.   Pulmonary artery adventitial fibroblasts were isolated from tissue explants of neonatal bovine calves exposed to 14 days of hypobaric hypoxia and cultured as previously described (5, 7). All animals were provided care under a protocol approved by the Institutional Animal Care and Use Committee, which operates under the Guiding Principles in the Care and Use of Animals of the American Physiological Society. Our laboratory (7) has previously demonstrated that fibroblasts from chronically hypoxic animals have exaggerated growth responses to hypoxia and may thus contribute to vascular remodeling in a distinct manner. Fibroblasts were used for experiments between passages 2 and 6. Cells were plated at a density of 5 x 105 cells/cm2, grown to 80% confluence, and then growth arrested in DMEM without serum for 3 days before hypoxic exposure. We have found that fibroblasts survive well in the absence of all serum and supplemental growth factors for 3 days. Under these conditions, basal activation of the signaling pathways of interest are at their lowest levels (not absent). This experimental system allows us to study the effects of hypoxia on cells in the absence of comitogens, which stimulate growth-related signaling pathways and thus confound the effects of hypoxia (7).

DNA synthesis.   DNA synthesis was determined by [methyl-3H]thymidine incorporation as described by Das et al. (6). Cells were incubated either with or without Egr-1 sense or antisense oligonucleotides (10 µM) (Integrated DNA Technologies, Coralville, IA) and labeled with 1 µCi of [methyl-3H]thymidine (NEN Life Science Products, Boston, MA). Cells were placed in a Bellco chamber for 24 h with either 1% O2 or 21% O2. At the end of this incubation, fibroblasts were washed and counted in a beta-scintillation counter (Beckman LS 6500).

Cell proliferation.   Cell proliferation was determined via a standard proliferation assay as described previously (6). Experimental cell cultures were incubated with or without sense or antisense oligonucleotides and placed in a Bellco chamber with either 1% or 21% O2 for a period of 48 h. After the incubations, cells were washed and counted.

Egr-1 analysis and oligonucleotide preparation.   Specified cell cultures were incubated with either sense or antisense Egr-1 oligonucleotides (10 µM) just before hypoxia or normoxia exposure. Egr-1 sense oligonucleotides were of the following sequence: 5'-AGTGTGCCCCTGCACCCCGC-3'. Egr-1 antisense oligonucleotides were arranged as follows: 5'-GCGGGGTGCAGGGGCACACT-3'. The oligonucleotides were obtained via an NCBI blast search for Mus musculus Egr-1 mRNA. Oligonucleotides were added to the cell cultures without modification or transfection reagent. The final concentration was determined via a dose-response experiment. Cells were placed in Bellco chambers under 1% or 21% O2 for times specified in the figure legends. At the termination of the experiments, cells were prepared for SDS-PAGE as previously described (11). Samples containing 20 µg of protein underwent electrophoresis, were transferred to a polyvinylidene difluoride membrane (Amersham, Piscataway, NJ) overnight, and then were probed with Egr-1, epidermal growth factor receptor (EGFR), or cyclin D primary antibodies under conditions recommended by the manufacturer (Santa Cruz Biotechnology, Santa Cruz, CA). Ponseau staining was used to confirm equal protein loading. Membranes were washed with Tris-buffered saline-Tween 20 and then incubated with peroxidase-conjugated secondary antibody under conditions specified by the manufacturer (Amersham). Immunoreactive bands were detected by chemiluminescence with the use of an enhanced luminol reagent kit (NEN Life Science Products), which was followed by exposure to hyperfilm (Amersham). We then quantitated protein bands using ImageJ densitometry.

Immunohistochemistry.   As per our previously described bovine model of chronic hypoxia, the pulmonary artery was harvested from neonatal calves at day 14 of life (27). Tissue specimens were frozen in OCT compound (Sakura Fine Technical, Torrance, CA), sectioned at 5 µm, and placed on glass slides for immunohistochemistry. Slides were stored at –70°C before they were stained. Slides were warmed to room temperature, fixed with 1% paraformaldehyde for 2 min, followed immediately by methanol (10 min at –20°C), and then air dried. The slides were rehydrated with PBS and then blocked with FBS-PBS (1:1) for 30 min. Egr-1 rabbit polyclonal primary antibody (Santa Cruz Biotechnology) was diluted 1:1,000 in 5% FBS in PBS and added to slides overnight in a humidified chamber at 4°C. On day 2, slides were washed with PBS before biotinylated secondary antibody (1:400, Vector Labs, Burlingame, CA) was added to the slides for 45 min at room temperature. Slides were again rinsed with PBS before the addition of streptavidin-conjugated Alexa-594 (1/2,000, Molecular Probes, Eugene, OR) for 45 min at room temperature. Slides were washed three times with PBS, rinsed with distilled H2O, dried, and embedded into Dappi-containing VectaShield mounting medium (Vector Laboratories). We obtained multichannel images with a Zeiss fluorescent microscope and the AxioVision digital imaging system.

Data analysis.   We quantified the density of bands using the NIH Image-J program. Data are expressed as means ± SE. Statistical significance was determined using Prism's one-way ANOVA followed by the Newman-Keuls test. All experiments were replicated three times (n = 3) unless otherwise specified.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Chronic hypoxia upregulates Egr-1 expression in the pulmonary artery adventitia.   We performed immunofluorescent staining on bovine lung tissue sections to investigate whether Egr-1 was upregulated within the pulmonary vasculature in response to hypoxia. Newborn calves were exposed to 14 days of hypoxia (12% oxygen) or normoxia (21% oxygen) before tissue collection. Consistent with our previous findings, we observed marked pulmonary artery adventitial thickening in animals exposed to hypoxia compared with animals maintained under normoxic conditions (Fig. 1). Egr-1 fluorescence was appreciated in endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts in lung tissue from hypoxic and normoxic animals. This observation is consistent with the ubiquitous expression of this transcription factor shown by other authors (11, 25). We observed a significant increase in Egr-1 protein expression within the distal pulmonary artery adventitia of hypoxic animals compared with normoxic controls. This increased fluorescence was particularly noteworthy in the fibroblasts surrounding sites of adventitial neovascularization (arrow).



View larger version (69K):
[in this window]
[in a new window]
  		Fig. 1. Chronic hypoxia exposure upregulates early growth response-1 (Egr-1) expression in pulmonary artery adventitial fibroblasts. Neonatal calves were exposed to either 21% or 12% oxygen for 14 days. Animals were killed, and pulmonary artery tissue was obtained and fixed as described in MATERIALS AND METHODS. Immunofluorescence for Egr-1 was performed on tissue from the distal pulmonary arteries of normoxic (A) and hypoxic (B) animals. Images are from a representative experimental and control animal. Egr-1 (red) fluorescence was increased in adventitial fibroblasts in hypoxic animals and was particularly noteworthy in fibroblasts surrounding adventitial neovascularization (arrow).

 
Hypoxia upregulates Egr-1 protein expression in cultured pulmonary artery fibroblasts.   Having demonstrated that Egr-1 protein is upregulated by hypoxia in pulmonary artery adventitial fibroblasts in vivo, we investigated whether hypoxia would induce increased Egr-1 protein expression in cultured pulmonary adventitial fibroblasts. Accordingly, we exposed cultured adventitial fibroblasts to 1% oxygen, 10% oxygen, or 21% oxygen for 6-h incubations. The respective partial pressures of oxygen in cell culture were ~6, 58, and 122 Torr, respectively. Protein expression was determined with Western blot analysis. We chose a 6-h incubation period based on earlier work with adventitial fibroblasts that showed maximal Egr-1 protein expression at between 4 and 6 h (13). As expected, Egr-1 protein expression was significantly increased in both 1% oxygen and 10% oxygen compared with 21% oxygen concentrations (Fig. 2). Thus exposure of cultured pulmonary artery adventitial fibroblasts to hypoxia results in a significant increase in the expression of Egr-1 protein.



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 2. Hypoxia upregulates Erg-1 protein expression in cultured pulmonary artery adventitial fibroblasts. Top: representative Western blot analysis. Growth-arrested adventitial fibroblasts were exposed to 1%, 10%, or 21% O2 for 6 h and prepared for analysis as described in MATERIALS AND METHODS. Equivalent amounts of cell protein were subjected to Western blot analysis with primary antibodies to Egr-1. Bottom: relative densitometry (NIH Image-J) comparing apparent protein productions from cells exposed to 21%, 10%, or 1% O2 for 6 h. Data represent means ± SE from 3 independent experiments. *P < 0.05: 21% O2 vs. 10% O2 and 21% O2 vs. 1% O2.

 
Egr-1 antisense oligonucleotides block Egr-1 protein production in adventitial fibroblasts exposed to hypoxia.   We sought to determine whether Egr-1 antisense oligonucleotides would inhibit hypoxia-induced upregulation of Egr-1 protein. We exposed adventitial fibroblasts to 1% or 21% oxygen for 48 h. Before hypoxic exposure, sense or antisense oligonucleotides (10 µM) were added to the media. As expected, 1% oxygen induced a significant increase in Egr-1 protein expression (Fig. 3). The addition of Egr-1 antisense oligonucleotides significantly inhibited the hypoxia-induced upregulation of Egr-1 protein. In fact, the level of Egr-1 protein expression after the addition of antisense oligonucleotides was similar to that observed in normoxic control cells. Egr-1 sense oligonucleotides did not affect hypoxia-induced upregulation of Egr-1 protein expression. There was no effect of the sense or antisense oligonucleotides on the baseline protein expression under normoxic conditions (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3. Erg-1 antisense oligonucleotides (oligos) block Erg-1 protein production in adventitial fibroblasts exposed to hypoxia. Top: representative Western blot analysis. Growth-arrested adventitial fibroblasts were exposed to 48 h of 21% or 1% O2 ± Egr-1 sense or antisense oligonucleotides. Bottom: relative densitometry comparing apparent protein productions. Data represent means ± SE from 3 independent experiments. *P < 0.05: 21% O2 vs. 1% O2 and 21% O2 vs. 1% O2 + Egr-1 sense oligonucleotides.

 
Egr-1 antisense oligonucleotides attenuate hypoxia-induced DNA synthesis and cell proliferation in pulmonary artery adventitial fibroblasts.   We next investigated the effect of Egr-1 antisense oligonucleotides on hypoxia-induced DNA synthesis. We added either Egr-1 sense or Egr-1 antisense oligonucleotides to pulmonary artery adventitial fibroblasts exposed to 1% or 21% oxygen for 24 h as per our previous [3H]thymidine protocol (7). The sense and antisense oligonucleotides (10 µM) had no effect on basal DNA synthesis at 21% oxygen in any experiment (data not shown). We found that 1% oxygen caused a significant increase in DNA synthesis in cultured fibroblasts compared with normoxic controls (Fig. 4). The addition of antisense Egr-1 oligonucleotides, but not sense oligonucleotides, significantly reduced hypoxia-induced DNA synthesis.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4. Erg-1 antisense oligonucleotides attenuate hypoxia-induced DNA synthesis in adventitial fibroblasts. Growth-arrested adventitial fibroblasts were exposed to 21% or 1% O2 in the presence of 1 µCi [3H]thymidine/well for 24 h. Specified cell cultures were preincubated with Egr-1 sense or antisense oligonucleotides. Incorporated radioactivity was determined in cell lysates as described in MATERIALS AND METHODS. Results are expressed as counts per minute (cpm)/10,000 cells. Data represent means ± SE from 3 independent experiments. *P < 0.05: 21% O2 vs. 1% O2, 21% O2 vs. 1% O2 + Egr-1 sense oligonucleotides.

 
We also tested whether the reduction in hypoxia-induced DNA synthesis caused by the addition of antisense oligonucleotides also reduced the hypoxia-induced increases in cell number. We found that cell number was significantly increased after 48-h incubations in 1% oxygen compared with 21% oxygen (Fig. 5). The addition of Egr-1 antisense oligonucleotides, but not sense oligonucleotides, significantly inhibited the increase in cell replication induced by exposure to 1% oxygen. These experiments suggest that Egr-1 plays a direct role in the autonomous growth phenotype exhibited by adventitial fibroblasts exposed to hypoxia in cell culture.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 5. Erg-1 antisense oligonucleotides attenuate hypoxia-induced cell proliferation in adventitial fibroblasts. Results are from cell proliferation studies. Growth-arrested adventitial fibroblasts were exposed to either 21% or 1% O2 for 48 h. Specified cells were preincubated with Egr-1 sense or antisense oligonucleotides. Cell proliferation was determined as described in MATERIALS AND METHODS. Data represent means ± SE from 6 independent experiments. *P < 0.05: 21% O2 vs. 1% O2, 21% O2 vs. 1% O2 + Egr-1 sense oligonucleotides.

 
Egr-1 antisense oligonucleotides block hypoxia-induced cyclin D and EGFR protein production.   Although it was clear that Egr-1 was upregulated in response to hypoxia and played a key role in the hypoxia-induced proliferation of adventitial fibroblasts, it was not clear how this response was orchestrated. Thus we looked at two key downstream regulators of cell proliferation: cyclin D and EGFR. The expression of these two proteins is thought to be controlled, at least in part, by Egr-1 (14, 18). Adventitial fibroblasts were incubated in either 1% or 21% oxygen for 48 h as in previous studies. As predicted, significant increases in the expression of these two downstream regulators of cell replication were appreciated in response to hypoxia (Figs. 6 and 7). The addition of Egr-1 antisense oligonucleotides caused a significant decrease in the expression of these proteins compared with that seen with hypoxia alone. The addition of sense oligonucleotides did not alter the expression of either protein compared with hypoxia alone. Thus, in our cells under our conditions tested, Egr-1 appears to induce hypoxic fibroblast proliferation, at least in part, via the downstream proteins EGFR and cyclin D.



View larger version (40K):
[in this window]
[in a new window]
  		Fig. 6. Erg-1 antisense oligonucleotides block hypoxia-induced epidermal growth factor receptor (EGFR) protein production. Top: representative bands from a Western blot analysis. Growth-arrested adventitial fibroblasts were exposed to 48 h of 21% or 1% O2 ± Egr-1 sense or antisense oligonucleotides. Bottom: relative densitometry comparing apparent protein productions. Data represent means ± SE from 3 independent experiments. *P < 0.05: 21% O2 vs. 1% O2, 21% O2 vs. 1% O2 + Egr-1 antisense oligonucleotides and 21% O2 vs. 1% O2 + Egr-1 sense oligonucleotides. **P < 0.05: 1% O2 vs. 1% O2 + Egr-1 antisense oligonucleotides.

 


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 7. Erg-1 antisense oligonucleotides block hypoxia-induced cyclin D protein production. Top: representative bands from a Western blot analysis. Growth-arrested adventitial fibroblasts were exposed to 48 h of 21% or 1% O2 ± Egr-1 sense or antisense oligonucleotides. Bottom: relative densitometry comparing apparent protein productions. Data represent means ± SE from 3 independent experiments. *P < 0.05: 21% O2 vs. 1% O2, 21% O2 vs. 1% O2 + Egr-1 antisense oligonucleotides and 21% O2 vs. 1% O2 + Egr-1 sense oligonucleotides. **P < 0.05: 1% O2 vs. 1% O2 + Egr-1 antisense oligonucleotides.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Previous studies in our laboratory (5, 13) have shown that hypoxia induces significant increases in Egr-1 protein and mRNA and DNA binding in cultured pulmonary artery adventitial fibroblasts. However, whether Egr-1 participates directly in the hypoxia-induced proliferative response remained unclear. Furthermore, little was known of the downstream targets of Egr-1 that contribute to the hypoxia-induced proliferative response. We found an increase in Egr-1 expression in the pulmonary artery adventitia of neonatal calves exposed to hypoxia for 2 wk. We confirmed that Egr-1 is significantly increased in cultured pulmonary artery adventitial fibroblasts in response to hypoxia (13). Utilizing an antisense strategy, we found that the direct inhibition of this transcription factor blocked hypoxia-induced adventitial cell proliferation significantly and reproducibly. Furthermore, we demonstrated that Egr-1 contributes to the proliferative phenotype demonstrated by the adventitial fibroblast in response to hypoxia, at least in part, by regulating expression of cyclin D and EGFR.

Several studies have demonstrated that Egr-1 is upregulated by hypoxia in a variety of cell types. Yan et al. (39) showed that exposure of cultured monocytes to hypoxia significantly increased Egr-1 mRNA expression. These investigators also determined that hypoxia-induced Egr-1 protein expression in smooth muscle cells and alveolar macrophages varied depending on the duration and degree of hypoxia (40). Increased expression of Egr-1 by a variety of lung cells has also been described in ischemia-reperfusion models (12, 38). In an in vitro model of adrenergic control during hypoxic stress, Wong et al. (37) found that Egr-1 controlled transcription of the pivotal enzyme, phenylethanolamine N-methyltransferase, responsible for epinephrine synthesis. Our present findings are in complete agreement with these previous studies. Thus it is clear that hypoxia consistently increases Egr-1 protein expression in many cell types, including pulmonary artery adventitial fibroblasts.

Egr-1 has been demonstrated to play an integral part in the proliferative response in numerous cell types. Santiago et al. (23) showed that they could inhibit serum-induced vascular smooth muscle growth by using anti-Egr-1 DNA enzymes. Furthermore, in rabbits, they showed that anti-Egr-1 DNA enzymes inhibited smooth muscle cell proliferation and neointimal formation after balloon-catheter injury (21). Similar observations have also been made in rodent and porcine models of catheter-induced vascular injury (9, 10, 15, 17, 24). In a hypercholesterolemic rabbit model, Ohtani et al. (19) found that Egr-1 antisense oligonucleotides inhibited neointimal formation after balloon injury. Similar results have also been reported in a murine model (34). In a primate heart transplant model, Wada et al. (35) found increased Egr-1 in smooth muscle cells of rejected hearts even before morphological changes such as intimal thickening were observed. In a mouse heart transplant model, Okada et al. (21) showed a dramatic reduction in coronary allograft vasculopathy and attenuated parenchymal rejection in homozygous Egr-1-null mice compared with wild-type animals. In a murine lung transplant model, these investigators (20) found that Egr-1 antisense oligonucleotides attenuated the pulmonary vascular injury induced by transplant and improved recipient survival by 56%. Thus our findings demonstrating that Egr-1 antisense oligonucleotides can inhibit hypoxia-induced fibroblast proliferation are consistent with the idea that Egr-1 plays a critical role in vascular cells in mediating the proliferative response to a variety of stressors.

The downstream modifiers that control the proliferative response orchestrated by Egr-1 remain incompletely understood. Wang et al. (36) found that that, in cell culture, the basic fibroblast growth factor gene is transcriptionally autoregulated by Egr-1. EGFR initiates the cellular response to epidermal growth factor, transforming growth factor-{alpha}, and other ligands (3, 33). Using a human osteosarcoma cell line, Nishi et al. (18) found that the upregulation of EGFR observed in response to hypoxia could be inhibited with the addition of Egr-1 antisense oligonucleotides. Our results in adventitial fibroblasts demonstrate that hypoxia-induced increases in EGFR can be attenuated by Egr-1 antisense oligonucleotides and are in complete agreement with the earlier studies. Together, these results suggest EGFR upregulation may play an important role in hypoxia-induced fibroblast proliferation.

Cyclin D is another well-described chaperone of cell proliferation (14). Recently, Baron et al. (1) showed that they could inhibit the tumor-associated increase in cyclin D and delay the occurrence of prostate tumors by injecting whole animals with Egr-1 antisense oligonucleotides. Our study also supports a role for Egr-1 in regulating cyclin D expression in hypoxic adventitial fibroblasts. Collectively, although our study suggests a role for EGFR and cyclin D in fibroblasts, other factors potentially downstream of Egr-1 need to be further defined. Indeed, in our study, the inhibition of EGFR and cyclin D with antisense Egr-1 was not as profound as what we achieved for Egr-1 itself. This suggests that Egr-1 is not the only transcription factor affecting the induction of these proteins under hypoxic conditions.

Egr-1 is a transcription factor that contributes to the orchestration of cellular responses to numerous insults, including hypoxia (22). To our knowledge, our report is the first to demonstrate that Egr-1 is directly involved in the hypoxia-induced proliferative responses of pulmonary artery adventitial fibroblasts. Moreover, Egr-1 remains a promising target for therapeutic intervention. Although it is clearly upregulated in response to a variety of stresses, it is not an essential transcription factor under normal conditions (27). Egr-1-null mice are phenotypically normal, save for a deficiency in luteinizing hormone (4). Inhibition of Egr-1 in a variety of animal models of heart and lung transplantation, tumor growth, and glomerulonephritis leads to an improved outcome (30, 31, 35, 40). Given the clear role that this master-switch transcription factor has in the pathological adaptations to stress, effective Egr-1 inhibition may significantly attenuate the pulmonary vascular remodeling induced by chronic hypoxic exposure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-57144 and Program Project Grant HL-14985.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Ivan McMurtry for critical reading of this manuscript. We are grateful to Jan Wenzlau and Stephen Hofmeister for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Stenmark, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Box B131, Denver, CO 80262 (E-mail: Kurt.Stenmark{at}UCHSC.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Baron V, Duss S, Rhim J, and Mercola D. Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice. Ann NY Acad Sci 1002: 197–216, 2003.[Abstract/Free Full Text]
  2. Belknap JK, Orton EC, Ensley B, Tucker A, and Stenmark KR. Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries. Am J Respir Cell Mol Biol 16: 366–371, 1997.[Abstract]
  3. Carpenter G and Cohen S. Epidermal growth factor. J Biol Chem 265: 7709–7712, 1990.[Free Full Text]
  4. Copland IB, Kavanagh BP, Engelberts D, McKerlie C, Belik J, and Post M. Early changes in lung gene expression due to high tidal volume. Am J Respir Crit Care Med 168: 1051–1059, 2003.[Abstract/Free Full Text]
  5. Das M, Bouchey DM, Moore MJ, Hopkins DC, Nemenoff RA, and Stenmark KR. Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 15631–15640, 2001.[Abstract/Free Full Text]
  6. Das M, Dempsey EC, Bouchey D, Reyland ME, and Stenmark KR. Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase C isozymes. Am J Respir Cell Mol Biol 22: 15–25, 2000.[Abstract/Free Full Text]
  7. Das M, Dempsey EC, Reeves JT, and Stenmark KR. Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia. Am J Physiol Lung Cell Mol Physiol 282: L976–L986, 2002.[Abstract/Free Full Text]
  8. Das M, Stenmark KR, and Dempsey EC. Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C. Am J Physiol Lung Cell Mol Physiol 269: L660–L667, 1995.[Abstract/Free Full Text]
  9. Fahmy RG and Khachigian LM. Antisense Egr-1 RNA driven by the CMV promoter is an inhibitor of vascular smooth muscle cell proliferation and regrowth after injury. J Cell Biochem 84: 575–582, 2002.[CrossRef][ISI][Medline]
  10. Fahmy RG and Khachigian LM. Locked nucleic acid modified DNA enzymes targeting early growth response-1 inhibit human vascular smooth muscle cell growth. Nucleic Acids Res 32: 2281–2285, 2004.[Abstract/Free Full Text]
  11. Fu M, Zhu X, Zhang J, Liang J, Lin Y, Zhao L, Ehrengruber MU, and Chen YE. Egr-1 target genes in human endothelial cells identified by microarray analysis. Gene 315: 33–41, 2003.[CrossRef][ISI][Medline]
  12. Fujita T, Asai T, Andrassy M, Stern DM, Pinsky DJ, Zou YS, Okada M, Naka Y, Schmidt AM, and Yan SF. PKC{beta} regulates ischemia/reperfusion injury in the lung. J Clin Invest 113: 1615–1623, 2004.[CrossRef][ISI][Medline]
  13. Gerasimovskaya EV, Ahmad S, White CW, Jones PL, Carpenter TC, and Stenmark KR. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J Biol Chem 277: 44638–44650, 2002.[Abstract/Free Full Text]
  14. Guillemot L, Levy A, Raymondjean M, and Rothhut B. Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells. J Biol Chem 276: 39394–39403, 2001.[Abstract/Free Full Text]
  15. Khachigian LM and Collins T. Inducible expression of Egr-1-dependent genes. A paradigm of transcriptional activation in vascular endothelium. Circ Res 81: 457–461, 1997.[Free Full Text]
  16. Khachigian LM, Lindner V, Williams AJ, and Collins T. Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science 271: 1427–1431, 1996.[Abstract]
  17. Lowe HC, Chesterman CN, and Khachigian LM. Catalytic antisense DNA molecules targeting Egr-1 inhibit neointima formation following permanent ligation of rat common carotid arteries. Thromb Haemost 87: 134–140, 2002.[ISI][Medline]
  18. Nishi H, Nishi KH, and Johnson AC. Early growth response-1 gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia. Cancer Res 62: 827–834, 2002.[Abstract/Free Full Text]
  19. Ohtani K, Egashira K, Usui M, Ishibashi M, Hiasa KI, Zhao Q, Aoki M, Kaneda Y, Morishita R, and Takeshita A. Inhibition of neointimal hyperplasia after balloon injury by cis-element "decoy" of early growth response gene-1 in hypercholesterolemic rabbits. Gene Ther 11: 126–132, 2004.[CrossRef][ISI][Medline]
  20. Okada M, Fujita T, Sakaguchi T, Olson KE, Collins T, Stern DM, Yan SF, and Pinsky DJ. Extinguishing Egr-1-dependent inflammatory and thrombotic cascades after lung transplantation. FASEB J 15: 2757–2759, 2001.[Free Full Text]
  21. Okada M, Wang CY, Hwang DW, Sakaguchi T, Olson KE, Yoshikawa Y, Minamoto K, Mazer SP, Yan SF, and Pinsky DJ. Transcriptional control of cardiac allograft vasculopathy by early growth response gene-1 (Egr-1). Circ Res 91: 135–142, 2002.[Abstract/Free Full Text]
  22. Pawlinski R, Pedersen B, Kehrle B, Aird WC, Frank RD, Guha M, and Mackman N. Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model. Blood 101: 3940–3947, 2003.[Abstract/Free Full Text]
  23. Santiago FS, Atkins DG, and Khachigian LM. Vascular smooth muscle cell proliferation and regrowth after mechanical injury in vitro are Egr-1/NGFI-A-dependent. Am J Pathol 155: 897–905, 1999.[Abstract/Free Full Text]
  24. Santiago FS, Lowe HC, Kavurma MM, Chesterman CN, Baker A, Atkins DG, and Khachigian LM. New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med 5: 1264–1269, 1999. [Corrigenda. Nat Med 5: December 1999, p. 1438.][CrossRef][ISI][Medline]
  25. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88: 1474–1480, 2000.[Abstract/Free Full Text]
  26. Semenza GL. Oxygen-regulated transcription factors and their role in pulmonary disease. Respir Res 1: 159–162, 2000.[CrossRef][Medline]
  27. Silverman ES and Collins T. Pathways of Egr-1-mediated gene transcription in vascular biology. Am J Pathol 154: 665–670, 1999.[Free Full Text]
  28. Stenmark KR, Bouchey D, Nemenoff R, Dempsey EC, and Das M. Hypoxia-induced pulmonary vascular remodeling: contribution of the adventitial fibroblasts. Physiol Res 49: 503–517, 2000.[ISI][Medline]
  29. Stenmark KR, Fasules J, Hyde DM, Voelkel NF, Henson J, Tucker A, Wilson H, and Reeves JT. Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4,300 m. J Appl Physiol 62: 821–830, 1987.[Abstract/Free Full Text]
  30. Stenmark KR, Frid M, Nemenoff R, Dempsey EC, and Das M. Hypoxia induces cell-specific changes in gene expression in vascular wall cells: implications for pulmonary hypertension. Adv Exp Med Biol 474: 231–258, 1999.[ISI][Medline]
  31. Stenmark KR, Gerasimovskaya E, Nemenoff RA, and Das M. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 122: 326S–334S, 2002.[Abstract/Free Full Text]
  32. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89–144, 1997.[CrossRef][ISI][Medline]
  33. Tikhomirov O and Carpenter G. Ligand-induced, p38-dependent apoptosis in cells expressing high levels of epidermal growth factor receptor and ErbB-2. J Biol Chem 279: 12988–12996, 2004.[Abstract/Free Full Text]
  34. Wada Y, Fujimori M, Suzuki J, Tsukioka K, Ito K, Sawa Y, Morishita R, Kaneda Y, Isobe M, and Amano J. Egr-1 in vascular smooth muscle cell proliferation in response to allo-antigen. J Surg Res 115: 294–302, 2003.[CrossRef][ISI][Medline]
  35. Wada Y, Suzuki J, Kawauchi M, Kurabayashi M, Tsukioka K, Zhang T, Endoh M, Takayama K, Nagai R, Takamoto S, Isobe M, and Amano J. Early growth-response factor 1 and basic transcriptional element-binding protein 2 expression in cardiac allografts. J Heart Lung Transplant 20: 590–594, 2001.[CrossRef][ISI][Medline]
  36. Wang D, Mayo MW, and Baldwin AS Jr. Basic fibroblast growth factor transcriptional autoregulation requires EGR-1. Oncogene 14: 2291–2299, 1997.[CrossRef][ISI][Medline]
  37. Wong DL, Tai TC, Wong-Faull DC, Claycomb R, and Kvetnansky R. Genetic mechanisms for adrenergic control during stress. Ann NY Acad Sci 1018: 387–397, 2004.[Abstract/Free Full Text]
  38. Yan SF, Fugita T, Jeishing L, Okada K, Zou YS, Mackman N, Pinsky DJ, and Stern DM. Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress. Nat Med 6: 1355–1361, 2000.[CrossRef][ISI][Medline]
  39. Yan SF, Lu J, Xu L, Zou YS, Tongers J, Kisiel W, Mackman N, Pinsky DJ, and Stern DM. Pulmonary expression of early growth response-1: biphasic time course and effect of oxygen concentration. J Appl Physiol 88: 2303–2309, 2000.[Abstract/Free Full Text]
  40. Yan SF, Lu J, Zou YS, Soh-Won J, Cohen DM, Buttrick PM, Cooper DR, Steinberg SF, Mackman N, Pinsky DJ, and Stern DM. Hypoxia-associated induction of early growth response-1 gene expression. J Biol Chem 274: 15030–15040, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. Nozik-Grayck, H. B. Suliman, S. Majka, J. Albietz, Z. Van Rheen, K. Roush, and K. R. Stenmark
Lung EC-SOD overexpression attenuates hypoxic induction of Egr-1 and chronic hypoxic pulmonary vascular remodeling
Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L422 - L430.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. Ngiam, M. Post, and B. P. Kavanagh
Early growth response factor-1 in acute lung injury
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1089 - L1091.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. L. Kramer, G. H. Deutsch, M. A. Sartor, W. D. Hardie, M. Ikegami, T. R. Korfhagen, and T. D. Le Cras
Perinatal increases in TGF-{alpha} disrupt the saccular phase of lung morphogenesis and cause remodeling: microarray analysis
Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L314 - L327.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
C.-J. Li, W. Ning, M. A. Matthay, C. A. Feghali-Bostwick, and A. M. K. Choi
MAPK pathway mediates EGR-1-HSP70-dependent cigarette smoke-induced chemokine production
Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1297 - L1303.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
H. Liao, M. C. Hyman, D. A. Lawrence, and D. J. Pinsky
Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1{alpha}, and C/EBP{alpha}
FASEB J, March 1, 2007; 21(3): 935 - 949.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
K. R. Stenmark, K. A. Fagan, and M. G. Frid
Hypoxia-Induced Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms
Circ. Res., September 29, 2006; 99(7): 675 - 691.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
P. R. Reynolds, M. G. Cosio, and J. R. Hoidal
Cigarette Smoke-Induced Egr-1 Upregulates Proinflammatory Cytokines in Pulmonary Epithelial Cells
Am. J. Respir. Cell Mol. Biol., September 1, 2006; 35(3): 314 - 319.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-J. Chen, H. Ning, W. Ishida, S. Sodin-Semrl, S. Takagawa, Y. Mori, and J. Varga
The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-beta and Mediates Stimulation of Collagen Gene Expression
J. Biol. Chem., July 28, 2006; 281(30): 21183 - 21197.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
T. Khomenko, S. Szabo, X. Deng, M. R. Jadus, H. Ishikawa, K. Osapay, Z. Sandor, and L. Chen
Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers
Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1211 - G1218.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
K. R. Stenmark, N. Davie, M. Frid, E. Gerasimovskaya, and M. Das
Role of the Adventitia in Pulmonary Vascular Remodeling
Physiology, April 1, 2006; 21(2): 134 - 145.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
L. M. Khachigian
Early Growth Response-1 in Cardiovascular Pathobiology
Circ. Res., February 3, 2006; 98(2): 186 - 191.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
E Sivridis, A Giatromanolaki, and M I Koukourakis
Proliferating fibroblasts at the invading tumour edge of colorectal adenocarcinomas are associated with endogenous markers of hypoxia, acidity, and oxidative stress
J. Clin. Pathol., October 1, 2005; 58(10): 1033 - 1038.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
G. C. Sieck
Commentary
J Appl Physiol, February 1, 2005; 98(2): 714 - 714.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
98/2/732    most recent
00821.2004v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Banks, M. F.
Right arrow Articles by Stenmark, K. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation