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Cardiovascular Research 2004 62(3):548-557; doi:10.1016/j.cardiores.2004.01.032
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Transcriptional regulation of FGF-2 gene expression in cardiac myocytes

Sarah K Jimeneza, Farah Sheikhb, Yan Jina, Karen A Detillieuxa, Jamit Dhaliwala, Elissavet Kardamic and Peter A Cattini*,a

aDepartment of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada R3E 3J7
bInstitute of Molecular Medicine, University of California San Diego, San Diego, CA, USA
cDepartment of Human Anatomy and Cell Science, University of Manitoba, Canada

* Corresponding author. Tel.: +1-204-789-3696; fax: +1-204-789-3934. Email address: peter_cattini{at}umanitoba.ca

Received 3 September 2003; revised 23 January 2004; accepted 28 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 
Objective: Fibroblast growth factor-2 (FGF-2) exerts its cardioprotective effect through cell surface receptor signaling and may play a role in the normal maintenance of a healthy myocardium. One mechanism of FGF-2 release from contracting cardiomyocytes is through transient sarcolemmal disruption, with accumulation in the extracellular matrix. Continuous FGF-2 release would require a link to synthesis and, thus, we examined regulation of FGF-2 promoter activity in cardiomyocytes as a potential target for optimizing cardioprotection. Methods and results: To investigate autoregulation, neonatal rat cardiomyocytes, (NRCM), were transfected with ~1 or 0.1 kb of rat FGF-2 promoter sequences linked to luciferase, –1058FGF–2p.luc and –110FGF–2p.luc, and treated with or without FGF-2. FGF-2 promoter activity was significantly increased ~2.5-fold with both genes. The proximal promoter region of rat FGF-2 contains putative binding sites for the early growth response-1 (Egr-1) and stimulating protein 1 (Sp1) transcription factors. Overexpression of Egr-1 and Sp1 increased –1058FGF–2p.luc expression by 4.4- and 8.7-fold, respectively. Mutation of Egr-1 and overlapping Sp1 sites did not blunt the response of –110FGF–2p.luc to FGF-2 treatment but did significantly reduce basal promoter activity. Transgenic mice expressing –1058FGF–2p.luc were treated with isoproterenol (IsP) to increase heart rate and endogenous FGF-2 release. FGF-2 promoter activity was stimulated significantly at 6 h, and increases in both FGF-2 and its receptor mRNA levels were also detected. In contrast, no effect of IsP was seen on –1058FGF–2p.luc or –110FGF–2p.luc in transfected NRCMs. Conclusions: FGF-2 released from cardiomyocytes may act to regulate its own synthesis at the transcriptional level. The mechanism does not appear to require an intact Egr-1 site in the proximal promoter region. This may, however, reflect redundancy in the control of FGF-2 promoter activity as our data support a stimulatory role for Egr-1 and Sp1.

KEYWORDS Cardioprotection; FGF-2; Gene expression; Growth factors; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Experimental procedures
 3. Results
 4. Discussion
 References
 
Fibroblast growth factor-2 (FGF-2) is both cardioprotective and angiogenic when used to treat mammalian hearts [1], or as a consequence of transgenic overexpression in cardiomyocytes [2]. FGF-2 exerts these effects by signaling through high-affinity FGF receptors (FGFRs), specifically, FGFR-1, of the tyrosine kinase family located on cardiomyocytes and endothelial cell plasma membranes [3]. This requires release of FGF-2 from cells for receptor interaction. Release of FGF-2 is well documented, although it does not possess a "classic" signal peptide [4]. Multiple mechanisms have been implicated in this process (reviewed in Ref. [5]). Previously, we proposed that FGF-2 and, specifically, its release during contraction might play a role in the normal maintenance of a healthy myocardium [1,6], presumably through autocrine/paracrine pathways. Recent gene ablation studies support this idea, suggesting that cardioprotection is compromised in FGF-2 deficient mice [7].

There is evidence that FGF-2 can function in an autocrine and paracrine manner in a number of different tissue and cell types including in the heart, often with effects on cell growth [8,9]. Autoregulation of FGF-2 synthesis has also been described in various tissue culture systems including cardiomyocytes, where FGF-2 addition increases FGF-2 messenger RNA levels [10–12]. The transcription factor early growth response-1 (Egr-1) has been implicated in the mechanism of FGF-2 autoregulation in astrocytes [11,13]; both human and rat FGF-2 genes contain Egr-1 and/or Egr-1-like sites in their proximal promoter regions [1,11,13,14]. We have used a combination of in vitro and in vivo studies to assess the possibility of FGF-2 autoregulation at the transcriptional level in cardiomyocytes. To look at the consequence of increased local availability of FGF-2 in the heart on FGF-2 synthesis, we have attempted to use the β-adrenergic agonist isoproterenol (IsP) to stimulate contractions in transgenic mice expressing a hybrid rat FGF-2 promoter–reporter gene to assess any effect on FGF-2 promoter in vivo. Our results indicate the capacity for multiple mechanisms controlling replacement synthesis of FGF-2 in cardiomyocytes.


   2. Experimental procedures
Top
 Abstract
 1. Introduction
 2. Experimental procedures
3. Results
 4. Discussion
 References
 
2.1. Plasmids
The hybrid rat FGF-2/luciferase genes –1058FGF–2p.luc (nucleotides –1058/+54), –110FGF–2p.luc (nucleotides –110/+42) and –110mFGF–2p.luc were described previously [1,14]. The expression vectors for Egr-1 (CMVp.Egr-1) and stimulating protein 1 (Sp1; CMVp.Sp1) were generous gifts from Dr. V.P. Sukhatme (Beth Israel Hospital, Boston, MA), and Dr. R. Tjian (UC Berkeley, CA), respectively. The "empty" expression vector pcDNA3 (Invitrogen, San Diego, CA), promoterless luciferase gene vector pxp1 [15] and the firefly luciferase gene directed by a minimal (–81/+53) thymidine kinase (TK) promoter [15] were used as controls.

2.2. Cell culture
All procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and conform to the NIH guidelines. Neonatal rat cardiomyocytes (NRCM) cultures were prepared as previously described [6] with modifications. Heart tissue was dissociated in collagenase (740 U/digestion; Worthington Biochemicals, NJ), trypsin (370 U/digestion), and DNase I (2880 U/digestion), and plated at a density of 0.85 x 106 cells per 35 mm dish in Ham's F10 medium containing 10% fetal bovine serum (FBS), 10% horse serum and antibiotic (1000 U/ml penicillin, 1 mg/ml streptomycin). Cultures are periodically assessed through specific labeling of cardiomyocytes (e.g., {alpha}-actinin) and evaluation of various labeling indices (e.g., bromodeoxyuridine or Ki67) using microscopy; cultures used are at least 90% cardiomyocytes at 5–7 days.

2.3. Transient gene transfer
NRCMs were transfected either by lipofection using Lipofectamine PLUS (Invitrogen; effect of IsP and FGF-2 studies), or by calcium phosphate DNA precipitation (transcription factor overexpression studies) as previously described [1]. For lipofection, NRCMs at 70–90% confluency (16–20 h after plating) were incubated in DMEM-F12 (10% FBS, antibiotics) for 4 h. For every 35-mm well, 2.25 µg DNA, 8.5 µl of PLUS reagent, and 8.75 µl of Lipofectamine were mixed with DMEM to a total of 2 ml. Following two calcium- and magnesium-free phosphate buffered saline (PBS-CMF) washes, the Lipofectamine mix was added and incubated at 37 °C for 16–20 h. Transfected myocytes were treated without or with 0.01–10 ng/ml recombinant human FGF-2 (Upstate, Lake Placid, NY), 0.01 mM IsP for 6, 24 or 48 h. Results are expressed as fold response of a promoter construct to FGF-2 or IsP treatment relative to control levels.

2.4. Transgenic mice
The generation and characterization of two independent transgenic mouse lines (P300 and P66) expressing –1058FGF–2p.luc was described elsewhere [6]. Adult animals (9–12 weeks) were injected intraperitoneally with a single dose of vehicle (saline) or IsP (80 mg/kg in saline). After 6 or 96 h, animals were euthanized and luciferase activity from isolated hearts was determined.

2.5. Reporter gene assays
Lysates from transfected NRCMs and transgenic mouse hearts were prepared as previously described [1,6]. Luciferase activity was normalized to protein (Bradford Assay, Bio-Rad Laboratories, ON, Canada) to give values of luciferase activity/µg protein as previously described [1].

2.6. RNA blotting
RNA isolation, electrophoresis and blotting (50 µg) were done as described previously [16]. Atrial natriuretic factor, ANF, FGF-2 and FGFR-1 cDNAs were radiolabeled and used as probes [16]. Autoradiographs were assessed by densitometry.

2.7. Electrophoretic gel mobility shift assay (EMSA)
Nuclear protein was isolated from whole mouse hearts by hypotonic cell lysis followed by high-salt protein extraction as described previously [16]. Electrophoretic gel mobility shift assay (EMSA) was done using an established protocol [1,16]. For competition, competitor double-stranded oligonucleotides (25–750 x mass excess) were added with nuclear extract for 10 min at room temperature, followed by radiolabeled probe for 20 min. The sequence of one strand from each of the DNA fragments, wild type (WT) and mutant (m), used as probes and/or competitors are shown in Table 1.


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  		Table 1 Oligonucleotide and primer sequences

 
2.8. Statistical analysis
Data presented in the text and figures are mean values from at least three independent experiments plus or minus standard error of the mean. Statistical analysis of the results was carried out using one-way analysis of variance and Tukey–Kramer Multiple Comparisons (post hoc) test, as well as the Student's t (parametric) or Mann–Whitney (non parametric) tests (InStat 3.0, GraphPad Software). In all cases p≤0.05 (*) was considered statistically significant; p<0.01 (**).


   3. Results
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
4. Discussion
 References
 
3.1. Exogenous FGF-2 addition increases FGF-2 promoter activity in transfected NRCMs
NRCMs were transfected with –1058FGF–2p.luc, and treated with 0 or 0.001–10 ng/ml FGF-2 for 48 h. A dose-dependent increase in promoter activity was detected with significant stimulations at 0.1 ng/ml FGF-2 or greater, and plateaued at 1 ng/ml with an ~2.5-fold overall increase (Fig. 1A). To assess the time of response, NRCMs were transfected with –1058FGF–2p.luc and treated with or without 1 ng/ml recombinant human FGF-2 for 6, 24 and 48 h. Although a modest stimulation of promoter activity was suggested at 24 h, a significant increase was only detected at 48 h (Fig. 1B).


Figure 1
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  		Fig. 1 NRCMs transfected with the –1058FGF–2p.luc gene, were treated without or with (A) 0.001, 0.01, 0.1, 1 and 10 ng/ml FGF-2 for 48 h and (B) 1 ng/ml FGF-2 for 6, 24 and 48 h. The results (luciferase activity/µg protein) are expressed as the mean fold effect of FGF-2 treatment. The values for vehicle in each experiment (A) (287±25) and (B) (6, 24 and 48 h: 220±54, n=3; 1484±341, n=18 and 447±58, n=3, respectively) have been arbitrarily set to 1.0.

 
3.2 Overexpression of Egr-1 or Sp1 increase –1058FGF–2p.luc expression in NRCMs
Examination of nucleotides –1058/+54 of the rat FGF-2 gene reveals the presence of three putative Egr-1 DNA elements [1]. These are localized to the proximal promoter region corresponding to nucleotides –7/+42 (Fig. 2). In the case of two of these elements, potential overlapping high-affinity sites for Sp1 binding (5'-GGCGG-3' at the core) are present (Fig. 2). To assess whether increased levels of Sp1 as well as Egr-1 [1] could influence FGF-2 promoter activity, NRCMs were transfected with –1058FGF–2p.luc and cotransfected with an Sp1 expression vector (CMVp.Sp1). Cardiomyocytes were also cotransfected with CMVp vector (pcDNA3, Cont.) and an Egr-1 expression vector (CMVp.Egr-1) as background and positive controls [1]. Significant stimulation of –1058FGF–2p.luc expression (p<0.05, n=4) was observed with either Sp1 or Egr-1 overexpression (Fig. 3).


Figure 2
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  		Fig. 2 Wild type (above) and mutated (below) sequences of the proximal promoter region of rat FGF-2 corresponding to nucleotides –7/+42. Disrupted sequences are indicated by bold type. Putative Egr-1 (small boxes) and overlapping Sp1 (large boxes) are indicated. The subfragments WT1, WT2 and WT3 used to assess binding of Egr-1 and Sp1 through EMSA competition, as well as the sequence of consensus Egr-1 and Sp1 elements are shown.

 

Figure 3
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  		Fig. 3 NRCMs were transfected with the –1058FGF–2p.luc gene and cotransfected with empty expression vector (Cont.) or expression vectors for Egr-1 or Sp1. Cells were harvested and luciferase activity was determined at 48 h. The results (luciferase activity/µg protein) are expressed as the mean fold effect on –1058FGF–2p.luc gene expression in the presence of Egr-1 or Sp1 overexpression. The value for "Cont." (369±53) has been arbitrarily set to 1.0 (n=3).

 
3.3. Characterization of Egr-1 and Sp1 binding sites
Specific Egr-1 and Sp1 protein/DNA complexes were identified by EMSA using consensus DNA elements for Egr-1 (Fig. 4A) and Sp1 (Fig. 4B) as probes with adult mouse heart nuclear extracts, and competition by wild type (Egr-1 and Sp1) vs. mutant (Egr-1m and Sp1m) oligonucleotide competitors (Table 1 and Fig. 2). To assess the relative binding of the putative Egr-1 and Sp1 elements located in the FGF-2 proximal promoter region, double-stranded oligonucleotides containing the putative sites (fragments WT1-3 in Fig. 2) were used as competitors of the radiolabeled consensus Egr-1 or Sp1 DNA elements (Fig. 4). WT1 but not WT2 or WT3 was able to compete effectively for Egr-1 binding (closed arrowhead, Fig. 4A). Two base pair mutant forms of WT1 and WT3 (WT1m and WT3m), which would be expected to interfere with both overlapping Egr-1/Sp1 sites, were generated (Table 1). When these were used as competitors, no efficient competition of the specific Egr-1 band was detected (Fig. 4A). In the case of Sp1, WT1 and to a lesser extent WT3 but not WT2 were able to compete effectively for the specific Sp1 complex (Fig. 4B). In contrast, WT1m and WT3m were not efficient competitors of the specific Sp1 complex.


Figure 4
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  		Fig. 4 Nuclear extract was isolated from adult mouse heart tissue and used in combination with a radiolabeled (A) Egr-1 or (B) Sp1 consensus DNA element (Table 1) as probes for EMSA. Multiple complexes are seen in the presence (+) vs. absence (–) of nuclear protein after nondenaturing gel electrophoresis and autoradiography. Specific Egr-1 and Sp1 complexes were identified by competition with increasing amounts of consensus Egr-1 (100, 500 and 750 x mass excess) and Sp1 (25, 50 and 100 x mass excess) vs. mutant (Egr-1m and Sp1m) DNA elements (Table 1). Closed and open arrowheads indicate the mobility of major high-affinity/specific Egr-1 (A) and Sp1 (B) complexes, respectively. The relative ability of oligonucleotides corresponding to WT1, WT2 and WT3, or mutated variants WT1m and WT3m (Table 1), to compete (25, 50 and 100 mass excess) for these specific complexes is shown.

 
Possible interaction of Egr-1 and Sp1 from mouse heart nuclear extracts with a radiolabeled rat FGF-2 (nucleotides –7/+42) probe was also assessed by EMSA. A low-mobility complex was observed and was competed with Sp1 but not Egr-1 oligonucleotides (Table 1) at relatively low (100 x) mass excess of probe (Fig. 5A). Some competition with Egr-1 oligonucleotide was seen at 500 x mass excess of probe (Fig. 5A) and was efficiently competed with a 750 x excess (data not shown). When WT1-3 were used as competitors, efficient competition was observed with WT1 but not WT3 (or WT2) (Fig. 5B).


Figure 5
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  		Fig. 5 Mouse heart nuclear extract (+) was used in combination with radiolabeled rat FGF-2 sequences (nucleotides –7/+42) as a probe for EMSA. A low mobility complex is evident (arrowhead). This complex was characterized by competition with (A) FGF-2 (–7/+42), Egr-1, or Sp1 oligonucleotides, and (B) WT1-3 fragments as indicated (100 and 200 x mass excess, plus 500 x for Egr-1).

 
3.4 Stimulation of –110FGF–2p.luc expression by FGF-2 treatment in transfected NRCMs
In an attempt to localize the FGF-2-responsive region, the –110FGF–2p.luc gene was tested for an effect of 1 ng/ml FGF-2 in transfected NRCMs (Fig. 6). A significant ~2 fold stimulation of –110FGF–2p.luc expression was detected with FGF-2 addition at 48 h (p<0.05, n=6).


Figure 6
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  		Fig. 6 NRCMs were transfected with hybrid luciferase (luc) genes directed by promoter regions –1058FGF–2p, –110FGF–2p, –110mFGF–2p and TKp or without a promoter (-p.), and treated with or without 1 ng/ml FGF-2 for 48 h. The results (luciferase activity/µg protein) are expressed as the mean fold effect of FGF-2 treatment. The value for vehicle has been arbitrarily set to 1.0 (n=4–6).

 
To determine the relative importance of the Egr-1 and/or overlapping Sp1 DNA elements in WT1-3 to FGF-2-responsiveness of the –110FGF–2p.luc gene (Figs. 2 and 4)Go, these sites were mutated (Fig. 2). The resulting mutated gene, –110mFGF–2p.luc, was then assessed for FGF-2 responsiveness (1 ng/ml) in transfected NRCMs after 48 h (Fig. 6). In addition, an unrelated minimal viral TK promoter (nucleotides –81/+53) fused to the luciferase gene (TKp.luc), as well as a promoterless luciferase gene (-p.luc) were included for comparison. Although lower (by ~35%) levels of activity were detected for the –110mFGF–2p.luc gene (basal activity 283±28) relative to the wild type –110FGF–2p.luc gene (basal activity 433±39), there was still a significant (p<0.05, n=6) effect of FGF-2 treatment on gene activity (Fig. 6). Similarly, a significant 1.7 fold (p<0.05, n=4) stimulation of TKp.luc gene expression (basal activity 142±15) was observed; however, no increase (p>0.05, n=5) in the activity of the promoterless-p.luc gene (basal activity 51±15) was detected (Fig. 6).

3.5. IsP treatment induces hypertrophic growth in the mouse heart
To determine whether IsP treatment was sufficient to increase contractile function and, presumably, FGF-2 export [17], adult mice were injected intraperitoneally with saline or 80 mg/kg IsP. The mice were weighed and euthanized 6 and 96 h after treatment. Hearts from saline- and IsP-treated mice were excised, atria removed, and weighed to determine heart weight-to-body weight (HW/BW) ratios (Fig. 7A). There was no significant difference between the HW/BW ratios for saline-treated mice (3.53±0.19 mg/g, n=9) and mice 6 h after IsP treatment (3.77±0.14 mg/g, n=9). In contrast, there was a significant 1.2-fold increase in HW/BW ratio between either saline-treated mice (p<0.01, n=9) or mice 6 h after IsP treatment (p<0.05, n=9) and mice 96 h after IsP treatment (4.35±0.13, n=9).


Figure 7
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  		Fig. 7 (A) Adult mice were injected intraperitoneally with 80 mg/kg IsP or saline vehicle. The mice were weighed and euthanized 6 and 96 h after treatment. Results (n=9) are expressed as the mean values of HW/BW ratio (mg/g). (B) Total RNA from ventricles was blotted and probed sequentially for ANF and GAPDH transcripts using radiolabeled cDNAs. Resulting autoradiographs are shown.

 
A fivefold increase (p<0.001, n=3) in the levels of 0.9 kb ANF transcript relative to GAPDH mRNA was observed in IsP vs. saline-treated hearts at 96 h but not 6 h (Fig. 7B).

3.6. IsP treatment increases FGF-2 promoter activity in the mouse heart but not transfected NRCMs
To assess whether the FGF-2 promoter responds to β-adrenergic stimulation in the heart in vivo, transgenic mice (P300 and P66 mice; 8–10 weeks) were injected intraperitoneally with 80 mg/kg IsP or saline. At 6 h, luciferase activity in hearts was determined. Significant 1.8-fold (p<0.05, n=6) and 3.3-fold (p<0.01, n=4) increases were observed following IsP treatment in both lines (Fig. 8A). The effect of IsP on FGF-2 promoter activity as well as endogenous P300 mouse FGF-2 mRNA accumulation were also assessed 96 h after treatment (Fig. 8B). Stimulation of FGF-2 promoter activity was decreased from 1.8- to 1.4-fold and was no longer significant (n=6); however, endogenous FGF-2 mRNA accumulation was increased 2.1-fold (p<0.05, n=3). Using RNA blotting and a radiolabeled mouse FGFR-1 probe, a significant 1.7-fold increase (p<0.05, n=3) in RNA levels was observed within 6 h of IsP treatment but did not persist to 96 h (Fig. 9).


Figure 8
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  		Fig. 8 (A) Adult P300 and P66 mice expressing the transgene –1058FGF–2p.luc were injected intraperitoneally with 80 mg/kg IsP or saline vehicle. Luciferase activity from isolated hearts was determined 6 h later (n=4–6). (B) Adult P300 mice were treated with IsP (+) or saline vehicle (–) for 96 h, and cardiac tissue was processed for both luciferase (–1058FGF–2p.luc) activity and endogenous mouse FGF-2 mRNA accumulation. For FGF-2 promoter activity, the results (luciferase activity/µg protein) are expressed as the mean fold effect of IsP. The value for saline (339±44) has been arbitrarily set to 1.0 (n=6). For endogenous FGF-2 RNA levels, total RNA blotted and probed sequentially for FGF-2 and GAPDH, and subjected to autoradiography (inset). The results are expressed as the mean fold effect of IsP vs. saline on levels of the 6.1 kb FGF-2 transcript, as assessed by densitometry of autoradiographs and standardization to GAPDH levels (n=3).

 

Figure 9
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  		Fig. 9 Adult mice (8–10 weeks) were injected intraperitoneally with saline for 6 h or 80 mg/kg IsP for 6 and 96 h. Total RNA from cardiac ventricles was blotted and probed sequentially for FGFR-1 and GAPDH transcripts using radiolabeled cDNAs and subjected to autoradiography (inset). The results are expressed as the mean fold effect of IsP vs. saline on levels of FGFR-1 transcript, as assessed by densitometry of autoradiographs and standardization to GAPDH levels (n=3).

 
To assess a direct effect of IsP on FGF-2 promoter activity, NRCMs were transfected with –1058FGF–2p.luc or –110FGF–2p.luc, and treated with or without 10 µM IsP for 6, 24 and 48 h. Although an increase in the level of contractions was evident in cultures treated with IsP, no significant increases in FGF-2 promoter activity were observed at any of the time points tested (Fig. 10).


Figure 10
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  		Fig. 10 NRCMs transfected with the (A) –1058FGF–2p.luc or (B) –110FGF–2p.luc gene, were treated without or with 10 µM IsP for 6, 24 and 48 h. The results (luciferase activity/µg protein) are expressed as the mean fold effect of IsP treatment. The value for vehicle in each experiment (A) (6, 24 and 48 h: 343±36, n=3; 1820±160, n=18 and 285±43, n=3, respectively) and (B) (6, 24 and 48 h: 166±18, n=3; 1089±203, n=18 and 249±28, n=3, respectively) has been arbitrarily set to 1.0.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
References
 
Using a combination of in vitro and in vivo gene transfer studies we have examined regulation of FGF-2 synthesis at the transcriptional level. Specifically, in the case of the β-agonist IsP, an indirect mechanism of control is suggested because IsP stimulated FGF-2 promoter activity in transgenic mouse hearts (in vivo) but not NRCMs (in vitro). FGF-2 autoregulation may provide this mechanism, based on the reported stimulation of FGF-2 release in response to increased heart rate in vivo [18,19], and our ability to increase FGF-2 promoter activity in FGF-2-treated NRCMs. However, unlike FGF-2 autoregulation in astrocytes [11,13], this does not appear to require an intact G/C-rich Egr-1 element in the proximal promoter region. Nonetheless, G/C-rich DNA elements may play a role; changes in both Egr-1 and Sp1 levels, which also bind G/C-rich DNA, were able to increase FGF-2 promoter activity. Thus, while the capacity for autoregulation of FGF-2 synthesis exists, our results are also consistent with the presence of multiple mechanisms to ensure constitutive FGF-2 promoter activity in the cardiomyocyte.

The region –7/+42 of the rat FGF-2 gene contains three putative Egr-1 elements (Fig. 2), and previous studies indicated that one or more of these sites binds Egr-1 [1]. Our current data suggest that only one of these sites (nucleotides +1/+9) in WT1 can bind Egr-1 by virtue of its ability to compete with a high affinity Egr-1 binding site (Fig. 4). The lack of Egr-1 binding at WT2 and WT3 is consistent with the presence of a base mismatch in the Egr-1 core (Fig. 2). Nonetheless, continued FGF-2 responsiveness was seen with disruption of the Egr-1 site in WT1 as well as the Egr-1 like sites in WT2 and WT3 (Fig. 6), using "GG" to "TA" dinucleotide mutations shown previously to interfere with binding [1]. Given the lack of any other consensus Egr-1 sites within nucleotides –110/+42 (or –1058/+54) of the rat FGF-2 promoter region, these data suggest that Egr-1 binding is not required for FGF-2 regulation of its own promoter in cardiomyocytes.

Neither human nor rat FGF-2 gene promoter regions contain conventional TATA sequences but are rich in GC sequences, which could bind Sp1 and are also characteristic of so-called "housekeeping" or constitutively active genes [20–22]. Certainly, the Sp family of transcription factors have been linked to expression of genes in the heart [23]. Due to similarity in their DNA elements, Sp1 and Egr-1 sites can overlap and result in mutually exclusive binding (Fig. 8A) [24,25]. This allows for a complex response based on displacement of one factor by the other. A role for Sp1 in FGF-2 promoter control is indicated by our ability to stimulate –1058FGF–2p.luc gene expression via Sp1 overexpression in transfected NRCMs (Fig. 3). We were also able to find evidence for high- and low-affinity Sp1 binding sites (nucleotides –3/+6 and +25/+33, respectively) overlapping two putative Egr-1 sites in oligonucleotides WT1 and WT3. This is based on efficient competition of a consensus Sp1 element with WT1 and, to a lesser extent, with WT3 sequences (Fig. 4), presumably, reflecting minor base pair differences in the Sp1 core (Fig. 2). Furthermore, WT1, but not WT3, was able to compete a low-mobility adult mouse heart nuclear protein/FGF-2 promoter DNA (nucleotides –7/+42) (Fig. 5). Interestingly, this complex could also be competed with Egr-1 oligonucleotide, but only at greater (500–750 x vs. 25–100 x mass) levels of oligonucleotide. Previously we reported that Egr-1 can be recruited to the FGF-2 promoter, based on competition of an induced NRCM nuclear protein/FGF-2 promoter (–7/+42) complex with an Egr-1 but not Sp1 antibody [1].

As with the Egr-1 site, however, mutation of these Sp1 sites in WT1 and WT 3 did not block FGF-2 responsiveness of the –110FGF–2p.luc gene (Fig. 6). The core of the consensus Sp1 element is shorter than that of the related Egr-1 element (Fig. 2) and overall is associated with more variability in terms of alternative binding sites, which include the related CACC-box [21,26]. Thus, given the G/C-rich nature of the FGF-2 promoter region (–110/+42 is 71% GC) and the ability of Sp1 to bind alternative sequences, it is not clear whether an additional site or sites might contribute to FGF-2 responsiveness. Consistent with this, the minimal thymidine kinase (TK) promoter in TKp.luc contains a single Sp1 element [27], and was stimulated significantly by FGF-2 treatment in transfected cardiomyocytes (Fig. 6). In terms of potential Sp1 regulatory elements, there are at least 11 Sp1 and CACC-box-related sequences within nucleotides –1058/+54 of the rat FGF-2 gene. Five of these are located in the –110/+42 promoter region. It would be difficult to determine the relative importance of these G/C-rich sequences for regulation given their role in basal promoter activity, particularly in the absence of a TATA sequence. FGF-2 promoter activity was reduced by ~35% with disruption of Sp1 elements in WT1 and WT3 alone. This value did not change significantly (~32%) when firefly luciferase values for each construct were corrected for DNA uptake using values obtained from cotransfection with a Renilla luciferase gene (data not shown).

The increases in endogenous rat [18] and mouse (Fig. 8) heart FGF-2 mRNA levels with IsP treatment suggest that the effect seen on FGF-2 promoter activity through –1058FGF–2p.luc transgene expression reflects a "normal" response (Fig. 8). Promoter–reporter gene studies are a well-recognized indicator of transcriptional control, and because the FGF-2 promoter–reporter gene assays mirror the effects seen in terms of FGF-2 mRNA levels in the heart, it is more likely that the increase is due to increased transcription as opposed to mRNA stability. There is limited evidence to suggest that FGF-2 mRNA stability plays a major role in increasing FGF-2 RNA. Although it is postulated that antisense transcripts can modulate FGF-2 mRNA stability in C6 glioma cells ([28] and references therein), their role in the myocardium and cardiomyocytes is unknown.

There was, however, a difference as to when a response was observed on heart FGF-2 mRNA levels vs. –1058FGF–2p.luc transgene expression after IsP treatment. In the case of FGF-2 transcripts, this was at 96 h and for –1058FGF–2p.luc expression this was at 6 h. This presumably reflects differences in the stage of the synthetic pathway assessed, specifically, mRNA accumulation vs. promoter activity, as well as the stability of the gene products concerned and sensitivity of the respective assays employed.

The 80 mg/kg dose of IsP used to treat transgenic mice was sufficient to increase heart rate, force of contraction and, thus, FGF-2 export. This is supported by evidence of resulting hypertrophy by 96 h posttreatment (significant increase in HW/BW ratio and ANF gene expression; Fig. 7), which would be expected with this stimulus. The fivefold stimulation of ANF RNA levels is similar to sixfold and sevenfold increases seen in angiotensin II- and pressure overload-induced hypertrophy [29,30].

Cardiomyocytes are capable of synthesizing FGF-2 in culture [31] and contracting on β-adrenergic stimulation [17]; however, IsP treatment failed to indirectly stimulate –1058FGF–2p.luc gene expression in NRCMs using the autoregulatory pathway suggested (Fig. 10). This may be related to a low dose of available FGF-2, resulting from the isolation procedure, culture-related depletion of intracellular stores, an inadequate (experimental) environment to trigger FGF-2 synthesis under and/or timeframe employed. The isolation would also separate myocytes from the surrounding extracellular matrix, a significant source of FGF-2 by virtue of its local export and heparan binding properties [32]. A response in culture may, therefore, require recovery of the myocytes both in terms of FGF-2 synthesis, intracellular and extracellular accumulation, as well as allow sufficient repair of damage to membranes. The requirement for a more prolonged FGF-2 (48 h) compared to IsP (6 h) treatment to detect a significant stimulation of FGF-2 promoter activity in transfected cultures vs. transgenic mice, respectively, is consistent with this notion (Figs. 1 and 8)Go. Of course, the possibility that these differences also reflect the developmental stage or maturity of the cardiomyocytes investigated cannot be ruled out.

The lack of IsP responsiveness of –1058FGF–2p.luc gene expression in transfected NRCMs is consistent with previous studies. Specifically, the effect of norepinephrine was examined with and without the β-antagonist atenolol (or the {alpha}-antagonist prazosin) on FGF-2 promoter activity [6]. Unlike {alpha}-adrenergic stimulation, no significant effect of β-adrenergic stimulation was observed and so this was not pursued in transgenic mice (in vivo).

FGF-2 is cardioprotective [5] and can be released from cardiomyocytes on contraction under normal physiological conditions [33]. This would be consistent with a role for FGF-2 in the normal maintenance of a healthy myocardium, perhaps by providing the heart with an increased capacity to function under greater range of conditions of stress, through its cardioprotective properties. Our results support this idea by providing a link between FGF-2 synthesis and release in the heart through possible autoregulation and/or redundancy through multifactorial control in cardiomyocytes. Sp1 might be important for constitutive FGF-2 expression as seen in several other TATA-less housekeeping genes [21,22,34], but also inducible expression because Sp1 activity is reported to increase under hypoxic conditions [20]. Egr-1 is a product of primary response or intermediate early genes, which might allow for additional control under periods of stress [13,35]. Although our mutation studies appear to rule out a role for Egr-1 in autoregulation in cardiomyocytes, the possibility of redundancy in the system makes this difficult to establish. Certainly, an increase in Egr-1 activity in cardiomyocytes will stimulate FGF-2 promoter activity (Fig. 3) [1], and paracrine release of FGF-2 from "scrape" injured bovine endothelial cells was shown to induce Egr-1 [36]. Cardioprotection by FGF-2 is mediated by FGFR-1 in the adult myocardium [37]. An increase in FGFR-1 mRNA levels was detected in IsP-treated hearts (Fig. 9), reflecting a direct or indirect response to β-adrenergic stimulation. The transient increase in receptor synthesis may be a component of the stress response and, more specifically, of cardioprotection via the FGF axis. Isolated hearts from transgenic mice overexpressing FGF-2 possess an increased capacity for FGF-2 accumulation, both intracellular and associated with the extracellular matrix, as well as cardioprotection in response to ischemic injury [2]. Thus, stimulation of FGF-2 synthesis and resulting accumulation may provide additional cardioprotective effects.


   Acknowledgements
 
We would like to acknowledge L.D. Norquay and M.E. Bock for their expertise and technical assistance. This work was supported by a grant from the CIHR (MT-12303). SKJ was the recipient of a HSC Studentship and a HSF Traineeship.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 

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