AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 278: H796-H805, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
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 (21)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saadane, N.
Right arrow Articles by Chalifour, L. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saadane, N.
Right arrow Articles by Chalifour, L. E.
Vol. 278, Issue 3, H796-H805, March 2000

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice

Nacéra Saadane1, Lesley Alpert2, and Lorraine E. Chalifour1,3

1 Lady Davis Institute for Medical Research and 2 Department of Pathology, Sir Mortimer B. Davis-Jewish General Hospital, Montreal H3T 1E2; and 3 Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 1A3


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar heart-to-body weight ratios but express lower amounts of atrial natriuretic factor (ANF), beta -myosin heavy chain (beta -MHC), skeletal actin, NGF1-A binding protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice. alpha -MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF, beta -MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1, GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+ mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump removal caused the agonist-treated hearts and gene expression to regress to control or near-control levels. Thus Egr-1 deficiency caused a blunted catecholamine-induced hypertrophy response and increased sensitivity to stress.

catecholamines; gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EARLY GROWTH RESPONSE-1 GENE (Egr-1; also termed NGF1-A, Zif/268, and Krox-24) was originally described as an immediate response gene (29). Depending on the cell and tissue source, Egr-1 has growth inhibitory (19, 25) or growth promoting activities (11), suggesting that the cellular context has a profound impact on the consequence of Egr-1 expression. Egr-1 encodes a Cys-2-His-2 zinc finger protein that shares strong sequence homology to a family of genes that includes NGFI-C (8), Krox-20 (7), Egr-3 (33), and Wilm's tumor proteins (reviewed in Ref. 12). The family members share homology in the zinc finger region but differ in their other domains and in their tissue distribution. Egr-1 expression is highest in brain and heart tissue, and Egr-1 overexpression induced differentiation of P19 cells to cardiac and neuronal cells in vitro (21). Egr-1-deficient mice are viable and display no life-threatening pathology (23, 47).

Egr-1 and the other family members bind to a GC-rich element, TGCGGGGGCG, and also to a TCCTCCTCCTCC element, present in >30 genes including angiotensin I-converting enzyme, tumor necrosis factor-alpha , alpha -myosin heavy chain (alpha -MHC), and Egr-1 itself (46; reviewed in Ref. 27). The GC-rich consensus sequence can overlap with the Sp1 consensus sequence GGGCGGG, and Egr-1 and Sp1 proteins compete for DNA binding (18, 26, 43). The cellular proteins NGF1-A binding protein (NAB)-1 and NAB-2 bind to the repressor domain 1 region of the Egr-1 protein (37, 45). NAB-1 is constitutively expressed, binds to Egr-1, and represses Egr-1-mediated transcription by inhibition of the general transcription machinery. NAB-2 expression is increased by some of the same stimuli that increase Egr-1 expression and binds to Egr-1; however, its mechanism of repression is unclear.

We identified Egr-1 as uniquely increased in expression in hypertrophied hearts of a transgenic mouse line that expressed the polyomavirus large T-antigen gene in cardiomyocytes (6, 16, 17). Subsequently, we showed that Egr-1 was overexpressed in hypertrophied hearts from mice treated with chronic infusion of alpha - and beta -adrenoreceptor agonists (39) or after damage caused by the cardiotoxic agent doxorubicin (38). Egr-1 expression is transiently increased in whole heart or neonatal cardiomyocytes after endothelin, adrenoreceptor, ANG II, doxorubicin, ischemia, or stretch treatment (5, 31, 38, 42, 48).

We hypothesized that a lack of Egr-1 expression might result in a blunted or absent response to hypertrophy-inducing agents in heart. In this paper we examine cardiac-specific genes, atrial natriuretic factor (ANF), alpha -MHC, beta -MHC, the cardiac-specific transcription factors GATA-4 and Nkx2.5, the immediate-early genes c-fos and c-jun, and the Egr-1 binding partners NAB-1, NAB-2, and Sp1 in male Egr-1 -/- and +/- mice as well as their wild-type (+/+) littermates. We describe our experiments on the expression of these genes in Egr-1 -/- mice and their +/+ littermates treated with adrenoreceptor agonists delivered chronically by osmotic minipumps. Furthermore, we describe the effect on gene expression of the removal of the pumps and hypertrophy regression. The results suggest that the lack of Egr-1 expression does not preclude a hypertrophy response to adrenoreceptor agonists. The data also suggest that the pattern of, and perhaps the pathway to, hypertrophy in Egr-1 -/- mice is not identical to that present in wild-type mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-Ascorbic acid, isoproterenol (Iso), and phenylephrine (PE) were purchased from Sigma. Mouse Egr-1 cDNA (GenBank/EMBL J04089) and mouse c-jun cDNA (GenBank/EMBL J04115) were purchased from American Type Culture Collection (ATCC). c-fos cDNA was a gift from Dr. John Hiscott (Lady Davis Institute, Jewish General Hospital, Montreal, Canada). Skeletal actin cDNA was a gift from Michael W. McBurney (University of Ottawa, Ottawa, Canada). ANF cDNA was a gift from Dr. Mona Nemer (Clinical Research Institute of Montreal, Montreal, Canada). Tubulin, Nkx2.5, GATA-4, Sp1, NAB-1, and NAB-2 cDNAs were generated by PCR, cloned into pBlueScript, and confirmed by sequencing.

Animals. We obtained two breeding pairs of mice as a generous gift from Dr. Jeffrey Milbrandt (Washington University School of Medicine, St. Louis, MO). Experiments were conducted in Egr-1 wild-type (+/+), heterozygous (+/-), and homozygous (-/-) male mice from progeny of these Egr-1-deficient mice. All experiments were performed according to the regulations of the Canadian Council of Animal Care and the Animal Care Committee of the Lady Davis Institute for Medical Research. Egr-1-deficient mice were genotyped with DNA obtained from tail docking using the Southern procedure (23).

Unmanipulated +/+, +/-, and -/- adult male mice were >6 wk of age at the time of death. Animals were killed by cervical dislocation. The mice were weighed, and their hearts were excised, rinsed in PBS, blotted dry with filter paper, and weighed. Heart weight-to-body weight ratios (HW/BW) were calculated and expressed as milligrams heart per gram per body weight.

Manipulated +/+ and -/- adult male mice harbored either vehicle- or agonist-loaded minipumps. Agonists, dissolved in vehicle (PBS containing 0.5 mM ascorbic acid), were administered by continuous subcutaneous infusion for 7 days via osmotic minipumps (model 2001, Alza, Palo Alto, CA). These pumps delivered a mix of Iso and PE (30 mg Iso + 29 mg PE per kg per day). Control animals received vehicle-loaded minipumps and were also treated for 7 days. Animals were anesthetized with Avertin (0.015 ml/g body wt, 2.5% solution in PBS, where 100% is 1 g of tribromoethanol dissolved in 1 ml of tert-amyl alcohol). Anesthesia was monitored by toe pinch. Minipumps were implanted dorsally through a 0.5-cm incision, and the wound was closed with Michel clips. Animals were housed in groups and given food and water ad libitum. To explore the regression of cardiac hypertrophy, vehicle- and Iso + PE-treated mice were reanesthetized and the minipumps removed 7 days after implantation. Animals were killed 7 days later and HW/BW determined as described above.

RNA preparation and analysis. RNA from ventricular tissues was extracted using TRIzol reagent (GIBCO-BRL) according to the manufacturer's instructions. The RNA pellets were resuspended in diethylpyrocarbonate-treated water, digested with RQ1 RNase-free DNase (Amersham Pharmacia Biotech), and incubated with proteinase K (Sigma). The enzymes were removed by phenol-chloroform extraction, and the DNA-free RNA was collected by ethanol precipitation. The RNA concentration was spectrophotometrically determined at 260 nm.

RT-PCR was performed using 3 µg of total RNA. The first-strand cDNA was synthesized using random primers and SuperScript II RNase H- reverse transcriptase (GIBCO-BRL). In control reactions, in which the reverse transcriptase was omitted, RNA samples did not generate amplified bands on PCR analyses, indicating the absence of contaminating DNA (data not shown). RT reactions proceeded according to instructions from the manufacturer and included RNase H digestion. Ten percent of the first-strand reaction was used for PCR using specific primers, 200 µM each of the four deoxynucleotide triphosphates (Amersham Pharmacia Biotech), and 1 unit of Taq DNA polymerase. PCRs were optimized with respect to magnesium concentration, and annealing temperature and all primer pairs showed linear kinetics when tested from 20 to 32 cycles of amplification (39). Positive control reactions contained genomic DNA as the substrate, and negative control reactions contained all reagents except cDNA. These parameters, the primer sequences, and the size of the amplified DNA fragments are given in Table 1. All PCRs were performed for 25 cycles, and then 20 µl of the PCR mix were electrophoresed through an agarose gel, stained with ethidium bromide, photographed, and transferred to Gene Screen Plus membranes (NEN) using the downward alkaline method (1). Membranes were prehybridized in 1% SDS-10% dextran sulfate-1 M NaCl at 65°C for 4 h, hybridized in the same buffer with radioactive gene-specific cDNA probes overnight, washed, and exposed to X-ray film. Densitometry of suitably exposed X-ray film was performed using an HP ScanJet 5100C and HP Precision Scan software (Hewlett-Packard). The areas under the peaks were quantified using ScionImage release beta 3 software (National Institutes of Health). Each series of RT-PCR was repeated at least three times on three separate occasions from RNA isolated from every animal.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   PCR conditions

Histological analysis. Hearts from one-half of the number of unmanipulated, vehicle-treated, or Iso + PE-treated Egr-1 +/+, +/-, and -/- mice were examined histologically. Horizontal slices of midventricle heart were fixed by immersion in neutral buffered Formalin, processed routinely, and embedded in paraffin. Sections were cut at 4 µm, stained with hematoxylin-eosin or Mason's trichome, and then examined in a blinded fashion using light microscopy.

Statistical analysis. All values are expressed as means ± SE. Comparisons among three or more groups were made by one-way ANOVA followed by Dunnett's modified t-test. A value of P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Unmanipulated Egr-1-deficient mice. We compared HW/BW of male Egr-1 +/+, +/-, and -/- mice. All these mice had undergone tail docking for genotype analyses, but no further surgery had been performed. There was no difference among HW/BW of -/-, +/-, and +/+ mice (Fig. 1). There was no difference in the body weights with genotype. Histological analysis of unmanipulated Egr-1 +/+, +/-, and -/- mice showed no differences among genotypes and no abnormalities (data not shown).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   Heart to body weight ratios (HW/BW) of early growth response-1 (Egr-1) +/+, +/-, and -/- mice. Adult male mice, >6 wk of age, of designated genotypes (n = 6 mice/genotype) were killed and their BW recorded. Hearts were removed, rinsed in PBS, blotted dry, and weighed. Values are means ± SE.

Gene expression was analyzed in untreated Egr-1 +/+, +/-, and -/- mice (n = 3 mice/genotype). We measured expression of ANF, alpha -MHC, beta -MHC, skeletal actin, NAB-1, NAB-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 (Fig. 2). We chose tubulin expression as a control of gene expression to minimize loading differences. We found that skeletal actin and ANF expression were significantly lower in Egr-1 -/- mice than in their +/+ and +/- littermates (P < 0.05 and P < 0.01, respectively). alpha -MHC expression was lower in -/- mice than in +/- mice (P < 0.05), but there was no difference compared with expression in +/+ mice. beta -MHC expression was decreased (P < 0.01) in both +/- (P < 0.01) and -/- (P < 0.05) Egr-1 mice compared with +/+ littermates. NAB-1 expression was similar in all genotypes; however, NAB-2 and Sp1 expression were significantly decreased in -/- (P < 0.05) compared with +/+ mice, and Sp1 expression was significantly different in +/- and -/- mice from that in +/+ mice. Expression of c-fos was significantly higher in +/- than in either -/- or +/+ mice (P < 0.05 or P < 0.01, respectively), whereas c-jun expression was similar in all genotypes. GATA-4 expression was similar in Egr-1 +/+ and +/- mice but was decreased in -/- mice (P < 0.05). Nkx2.5 expression was decreased in -/- mice compared with that in +/+ mice (P < 0.01). Overall, the data suggest a decrease in cardiac-specific and immediate-early gene expression in -/- mice that is gene specific. The data also suggest that the cardiac-specific gene expression in -/- mice is compromised.



View larger version (4132K):
[in this window]
[in a new window]
  		Fig. 2.   Gene expression in unmanipulated Egr-1 +/+, +/-, and -/- mice. RNA was extracted from ventricular heart and purified. Three micrograms of DNA-free RNA were included in RT reactions with random primers, and 10% of first-strand reaction was then amplified with gene-specific primers for 25 cycles. Equal amounts from each reaction were electrophoresed through an agarose gel, and DNA was transferred to membranes and probed with radioactive gene-specific cDNAs. A: expression of muscle gene skeletal (sk) actin, alpha -myosin heavy chain (alpha -MHC), beta -MHC, and atrial natriuretic factor (ANF). B: expression of Egr-1 partner genes NGF1-A binding protein (NAB)-1, NAB-2, and Sp1. C: expression of immediate-early genes c-fos and c-jun. D: expression of cardiac-specific transcription factor genes GATA-4 and Nkx2.5. Tubulin expression was used as a control in A-D. Top: representative Southern blots from RT-PCR of RNA from +/+, +/- and -/- mice in Fig. 1 with genes indicated at left of blot. Bottom: bar graphs of densitometry of Southern blots from 2 separate experiments. Expression of +/+ was artificially designated as 1. Values are means ± SD. * P < 0.05; black-diamond  P < 0.01. 

Adrenoreceptor stimulation of Egr-1 +/+ and -/- mice. We treated +/+ and -/- male mice with a combination of the beta -adrenoreceptor agonist Iso and the alpha -adrenoreceptor agonist PE or with vehicle (0.5% ascorbic acid in PBS) for 7 days. A previous study indicated that this dose and time is adequate to produce an increase in HW/BW in male C57Bl/6 mice (39). HW/BW of vehicle-treated -/- mice and +/+ littermates was similar after 7 days of continuous treatment (Fig. 3). In Iso + PE-treated +/+ and -/- littermates, HW/BW were significantly increased (P < 0.01) after 7 days of continuous treatment compared with ratios in vehicle-treated animals.



View larger version (14117K):
[in this window]
[in a new window]
  		Fig. 3.   HW/BW and histology of vehicle- or adrenoreceptor-stimulated +/+ and -/- mice. Top: minipumps loaded with either vehicle or isoproterenol plus phenylephrine (Iso + PE) were implanted into adult male mice of designated genotypes. Mice (minimum: n = 4 mice/treatment) were killed 7 days later. In a second group, mice were anesthetized, minipumps were removed, and mice were allowed to recover for 7 days before death. HW and BW were recorded as described in MATERIALS AND METHODS and Fig. 1 legend. Values are means ± SD. black-diamond  P < 0.01. Bottom: histology of adrenoreceptor damage. Representative midventricular hematoxylin and eosin-stained cross sections of hearts from vehicle-treated +/+ (A), vehicle-treated -/- (B), or Iso + PE-treated -/- mice (C). Arrows, regions of fibrosis in hearts; scale bar, 150 µm.

Histological analysis showed normal architecture and no evidence of fibrosis in the vehicle-treated +/+ hearts, whereas there was evidence of significant foci of fibrosis in five of six Iso + PE-treated +/+ hearts examined (Fig. 3). In contrast, heart sections from both vehicle- and Iso + PE-treated -/- mice showed multiple foci of fibrosis in three of four and in all five mice examined, respectively. The data suggest that a lack of Egr-1 expression does not preclude physiological hypertrophy with adrenoreceptor stimulation. The data also suggest that vehicle-treated -/- hearts were under sufficient stress to form fibrotic lesions.

Reversal of induced cardiac hypertrophy. To examine HW/BW, histology, and gene expression in +/+ and -/- mice during hypertrophy regression, we treated mice of each genotype for 7 days with either vehicle or Iso + PE. After the 7 days of drug treatment, we removed the minipumps and allowed the mice to recover for another 7 days (Fig. 3). Cardiac mass decreased to control levels within 7 days after drug withdrawal in all treated animals, regardless of genotype. HW/BW in Iso + PE-treated -/- mice and their +/+ littermates were similar to the ratios found in vehicle-treated -/- mice and their +/+ littermates. The data suggest that Egr-1 expression is not required for regression of hypertrophy.

Muscle gene expression: ANF, alpha -MHC, beta -MHC, and skeletal actin. We measured expression of the muscle genes in +/+ and -/- mice treated with either vehicle or Iso + PE for 7 days. We examined RNA samples prepared from ventricular heart for tubulin, ANF, alpha -MHC, beta -MHC, and skeletal actin (Fig. 4). Stimulation of adrenoreceptors in -/- mice and their +/+ littermates did not affect the expression of tubulin or alpha -MHC. Iso + PE induction of beta -MHC in +/+ mice was fourfold higher than the level found in vehicle-treated +/+ mice (P < 0.01). beta -MHC expression in Iso + PE-treated -/- mice was also higher than in vehicle-treated -/- mice. However, the magnitude of the response in Iso + PE-treated -/- mice was lower, and beta -MHC was only induced at a level 1.5-fold higher than the level found in vehicle-treated -/- mice. ANF induction was 3.4-fold higher in Iso + PE- than in vehicle-treated +/+ mice (P < 0.01) but did not increase significantly in Iso + PE- compared with vehicle-treated -/- mice. Thus the pattern of ANF and beta -MHC induction is dissimilar. In contrast, although we found that Iso + PE-treated +/+ mice had a 35-fold higher induction of skeletal actin gene expression than vehicle-treated +/+ mice (P < 0.01), there was no observable increase in skeletal actin induction in Iso + PE-treated -/- mice. These data suggest that the response to adrenoreceptor stimulation in -/- mice is diminished or absent and that this is a gene-specific phenomenon.


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4.   Muscle-specific gene expression in vehicle- or adrenoreceptor-stimulated +/+ and -/- mice. DNA-free RNA was extracted and RT-PCR performed as described in MATERIALS AND METHODS and Fig. 2 legend. Top: representative Southern blots from triplicate experiments with gene amplified indicated at left. Mice in hypertrophy group (A) were treated for 7 days; mice in regression group (B) were treated for 7 days, and then minipumps were removed and animals were allowed to recover for 7 days. Lane 1: vehicle-infused +/+ mouse. Lane 2: Iso + PE-infused +/+ mouse. Lane 3: vehicle-infused -/- mouse. Lane 4: Iso + PE-infused -/- mouse. Bottom: bar graphs of densitometry from Southern blots from 3 separate experiments. Levels of expression in agonist-treated samples were compared with levels found in vehicle-treated mice of each specific genotype, which were artificially designated as 1. Values are means ± SD. * P < 0.05; black-diamond  P < 0.01.

To study the gene expression found after regression of induced cardiac hypertrophy, +/+ and -/- littermates were continually infused with vehicle or Iso + PE for 7 days, and then the minipumps were removed and the animals allowed to recover for 7 days before death. In Iso + PE-treated +/+ mice, gene expression levels of tubulin, ANF, alpha -MHC, and beta -MHC were not significantly different from those found in vehicle-treated mice 7 days after minipumps were removed (Fig. 4). Expression of skeletal actin was not significantly elevated in Iso + PE-treated +/+ mice compared with that in vehicle-treated +/+ mice. Expression of tubulin, alpha -MHC, and skeletal actin was similar in Iso + PE-treated -/- mice allowed to recover from the agonist-treatment. However, the expression of ANF (P < 0.05) and beta -MHC (P < 0.05) remained higher than that in vehicle-treated -/- mice. Thus the increase in ANF and beta -MHC was absent or lower in -/- mice than in +/+ mice, but the higher levels were maintained longer after the Iso + PE stimulus was removed.

Egr-1 partner gene expression: NAB-1, NAB-2, and Sp1. Our next series of experiments was designed to measure the expression of NAB-1 and NAB-2, transcriptional repressors of Egr-1, and Sp1, a competitive inhibitor of Egr-1 DNA binding. Iso + PE infusion induced an increase in Sp1 expression in +/+ (P < 0.01) and -/- (P < 0.01) compared with expression in respective vehicle-treated control mice (Fig. 5). Sp1 induction was higher in Iso + PE-treated +/+ mice (3.5-fold increase, P < 0.01) than in Iso + PE-treated -/- mice (2.2-fold increase, P < 0.01). NAB-1 expression was moderately induced in Iso + PE-treated +/+ mice (2-fold increase, P < 0.05) but was not increased in Iso + PE-treated -/- mice. NAB-2 expression was induced in Iso + PE-treated +/+ mice (4-fold increase, P < 0.01) compared with a moderate induction in Iso + PE-treated -/- mice (1.5-fold increase, P < 0.05). In all cases, the response of the agonist-treated -/- mice was less than that of the agonist-treated +/+ mice.


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5.   Egr-1 competitive inhibitor Sp1 and binding partners NAB-1 and NAB-2 in vehicle- or adrenoceptor-stimulated +/+ and -/- mice. DNA-free RNA was extracted, RT-PCR was performed, and data were analyzed as described in MATERIALS AND METHODS and legends to Figs. 2 and 4. Top: representative Southern blots from triplicate experiments with gene amplified indicated at left. Mice in hypertrophy group (A) were treated for 7 days; mice in regression group (B) were treated for 7 days, and then pumps were removed and animals were allowed to recover for 7 days. Lane 1: vehicle-infused +/+ mouse. Lane 2: Iso + PE-infused +/+ mouse. Lane 3: vehicle-infused -/- mouse. Lane 4: Iso + PE-infused -/- mouse. Bottom: bar graphs of densitometry from Southern blots from 3 separate experiments. Levels of expression in agonist-treated samples were compared with levels found in vehicle-treated mice of each specific genotype, which were artificially designated as 1. Values are means ± SD. * P < 0.05; black-diamond  P < 0.01.

Expression of Sp1, NAB-1, and NAB-2 in hearts 7 days after minipump was removed was similar in Iso + PE-treated and control vehicle-treated +/+ mice (Fig. 5). Likewise, expression of Sp1, NAB-1, and NAB-2 was similar in the Iso + PE- and control vehicle-treated -/- mice.

Immediate-early gene expression: c-jun and c-fos. Fos and Jun form the transcription factor AP-1, and overexpression of c-jun and c-fos is a common feature of rodent hypertrophy. We previously showed that both genes are overexpressed in C57Bl/6 mice treated with Iso + PE (39). Egr-1 +/+ and -/- mice treated with Iso + PE for 7 days had increased expression of c-fos (20-fold, P < 0.01 and 25-fold, P < 0.01, respectively) (Fig. 6). Iso + PE-treated +/+ mice had increased expression of c-jun (4.5-fold, P < 0.01) over that present in vehicle-treated +/+ mice. However, although Iso + PE treatment of -/- mice led to an increase in c-jun expression (2-fold, P < 0.01) compared with vehicle-treated -/- mice, this induction was reduced compared with that found in +/+ mice. Thus, whereas c-fos expression was increased to equivalent levels in both +/+ and -/- mice, c-jun expression in -/- mice was blunted.


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 6.   Expression of immediate early genes c-fos and c-jun in vehicle- or adrenoreceptor-stimulated +/+ and -/- mice. DNA-free RNA was extracted, RT-PCR was performed, and data were analyzed as described in MATERIALS AND METHODS and legends to Figs. 2 and 4. Top: representative Southern blots from triplicate experiments with gene amplified indicated at left. Mice in hypertrophy group (A) were treated for 7 days; mice in regression group (B) were treated for 7 days, and then pumps were removed and animals were allowed to recover for 7 days. Lane 1: vehicle-infused +/+ mouse. Lane 2: Iso + PE-infused +/+ mouse. Lane 3: vehicle-infused -/- mouse. Lane 4: Iso + PE-infused -/- mouse. Bottom: bar graphs of densitometry from Southern blots from 3 separate experiments. Levels of expression in agonist-treated samples were compared with levels found in vehicle-treated mice of each specific genotype, which were artificially designated as 1. Values are means ± SD. * P < 0.05; black-diamond  P < 0.01.

Seven days after minipumps were removed, the expression of c-fos in Iso + PE-treated +/+ and -/- mice was reduced to that found in vehicle-treated mice (Fig. 6). The amount of c-jun in Iso + PE-treated +/+ mice was reduced 0.65-fold (P < 0.05) compared with the level in vehicle-treated +/+ mice. Expression in Iso + PE- and vehicle-treated -/- mice was similar.

Cardiac-specific transcription factor gene expression: GATA-4 and Nkx2.5. We found increased expression of GATA-4 and Nkx2.5 after adrenoreceptor stimulation of C57Bl/6 mice (39). Iso + PE treatment of +/+ mice induced increased expression of GATA-4 (7-fold, P < 0.01) and Nkx2.5 (25-fold, P < 0.01) compared with the level found in vehicle-treated +/+ control mice (Fig. 7). Surprisingly, there was no induction of expression of GATA-4 or Nkx2.5 in Iso + PE-treated -/- mice compared with vehicle-treated -/- mice (P = not significant). The expression of GATA and Nkx2.5 in vehicle- and Iso + PE-treated -/- mice was at levels comparable to those found in Iso + PE-treated +/+ mice. These data suggest that although GATA-4 and Nkx2.5 induction is not present in adrenoreceptor-stimulated -/- mice, the levels of GATA-4 and Nkx2.5 are increased in both vehicle- and Iso + PE-treated -/- mice.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 7.   Expression of cardiac-specific genes Nkx2.5 and GATA-4 in vehicle- or adrenoreceptor-stimulated +/+ and -/- mice. DNA-free RNA was extracted, RT-PCR was performed, and data were analyzed as described in MATERIALS AND METHODS and legends to Figs. 2 and 4. Top: representative Southern blots from triplicate experiments with gene amplified indicated at left. Mice in hypertrophy group (A) were treated for 7 days; mice in regression group (B) were treated for 7 days, and then pumps were removed and animals were allowed to recover for 7 days. Lane 1: vehicle-infused +/+ mouse. Lane 2: Iso + PE-infused +/+ mouse. Lane 3: vehicle-infused -/- mouse. Lane 4: Iso + PE-infused -/- mouse. Bottom: bar graphs of densitometry from Southern blots from 3 separate experiments. Levels of expression in agonist-treated samples were compared with levels found in vehicle-treated mice of each specific genotype, which were artificially designated as 1. Values are means ± SD. black-diamond  P < 0.01.

Expression of both GATA-4 and Nkx2.5 in Iso + PE-treated +/+ and -/- mice was reduced to that found in vehicle-treated +/+ and -/- control mice 7 days after minipumps were removed (Fig. 7). The expression of GATA and Nkx2.5 in +/+ and -/- mice was similar regardless of the treatment regimen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inactivation of Egr-1 through insertion of a neo cassette at the Nde I site 5' of the DNA binding domain did not affect the growth and differentiation of embryonic stem cells lacking Egr-1 (23). Egr-1-deficient mice lacked demonstrable Egr-1 mRNA and protein expression and had normal growth without any obvious defects in cellular differentiation (23, 24). However, Lee et al. (22) found that Egr-1 -/- male mice were fertile, whereas female -/- mice were infertile. Fertility in female mice could be restored with luteinizing hormone injections. Using a similar deletion strategy, Topilko et al. (47) also created Egr-1-deficient mice. They found that both male and female Egr-1 -/- mice were infertile and that the female infertility could not be improved by luteinizing hormone injections. Furthermore, they found a smaller-than-normal body size that they attributed to a reduction in growth hormone secretion. Regardless of the severity of the phenotype, evidently Egr-1 expression is not required for major organogenesis, including heart development, and for passage to adult life.

When we compared HW/BW of unmanipulated Egr-1 +/+, +/-, and -/- progeny derived from the mice of Lee et al. (23), we did not find a significant difference in HW/BW in the various genotypes. Although normal in size, the -/- hearts showed a decrease in basal expression of ANF, alpha -MHC, beta -MHC, skeletal actin, NAB-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 compared with these expression levels in +/+ and +/- hearts. All hearts had similar levels of tubulin and NAB-1 regardless of genotype. These results suggest that the low levels of expression in the first set of genes is gene specific and that the lack of Egr-1 does not cause a global decrease in gene expression in heart tissue. Except for c-fos, the gene changes indicate a similar or slightly decreased expression in the +/- mice, with a more pronounced decrease in the -/- mice. This suggests an involvement for Egr-1, either directly or indirectly, in c-fos regulation and suggests that this regulation might involve both stimulatory and inhibitory effects on c-fos transcription. In other studies we could not detect differences in tubulin or alpha -MHC expression in vehicle- versus adrenoreceptor-stimulated C57Bl/6 male mice (39) or in saline- versus doxorubicin-treated CD-1 female mice (38), suggesting that expression of tubulin and alpha -MHC is constant in these mouse hearts.

ANF expression is controlled by GATA-4 and Nkx2.5 through GATA and NKE sites, by Fos and Jun through an AP-1 site, and by Egr-1/Sp1 through the presence of an Sp1 site in the ANF promoter (10, 36, 41). beta -MHC and skeletal actin are thought to be regulated through Sp1, M-CAT, and CArG sites (9, 35), with the GATA motif required for induced, but not basal, levels of expression (14). However, a lack of Nkx2.5 expression, although lethal, did not prevent expression of most muscle-specific genes including beta -MHC in Nkx2.5- or GATA-4-deficient embryos (28, 30). Similarly, c-fos- or Egr-1-deficient mice develop relatively normal hearts (23, 50). It is likely that the low levels of ANF, beta -MHC, and skeletal actin expression in -/- mice here is a consequence of the combined low levels of c-fos, c-jun, GATA-4, and Nkx2.5 and the complete lack of Egr-1, whereas lack of a single factor is less effective.

Adrenoreceptor stimulation induces rodent cardiac hypertrophy through interaction with alpha - and beta -adrenoreceptors present on cardiomyocytes and the vasculature (20, 52). Associated with beta -adrenoreceptor agonist stimulation are increases in heart rate, arterial blood pressure, and force of contraction (reviewed in Ref. 32). An increase in heart rate and arterial blood pressure was also found in mice infused with adrenoreceptor agonists (39). Increased expression of immediate-early genes such as c-fos, c-jun, junB, and Egr-1 was found in hearts within hours of a single bolus injection of catecholamine (3, 4, 51; reviewed in Ref. 34). To more closely mimic the chronic high levels of catecholamine found in heart failure (32), we infused Iso, PE, or both into C57Bl/6 mice using minipumps. We previously showed a stable increase in HW/BW with increased expression of ANF, beta -MHC, Egr-1, c-fos, c-jun, GATA-4, and Nkx2.5 maintained in C57Bl/6 mice infused with adrenoreceptor agonists for <= 14 days (39). Similar results were shown in the present study in that high levels of ANF, beta -MHC, c-fos, c-jun, GATA-4, and Nkx2.5, with no change in tubulin or alpha -MHC expression, were present in Iso + PE-stimulated +/+ mice. The Egr-1-deficient mice have a 129/C57Bl/6 genetic background. Infusion of Iso into C3Heb/FeJ, SJL/J, and SWR/J mice led to cardiac hypertrophy in all lines (43). The data suggest that chronic infusion of catecholamines will reproducibly stimulate a cardiac hypertrophy with increased expression of muscle-specific and immediate-early genes in mice.

The availability of Egr-1-deficient mice offered an opportunity to determine whether expression of this gene is essential for catecholamine-induced hypertrophy initiation, maintenance, and/or regression. We found that Egr-1 deficiency did not preclude a similar increase in HW/BW in +/+ or -/- mice, suggesting that adaptive responses to catecholamine excess do not absolutely depend on Egr-1. The adaptive response, though, was not achieved with a wild-type pattern of muscle-specific expression. Although beta -MHC expression was increased, neither ANF nor skeletal actin expression was induced. alpha -MHC was not increased to compensate for the lower beta -MHC expression in stimulated -/- mice. Increased expression of ANF and beta -MHC is found in many experimental systems (reviewed in Ref. 7), and we were surprised to find ANF absent and little induction of beta -MHC in these agonist-stimulated -/- mice.

As mentioned above, expression of ANF, both MHCs, and skeletal actin is controlled by multiple transcription factors that include the AP-1 factors, Fos and Jun, Sp1, and also GATA-4 and Nkx2.5. Induced expression of beta -MHC was dependent on GATA sequences (14). Both c-fos and, to a lesser extent, c-jun are stimulated in the two genotypes, suggesting that a lack of Egr-1 does not prevent their stimulation and that a lack of Fos or Jun is unlikely to be responsible for lower beta -MHC or absent ANF and skeletal actin induction. However, the response of GATA-4 and Nkx2.5 to the experimental protocol is genotype dependent. No induction of GATA-4 or Nkx2.5 was found when levels in the vehicle- versus Iso + PE-treated -/- mice were compared. This is probably because Nkx2.5 and GATA-4 were increased in both vehicle- and Iso + PE-treated -/- mice to levels similar to those found in Iso + PE-stimulated +/+ mice. It is unlikely that the lower level of beta -MHC induction can be attributed to low levels of Nkx2.5 or GATA-4.

In untreated and unmanipulated mice, GATA-4 and Nkx2.5 were at lower levels in -/- mice compared with +/+ mice, yet these genes were overexpressed in vehicle-treated -/- mice. The difference in expression between unmanipulated -/- mice and vehicle-treated -/- mice suggests that the surgery required to implant the pumps and/or the stress of maintaining the pump weight, or another factor, was sufficient to stimulate GATA-4 and Nkx2.5 expression and that this stimulation was comparable to the stress of catecholamine excess. Similarly, although to a lesser extent, Sp1 expression was also lowest in the untreated and unmanipulated -/- mice yet was increased in vehicle-treated -/- hearts compared with vehicle-treated +/+ hearts. Increased Sp1 was previously found in hypertrophied hearts of pressure overload-induced mice and was thought to influence the conversion from a fatty acid oxidation to a glycolysis program of energy metabolism (40). We suspect that expression of Nkx2.5, GATA-4, and/or Sp1 genes may increase in response to stress as well as hypertrophy-inducing agonists, and we suggest that a lack of Egr-1 increases this stress response. Histological data corroborate the biological data. Most vehicle- and Iso + PE-treated -/- hearts showed fibrotic foci, whereas unmanipulated -/- hearts did not. This suggests that the hearts were not damaged until after the pumps were implanted. Once the pumps were removed and the mice allowed to recover for 7 days, the levels of expression in vehicle-treated hearts more closely resembled those found in untreated and unmanipulated mice. This further suggests that it was the presence of the minipump that was the stressor and that a lack of Egr-1 sensitized the hearts to this stress.

Egr-1 overexpression may be growth inhibitory or growth stimulatory depending on the cell context (11, 19, 25; reviewed in Ref. 27). Supporting the idea that Egr-1 overexpression was crucial to prostate cancer cell growth was the finding that transfection of a portion of chromosome 12, which contains the NAB-2 repressor gene, reduced prostate cancer cell proliferation (2). In other studies using these Egr-1-deficient mice, macrophage differentiation and activation were shown to be indistinguishable between +/+ and -/- genotypes (22). Similarly, postnatal neurogenesis was equivalent in +/+ and -/- mice of the Topilko lineage (13). In contrast, lungs from Egr-1-deficient mice subjected to oxygen deprivation were unable to respond normally (50). Thus the fate of Egr-1 overexpression or a lack of Egr-1 expression may have different consequences depending on the cell context. This fate may depend on expression of other members of the Egr-1 family, Sp1, the NAB-1 or NAB-2 repressors, or some as yet undetermined element.

NAB-1, constitutively expressed, and NAB-2, inducible, are related proteins important for Egr-1 transcription repression (45). We found an increase in NAB-1 and NAB-2 expression in Iso + PE-treated +/+ mice. These data suggest that NAB-1 and NAB-2 may be positively regulated in the heart. NAB-1 was shown to interact with Egr-1 only when Egr-1 was bound to DNA (45). The neo/stop codon cassette was inserted before the DNA binding region of Egr-1, interrupting the Egr-1 coding sequence (23). It is therefore unlikely that NAB-1-Egr-1-DNA interactions occur in -/- mice, but this interaction may be present in +/+ animals. NAB-1 and NAB-2 bind to and repress transcription from Krox-20 and Egr-3 (45). However, neither Egr-3 nor Krox-20 expression is detectable in the adult heart (7, 33), and thus it is unlikely that either would be replacing Egr-1 in the heart or function as NAB binding partners in the heart. We found that NAB-1 and NAB-2 expression increased in Iso + PE-treated +/+ hearts, but only NAB-2 was significantly increased in stimulated -/- hearts. Serum response elements are binding targets of Nkx2.5, presumably through Nkx2.5-serum response factor complexes (40). The suggestion is that NAB-2 expression could be controlled by Nkx2.5 and serum response factor in the normal heart. In the absence of Egr-1 we found no induction of NAB-1 and lower induction of NAB-2 even when Nkx2.5 was increased. This suggests that Egr-1, directly or indirectly, may also play a role in regulating NAB gene expression.

In conclusion, we have shown that Egr-1 expression is not obligatory for catecholamine-induced hypertrophy. However, the lack of Egr-1 expression had a profound effect on the gene expression pattern, and a blunted or absent induction of muscle-specific genes was found. We suspect that this lower induction is caused by lower levels of Sp1, c-jun, and c-fos as well as the cardiac-specific regulators Nkx2.5 and GATA-4. The Egr-1-deficient mice appear to be hyperresponsive to stress.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the photoprinting provided by Carole Querillon.


    FOOTNOTES

This work was funded by grants from the Heart and Stroke Foundation of Quebec and the Medical Research Council of Canada (to L. E. Chalifour).

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

Address for reprint requests and other correspondence: L. E. Chalifour, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 chemin Côte Sainte Catherine, Montréal, Québec, Canada H3T 1E2 (E-mail: CZLC{at}MusicA.McGill.CA).

Received 1 March 1999; accepted in final form 16 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. Current Protocols in Molecular Biology. Toronto: Greene, 1997.

2.   Berube, N. G., M. D. Speevak, and M. Chevrette. Suppression of tumorigenicity of human prostate cancer cells by introduction of human chromosome del(12)(q13). Cancer Res. 54: 3077-3081, 1994[Abstract/Free Full Text].

3.   Boluyt, M. O., X. Long, T. Eschenhagen, U. Mende, W. Schmitz, M. T. Crow, and E. G. Lakatta. Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart. Am. J. Physiol. Heart Circ. Physiol. 269: H638-H647, 1995[Abstract/Free Full Text].

4.   Brand, T., H. S. Sharma, and W. Schaper. Expression of nuclear proto-oncogenes in isoproterenol-induced cardiac hypertrophy. J. Mol. Cell. Cardiol. 25: 1325-1337, 1993[ISI][Medline].

5.   Bruneau, B. G., L. A. Piazza, and A. J. DeBold. Alpha(1)-adrenergic stimulation of isolated rat atria results in discoordinate increases in natriuretic peptide secretion and gene expression and enhances Egr-1 and c-myc expression. Endocrinology 137: 137-143, 1996[Abstract].

6.   Chalifour, L. E., M. L. Gomes, N.-S. Wang, and A.-M. Mes-Masson. Polyomavirus large T-antigen expression in heart of transgenic mice causes cardiomyopathy. Oncogene 5: 1719-1726, 1990[ISI][Medline].

7.   Chavrier, P., M. Zerial, P. Lemaire, J. Almendral, R. Bravo, and P. Charnay. A gene encoding a protein with zinc fingers is activated during G transition in cultured cells. EMBO J. 7: 29-35, 1988[ISI][Medline].

8.   Crosby, S. D., J. J. Peutz, K. S. Simburger, T. Fahrner, and J. Milbrandt. The early response gene NGFI-C encoded a zinc finger transcriptional activator and is a member of the GCGGGGGCG (GSG) element-binding protein family. Mol. Cell. Biol. 11: 3835-3841, 1991[Abstract/Free Full Text].

9.   Davey, H. W., J. K. Kelly, and A. G. Wildeman. The nucleotide sequence, structure and preliminary studies on the transcriptional regulation of the bovine skeletal actin gene. DNA Cell. Biol. 14: 609-618, 1995[ISI][Medline].

10.   Durocher, D., F. Charron, R. Warren, R. J. Schwartz, and M. Nemer. The cardiac transcription factors Nkx2.5 and GATA-4 are mutual cofactors. EMBO J. 16: 5687-5696, 1997[ISI][Medline].

11.   Eid, M. A., M. Vijay, K. A. Iczkowski, D. G. Bostwick, and D. J. Tindall. Expression of early growth response genes in human prostate cancer. Cancer Res. 58: 2461-2468, 1998[Abstract/Free Full Text].

12.   Gashler, A., and V. P. Sukhatme. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog. Nucleic Acid Res. Mol. Biol. 50: 191-224, 1995[ISI][Medline].

13.   Gorbel, M., I. Seugnet, N. Hadj-Sahrsoui, P. Topilko, G. Levi, and B. Demeneix. Thyroid hormone effects of Krox-24 transcription in the postnatal mouse brain are developmentally regulated but are not correlated with mitosis. Oncogene 18: 917-924, 1999[ISI][Medline].

14.   Hasegawa, K., S. J. Lee, S. M. Jobe, B. E. Markham, and R. N. Kitsis. cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction. Circulation 96: 3833-3835, 1997.

15.   Herzig, T. C., S. M. Jobe, H. Aoki, J. D. Molkentin, A. W. Cowley, Jr., S. Izumo, and B. E. Markham. Angiotensin II type 1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload. Proc. Natl. Acad. Sci. USA. 94: 7543-7548, 1997[Abstract/Free Full Text].

16.   Holder, E. L., B. Mitmaker, L. Alpert, and L. E. Chalifour. Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice. Am. J. Physiol. Heart Circ. Physiol. 269: H86-H95, 1995[Abstract/Free Full Text].

17.   Holder, E. L., A.-E. Al Moustafa, and L. E. Chalifour. Molecular remodelling in hypertrophied hearts from polyomavirus large T-antigen transgenic mice. Mol. Cell. Biochem. 152: 131-141, 1995[ISI][Medline].

18.   Huang, R.-P., Y. Fan, Z. Ni, D. Mercola, and E. D. Adamson. Reciprocal modulation between Sp1 and Egr-1. J. Cell. Biochem. 66: 489-499, 1997[ISI][Medline].

19.   Huang, R.-P., Y. Fan, C. Niemeyer, M. M. Gottardis, D. Mercola, and E. Adamson. Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlated with tumor formation. Int. J. Cancer 72: 102-109, 1997[ISI][Medline].

20.   Kobilko, B. Adrenergic and muscarinic receptors of the heart. In: Molecular Basis of Cardiology, edited by R. Roberts. Oxford, UK: Blackwell Scientific, 1993, p. 193-210.

21.   Lanoix, J., A. Mullick, Y. He, R. Bravo, and D. Skup. Wild-type egr-1/Krox24 promotes and dominant-negative mutants inhibit, pluripotent differentiation of p19 embryonal carcinoma cells. Oncogene 17: 2495-2504, 1998[ISI][Medline].

22.   Lee, S. L., Y. Sadovsky, A. H. Swirnoff, J. A. Polish, P. Goda, G. Gavrilina, and J. Milbrandt. Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGF1-A (Egr-1). Science 273: 1219-1221, 1996[Abstract].

23.   Lee, S. L., L. C. Tourtellotte, R. L. Wesselschimdt, and J. Milbrandt. Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A. J. Biol. Chem. 270: 9971-9977, 1995[Abstract/Free Full Text].

24.   Lee, S. L., Y. Wang, and J. Milbrandt. Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transcription factor NGF1-A (Egr-1). Mol. Cell. Biol. 16: 4566-4572, 1996[Abstract].

25.   Levin, W. J., M. F. Press, R. B. Gaynor, V. P. Sukhatme, T. C. Boone, P. T. Reissmann, R. A. Figlin, E. C. Holmes, L. A. Souza, D. J. Slamon, and the Lung Cancer Study Group.. Expression patterns of immediate early transcription factors in human non-small cell lung cancer. Oncogene 11: 1261-1269, 1995[ISI][Medline].

26.   Lin, J.-X., and W. J. Leonard. The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta -chain promoter through noncanonical Egr and Sp1 binding sites. Mol. Cell. Biol. 17: 3714-3722, 1997[Abstract].

27.   Liu, C., V. M. Rangnekar, E. Adamson, and D. Mercola. Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther. 5: 3-28, 1998[ISI][Medline].

28.   Lyons, I., L. M. Parsons, L. Hartley, R. Li, J. E. Andrews, L. Robb, and R. P. Harvey. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2.5. Genes Dev. 9: 1654-1666, 1995[Abstract/Free Full Text].

29.   Milbrandt, J. A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 238: 797-799, 1987[Abstract/Free Full Text].

30.   Narita, N., M. Biehinska, and D. B. Wilson. Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells. Development 124: 3755-3764, 1997[Abstract].

31.   Neyes, L., J. Nouskas, J. Luyken, S. Fronoff, S. Oberdorf, U. Pfeifer, R. S. Williams, V. P. Sukhatme, and H. Vetter. Induction of immediate-early genes by angiotensin II and endothelin-1 in adult rat cardiomyocytes. J. Hypertens. 11: 927-934, 1993[ISI][Medline].

32.   Packer, M. beta -Blockade in heart failure: basic concepts and clinical results. Am. J. Hypertens. 11: 23S-37S, 1998[ISI][Medline].

33.   Patwardhan, S., A. Gashler, M. G. Siegel, L. C. Chang, L. J. Joseph, T. B. Shows, M. M. LeBeau, and V. P. Sukhatme. EGR3, a novel member of the Egr family of genes encoding immediate-early transcription factor. Oncogene 6: 917-928, 1991[ISI][Medline].

34.   Pollak, P. S. Proto-oncogenes and the cardiovascular system. Chest 107: 826-835, 1995[Free Full Text].

35.   Rindt, H., J. Gulick, S. Knott, J. Neumann, and J. Robbins. In vivo analysis of the murine beta -myosin heavy chain gene promoter. J. Biol. Chem. 268: 5332-5338, 1993[Abstract/Free Full Text].

36.   Rosenzweig, A., T. D. Halazonetis, J. G. Seidman, and C. E. Seidman. Proximal regulatory domains of rat atrial natriuretic factor gene. Circulation 84: 1256-1265, 1991[Abstract/Free Full Text].

37.   Russo, M. W., B. R. Sevetson, and J. Milbrandt. Identification of NAB1, corepressor of NGF1-A- and Krox20-mediated transcription. Proc. Natl. Acad. Sci. USA 92: 6873-6877, 1995[Abstract/Free Full Text].

38.   Saadane, N., L. Alpert, and L. E. Chalifour. TAFII250, Egr-1, and D-type cyclin expression in mice and neonatal rat cardiomyocytes treated with doxorubicin. Am. J. Physiol. Heart Circ. Physiol. 276: H803-H814, 1999[Abstract/Free Full Text].

39.   Saadane, N., L. Alpert, and L. E. Chalifour. Expression of immediate early genes, GATA-4, and Nkx2.5 in adrenergic-induced cardiac hypertrophy and during regression in adult mice. Br. J. Pharmacol. 127: 1165-1176, 1999[ISI][Medline].

40.   Sack, M. N., D. L. Disch, H. A. Rockman, and D. P. Kelly. A role for Sp and nuclear receptor transcription in a cardiac hypertrophic growth program. Proc. Natl. Acad. Sci. USA 94: 6438-6443, 1997[Abstract/Free Full Text].

41.   Sepulveda, J. L., N. Belaguli, V. Nigam, C. Y. Chen, M. Nemer, and R. J. Schwartz. GATA-4 and Nkx2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression. Mol. Cell. Biol. 18: 3405-3415, 1998[Abstract/Free Full Text].

42.   Sharma, H. S., H. A. A. Vanheugten, M. A. Goedbloed, P. D. Verdouw, and J. M. J. Lamers. Angiotensin II induced expression of transcription factors precedes increase in transforming growth factor beta-1 mRNA in neonatal cardiac fibroblasts. Biochem. Biophys. Res. Commun. 205: 105-112, 1994[ISI][Medline].

43.   Silverman, E. S., L. M. Hhachigian, V. Lindner, A. J. Williams, and T. Collins. Inducible PDGF A-chain transcription in smooth muscle cells is mediated by Egr-1 displacement of Sp1 and Sp3. Am. J. Physiol. Heart Circ. Physiol. 273: H1415-H1426, 1997[Abstract/Free Full Text].

44.   Soonpaa, M. H., and L. J. Field. Assessment of cardiomyocyte DNA synthesis during hypertrophy in adult mice. Am. J. Physiol. Heart Circ. Physiol. 266: H1439-H1445, 1994[Abstract/Free Full Text].

45.   Svaren, J., B. R. Sevetson, E. D. Apel, D. B. Zimonjic, N. C. Popescu, and J. Milbrandt. NAB2, a corepressor of NGF1A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol. Cell. Biol. 16: 3545-3553, 1996[Abstract].

46.   Swirnoff, A. H., and J. Milbrandt. DNA-binding specificity of NGF1-A and related zinc finger transcription factors. Mol. Cell. Biol. 15: 2275-2287, 1995[Abstract].

47.   Topilko, P., S. Schneider-Maunoury, G. Levi, A. Trembleau, D. Gourdji, M.-A. Driancourt, C. H. V. Rao, and P. Charnay. Multiple pituitary and ovarian defects in Krox24 (NGF1-A, Egr-1)-targeted mice. Mol. Endocrinol. 12: 107-122, 1998[Abstract/Free Full Text]<