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Departments of 1Surgery and 2Pediatrics, University of California, San Francisco, San Francisco, California
Submitted 12 December 2005 ; accepted in final form 7 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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promoter function; transcription factors; cardiac differentiation; cell specification; cardiac muscle hypertrophy
We use the cardiac troponin T (cTnT) gene promoter as a model of cardiac-specific gene expression during myocardial differentiation (3, 5, 22, 27, 29). The cTnT promoter is developmentally regulated by numerous promoter motifs and trans-acting factors, including MCAT or transcription enhancer factor-1 (TEF-1) binding sites, AT-rich/monocyte enhancer factor-2 (MEF-2) binding sites, GATA motifs, and Nkx2.5. The significance of the GC box for cTnT promoter function and the role of the Sp family of transcription factors in the regulation of cTnT promoter activity are not known. In this study, we use transient cotransfections of Sp expression constructs with cTnT promoter constructs in embryonic cardiomyocytes to define the trans-activating functions of Sp1 and Sp3. We show that Sp1 activates, whereas Sp3 represses, transiently transfected cTnT promoter constructs in cardiomyocyte cultures. Although the presence of cis elements in the regulation of promoter function has been well established in the avian embryonic cardiomyocyte system, we found it necessary to determine in vivo correlates between Sp1/Sp3 and sarcomeric protein expression. To determine the in vivo relevance of Sp factors to myocardial gene expression, we report their chromatin binding and the changing expression patterns of sarcomeric proteins and Sp factors in a clinically relevant and novel model of pathological cardiac hypertrophy.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Oligonucleotides were purchased from Operon Technologies (Alameda, CA) or Invitrogen (Carlsbad, CA). Media, serum, enzymes, reagents, and materials for tissue culture were purchased from the Cell Culture Facility at the University of California, San Francisco.
Plasmids and Constructs
The promoter/reporter constructs consist of 268 nucleotides of the chicken cTnT promoter upstream of the transcription initiation site (4). These promoter constructs were originally generated by nested deletions of the chicken cTnT gene in Bluescript KS+ linked to the chloramphenicol acetyltransferase reporter gene (pBS.CAT) containing +38 nucleotides of exon 1. Luciferase reporter constructs were created by digestion of the above-mentioned promoter inserts with XhoI, which is located in the polylinker, and NaeI, which cuts at nucleotide +15 of exon 1, and subcloned into the plasmid pGL2 (Promega, Madison, WI). Mutant cTnT promoter constructs were made using the Quick-Change site-directed mutagenesis kit (Stratagene) with each of the following forward primers and their antisense oligonucleotides as reverse primers: wild-type GC box primer (5'-GCTGCTCTGCCCGCCCCGGGGTGGGC-3') and mutant GC box primer (5'-GCTGCTCTGCAGATCTCGGGGTGGGC-3').
Briefly, 25 ng of double-strand DNA template (–268cTnTpGL2) and 125 ng of each respective oligonucleotide were used in the PCR according to the manufacturer's instructions. After temperature cycling (model PTC-200, MJ-Research, Boston, MA), parental DNA was cleaved by treatment with DpnI to improve the efficiency of mutant plasmid screening. The reaction mixture was transferred into one-shot competent cells (Invitrogen) on LB ampicillin plates.
Sp1 (pEVR2), Sp2 (pcCMV SPORT6), and Sp3 (pRC/CMV) expression constructs for cardiomyocyte cultures were kind gifts from Dr. Guntram Suske (Institut fur Molekular Biologie und Tumorforschung, Philipps-Universitaet Marburg). All constructs were confirmed by standard sequencing methods.
Nuclear Extracts
All tissues (cardiac muscle, skeletal muscle, fibroblasts, lung, liver, kidney, brain, and gizzard) were harvested from embryonic day 12 chicks at 4°C. Nuclear extracts were prepared using standard methods (Active Motif, Carlsbad, CA).
Protein Expression/Drosophila Schneider Cell Culture
Schneider cells were cultured according to previously reported techniques (28) and the manufacturer's protocol (Invitrogen). Sp1 (pPAC) and Sp3 (pPACUbx) constructs for expression in Drosophila SL2 cells were kind gifts from Dr. Guntram Suske. Schneider cell nuclear protein was prepared using the nuclear extract kit according to the manufacturer's protocols (Active Motif).
Mobility Shift, Competition, and Supershift Assays
Gel shift probes and competitors were made from 26-mer oligonucleotide fragments of wild-type cTnT-GC-box oligonucleotide (5'-GCTGCTCTGCCCGCCCCGGGGTGGGC-3') and mutant GC-box oligonucleotide (5'-GCTGCTCTGCAGATCTCGGGGTGGGC-3'). Probe labeling, DNA-protein binding reactions, and gel shifts were performed as previously described (14). Supershift assays were performed using rabbit anti-Sp1, -Sp2, -Sp3, and -Sp4 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).
Western Analysis
Total protein concentration in the samples was quantitated using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). An equal amount of protein was loaded in each lane for Western blots: nuclear proteins (10 µg) were separated by SDS-PAGE on a 10% gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in 130 mM NaCl and 25 mM Tris (pH 7.5) for 1 h at room temperature. After they were washed in Tris-buffered saline and 0.05% Tween 20 (TBST), the membranes were incubated in antibody (0.5–2.0 µg IgG/ml). The immunoblot was incubated at 4°C overnight and then washed in TBST. Goat anti-rabbit IgG peroxidase in TBST-2% goat serum was added to the incubation for 1 h at 4°C, and the immunoblot was washed in TBST. Chemiluminescence was used for visualization of bands. Parallel SDS gels and immunoblots of 
-actin and GAPDH controls were carried out to verify sample integrity and quantity.
To identify proteins in gel shift complexes (14), the mobility shift reactions were scaled up fivefold in a final volume of 20 µl. After electrophoresis, the gels were soaked in 2% SDS, 62.5 mM Tris, and 25 mM dithiothreitol and then dried. The DNA-protein complexes were identified by autoradiography, and the band of interest was excised and loaded onto a 10% SDS-polyacrylamide gel. The immunoblot was then carried out as described above.
Coimmunoprecipitation
The immune complexes were prepared using the Seize classic (A) immunoprecipitation kit (Pierce Biotechnology, Rockford, IL). Nuclear extracts or whole cell lysates were incubated with 5 µl of anti-Sp1 or -Sp3 antibody, control IgG or preimmune serum, or no antibody/antiserum in binding buffer overnight at 4°C (4). Membranes were probed separately with TEF-1 antibody (BD Transduction Laboratories), anti-divergent TEF-1 (DTEF-1) antiserum (Anaspec, San Jose, CA), poly(ADP ribose) polymerase (PARP) antibody, GATA antibody, MEF-2 antibody, Nkx2.5 antibody (Santa Cruz Biotechnology), and preimmune antiserum (AnaSpec).
Tissue Culture, Cell Transfection, and Reporter Gene Assay
Hearts were harvested from embryonic day 6–12 chicks, and cardiomyocyte cultures were established using standard techniques (5, 13–15, 18–22, 26). The differentiation state of cardiomyocytes was determined by light microscopy, presence of beating cardiomyocytes, immunohistochemistry for anti-sarcomeric myosin antibody MF-20, and Western blotting for myosins, cTnT, GATA-4, MEF-2, and Nkx2.5. Plasmid constructs were transfected according to the manufacturer's protocol (Effectene transfection kit, Qiagen) with minor modifications. Briefly, cultured cells were washed and resuspended in serum-free medium. –268cTnTpGL2 promoter constructs (300 ng) with wild-type and mutated GC box were transfected. Sp1, Sp2, and Sp3 expression constructs (see above) were cotransfected at concentrations of 2–2,000 ng in a total volume of 200 µl. cAMP-mediated activation of –268cTnT promoter constructs was determined by addition of 20 µM forskolin for 60 min to cardiomyocytes in culture. Reporter gene activity was determined using the luciferase reporter assay (Promega). Firefly light intensity was read on a luminometer (model TD-20/20, DLR Ready, Turner Designs Instrument, Sunnyvale, CA). Cotransfection with TK reporter was used to standardize for transfection variability. Cotransfection efficiency of Sp expression constructs was verified using pSV--Gal (Promega) and/or pAAV-GFP staining. Reporter gene activity data are expressed as means ± SD. Statistical comparisons were performed using paired t-tests, with level of significance set at 0.01.
Chromatin Immunoprecipitation
All chromatin immunoprecipitation (ChIP) assays were performed according to the protocols of the ChIP assay kit (Upstate, Lake Placid NY) (4). Five micrograms of primary antibody against Sp1, DTEF-1, GATA-4, and PARP were added to a 500-µl chromatin sample for the immunoprecipitation reaction. ChIP dilution buffer and preimmune rabbit serum were used as controls for nonspecific interactions and DNA contamination. Anti--actin and anti-cTnT antibodies were used as negative controls.
PCR
After DNA purification, samples were subjected to PCR with primers designed for the chick heart cTnT promoter (cTnT-268) as follows: 5'GCTGGCTGGCTTGTGTCA-3' (upper primer) and 5'-CTTGGGGGGCAGAGGCTTT-3' (lower primer). The primers used for PCR were designed by primer analysis software (Oligo 6.8, Molecular Biology Insights, Cascade, CO). The amplified PCR product is 265 bp.
Immunohistochemistry
Embryonic chick cardiomyocytes were prepared as described elsewhere (15). For immunofluorescent staining, the cells were plated in rat-tail collagen-coated (Roche Diagnostics, Indianapolis, IN; 50 mg/ml in 0.2% acetic acid) two-well chamber slides (4.2 cm2/well, Nalgene Nunc, Naperville, IL) at a seeding density of 0.1 x 106 cells and then cultured in a 5% CO2 incubator at 37°C for 72–96 h. After fixation in 0.2% paraformaldehyde for 10 min at room temperature, the cells were washed in PBS and then blocked in goat serum (Vector Laboratories, Burlingame CA) for 20 min at 37°C. The cells were incubated in primary antibody for 60 min in PBS with 1.5% normal blocking serum at 4°C overnight, washed, and then incubated with secondary antibody [goat anti-rabbit green fluorescent protein (Alexa Fluor 488), goat anti-mouse red fluorescent protein (Alexa Fluor 555), or rabbit anti-goat red fluorescent protein (Alexa Fluor 555) IgG antibodies, Molecular Probes, Eugene, OR], diluted in PBS with 1.5–3% normal blocking serum at 37°C for 1 h. The coverslip was mounted with aqueous mounting medium or 90% glycerol in PBS. Primary antibodies were as follows: rabbit anti-rat Sp1 (catalog no. sc-59), anti-human Sp2 (catalog no. sc-643), and anti-human Sp3 (catalog no. sc-644) polyclonal antibodies (Santa Cruz Biotechnology); mouse anti-chicken MF-20 antibody (Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-chick DTEF polyclonal antibody (AnaSpec); and goat anti-human cytokeratin (epithelial/preproepicardial cells) polyclonal antibody, goat anti-human caldesmon (smooth muscle) polyclonal antibody, and goat anti-human vimentin (fibroblasts and mesenchyme) polyclonal antibody (Santa Cruz Biotechnology).
Surgical Preparation and Care
Ewes. Five pregnant, mixed-breed Western ewes (135–140 days gestation, full term = 145 days) were operated on under sterile conditions. Through a left lateral fetal thoracotomy, an 8.0-mm Gore-Tex vascular graft (2 mm long; W. L. Gore, Milpitas, CA) was sewn between the ascending aorta and the main pulmonary artery (53). After surgery, the ewes were returned to their cages and allowed free access to food and water. The ewes were treated with 1 g of cefazolin and 100 mg of gentamicin sulfate intraoperatively and daily thereafter until 2 days after spontaneous delivery of the lambs.
Lambs. At 2 wk after spontaneous delivery, weight, respiratory rate, and heart rate were monitored daily. At 2 wk of life, 10 lambs (5 shunted and 5 age-matched controls) were sedated, induced [ketamine hydrochloride (0.3 mg·kg–1·min–1), diazepam (0.002 mg·kg–1·min–1), and fentanyl citrate (1.0 µg·kg–1·h–1)], intubated, and mechanically ventilated. The lambs were instrumented for measurement of cardiopulmonary dynamics as previously described (53).
All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco. All animals were euthanized using appropriate methods as described in the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals.
Heart Tissue Preparation
Whole heart and chamber-specific weights were measured in all shunted and control specimens. Samples of right atrium, right ventricle, left atrium, left ventricle, and ventricular septum were excised, weighed, and used to make fresh nuclear preparations. Whole tissue and excess nuclear preparations were snap frozen in liquid N2. Samples were stored at –70°C.
Determination of DNA Content
DNA content was determined using standard methods (53). Values were normalized to gram of cardiac muscle.
Statistical Analysis
Comparisons between shunt and age-matched controls were made by an unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Maximal expression of reporter gene driven by cTnT promoter constructs in vitro requires cis-elements within the 268-nt region upstream of the transcription initiation site (Fig. 1A). Within the –268cTnT promoter, tandem MCAT sites and an AT-rich/MEF-2 binding site have been shown to be essential for promoter activity. We sought to determine the contribution of the proximal GC box (nt –49 to –57), a putative Sp binding site, to promoter activity. Site-directed mutagenesis was used to generate promoter constructs with a mutated GC box. Transient transfections of embryonic cardiomyocyte cultures with mutated promoter constructs resulted in an 11-fold reduction in promoter activity relative to wild-type constructs that was statistically significant: 100% for –268cTnT and 9% for –268cTnT GC-box mutation (n = 12, P < 0.0001). Forskolin has a unique ability to stimulate adenylate cyclase activity and increase intracellular cAMP, thus activating cAMP-dependent protein kinase and other cAMP receptor proteins. Addition of forskolin results in a four- to fivefold increase in reporter gene activity. cAMP-mediated activation of the cTnT promoter requires an intact GC box, because addition of forskolin to GC-box-mutated promoters did not increase promoter activity (Fig. 1B).
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Gel retardation and supershift assays were performed using embryonic chick cardiac nuclear extracts, GC-box DNA probes (26-mer), and antibodies specific to Sp1, Sp2, Sp3, and Sp4. The cTnT GC box binds cardiac nuclear protein activities, generating two mobility shift complexes: C1 and C2 (Fig. 2, lane 2). Addition of 50- to 100-fold excess of wild-type unlabeled DNA results in competition of the radiolabeled complexes (Fig. 2, lanes 3 and 4). There is no competition when excess mutant DNA competitor is added to the DNA-protein binding reaction (Fig. 2, lanes 5 and 6), suggesting that GC-box activities bind the cTnT promoter motif in a sequence-specific fashion. Addition of Sp1, Sp2, or Sp3 antibody (Fig. 2, lanes 7–9) results in supershifting of complexes, whereas addition of Sp4 antibody to the binding reaction does not (Fig. 2, lane 10).
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Expression Patterns of Sp Factors in Embryonic Chick Nuclear Extracts
The protein expression profiles of Sp family members in avian embryonic tissues were determined using antibodies specific for Sp1, Sp2, Sp3, and Sp4 (Fig. 5). Sp1 is expressed as a 95- or 106-kDa isoprotein, and each is widely distributed in various embryonic tissues, including heart, skeletal muscle, skin, lung, liver, gizzard, kidney, and brain. In heart and skeletal muscle, the 95-kDa isoform is expressed at higher levels than the 106-kDa protein. In gizzard, the 106-kDa isoform is highly expressed and the 95-kDa isoform is not detected. Expression of Sp2 is tissue restricted. Sp2 is expressed as an 80-kDa protein in heart, fibroblasts, and gizzard but is not detected in other avian embryonic nuclear extracts.
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Sp1 Binds the cTnT Promoter "In Vivo" Within the Context of Chromatin
Having shown that Sp1 is present in cardiac nuclear extracts and contributes to GC-box binding activities, we sought to determine whether Sp1 binds the endogenous or chromosomal cTnT promoter in vivo within the context of chromatin (Fig. 6). After it was subjected to cross-linking and shearing, chromatin from embryonic chick heart was immunoprecipitated with Sp antibody and DTEF, GATA, and PARP antibodies (positive controls), as well as troponin and -actin antibody and preimmune serum (negative controls). The immunoprecipitated complexes were eluted, and protein DNA cross-links were reversed. DNA from the immunoprecipitated sample was then purified and subjected to PCR using primers specific to the cTnT promoter (265 bp). Antibody to Sp1, DTEF-1, PARP, and GATA factors immunoprecipitates chromatin from cardiac nuclear extracts, which produces a PCR fragment spanning the 265-bp region upstream of the transcription initiation site, implying that these factors bind the cTnT promoter in vivo.
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To determine the transcriptional effects of Sp factors on cTnT promoter activity, Sp expression constructs and cTnT promoter constructs were subjected to cotransfections in embryonic chick cardiomyocytes (Fig. 7). Cotransfection of Sp1 (5 ng, n = 22) resulted in a moderate, statistically significant superactivation of promoter activity above baseline: 100% for 268 cTnT and 171% for 268 cTnT + Sp1 (n = 22, P 
0.0001). Sp3, however, had an inhibitory effect on promoter activity, whereas Sp2 also produced a moderate increase in promoter activity: 100% for 268cTnT and 32% for 268cTnT + Sp3 (n = 10, P < 0.001) and 149% for 268cTnT + Sp2 (n = 18, P = 0.01). The activating effect of Sp1 was inhibited by cotransfection with Sp3 (Fig. 7A): 171% for –268cTnT + Sp1 (n = 22) and 89% for –268cTnT + Sp1 + Sp3 (n = 12). Increasing doses of Sp3 expression construct resulted in greater degrees of inhibition (Fig. 7B).
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Western blot and electrophoretic mobility shift analyses demonstrate that Sp3 is expressed in whole embryonic chick hearts. Paradoxically, expressed Sp3 inhibits reporter gene expression driven by cTnT promoter constructs that are transiently cotransfected in embryonic chick cardiomyocytes. Numerous cell types, including myocytes, fibroblasts, endothelial cells, epicardial-epithelial cells, and smooth muscle cells, populate the heart. To determine the cell-specific expression of Sp3, embryonic cardiomyocytes in cell culture were stained for immunocytochemistry using various cell-specific antibodies, including MF-20, vimentin, cytokeratin, caldesmon, and PCAM, and the results correlated with cellular colocalization of Sp1/Sp3 immunostaining (Fig. 8).
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In Vivo Model of Pathological Cardiac Hypertrophy: Hemodynamics
Hemodynamic parameters for control and shunted lambs at 2 wk of postnatal life are summarized in Table 1. In 2-wk-old lambs with large left-to-right shunts, pulmonary artery, right atrial, and left atrial pressures were significantly elevated. The overall cardiac output in shunted animals was also significantly increased. Mean blood pressure was comparable in both groups.
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Hearts were significantly larger in shunted animals than in nonshunted controls (Fig. 10). The right atrium (P = 0.003), left atrium (P = 0.001), right ventricle (P = 0.004), and left ventricle (P = 0.02) were significantly larger in shunted than in control animals. Hearts of shunted animals weighed nearly twice as much as those of controls. Septal weight and size did not significantly differ between groups. Western blot analysis of chamber-specific tissue was performed for shunted (n = 5) and control hearts (n = 5), and band density was scanned to assess relative protein levels of myosins, cTnT, and Sp factors. In a Western blot of sarcomeric and Sp protein levels in atria and ventricles, no significant difference in GAPDH or -actin expression levels was shown between control and shunted animals (Fig. 11). Myosin expression was significantly increased in shunted hearts within atria and ventricles. cTnT protein levels were also significantly increased in atria and ventricles. In addition to increased cTnT protein levels, the embryonic/fetal high-molecular-weight isoform was upregulated in the shunted hypertrophied hearts.
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Densitometric analysis of Sp1 protein levels were significantly increased in all chambers (except the septum) after shunting, volume loading of the heart, and development of pathological cardiac hypertrophy. Concomitant with an increase in Sp1 protein levels, Sp3 expression levels decreased in all four cardiac chambers.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Sp1 Activates the –268cTnT Promoter
The necessity of the GC box for cTnT promoter activity is consistent with other reports in the literature which suggest that cardiac promoters require Sp1 binding elements for activity. The human and murine cardiac troponin I promoters require their respective GC elements for promoter activity (9, 12). Mutation of murine GC/GA elements within the cardiac troponin I promoter produces a four- to fivefold reduction of promoter activity comparable to that seen for the avian cTnT promoter. Putative Sp1 promoter elements have also been shown to be required for optimal activity of cardiac and skeletal 
-actin, sarco(endo)plasmic Ca2+-ATPase, and Na+-K+-ATPase 1-subunit genes in cultured cells (6, 16, 24, 31, 33). These studies do not show direct trans-activation of promoter constructs cotransfected with expressed Sp factors. In the present study, we show that the GC box within the cTnT promoter is necessary for basal and cAMP-mediated promoter activity and bound by Sp factors within the context of chromatin in vivo. The cAMP responsiveness of cTnT promoter constructs is abolished by mutation of the Sp1 binding site, similar to that seen for the CYP11A gene (1). We extend the evidence supporting a role for Sp1 in cardiac muscle gene activation by demonstrating that Sp1 directly activates cTnT promoter constructs when cotransfected in primary cardiomyocyte cultures. Control of tissue-specific activation of the cardiac genes probably requires combinatorial cis-acting elements and their respective trans-acting factors. Gel shift and coimmunoprecipitation experiments suggest protein interactions between Sp and other cardiac transcription factors. Elution of proteins from mobility shift complexes formed by the binding reaction between cardiac nuclei and GC-box DNA elements shows that TEF-1, PARP, and Nkx2.5 contribute to GC-box binding activities, probably through their interaction with Sp1. Coimmunoprecipitation experiments confirmed interactions between Sp1 and DTEF-1, PARP, and Nkx2.5 (data not shown) but not MEF-2 or GATA-4. The protein interactions are predictable on the basis of the proximity of the respective cis elements within the promoter to each other. The cis elements for TEF-1, Nkx2.5, PARP, and Sp1 are close to each other, supporting any potential protein-protein interactions. The binding sites for MEF-2 and GATA factors are more distant (
200 bp away) within the promoter.
Sp3 Represses –268cTnT Promoter Activity
Reports on the transcriptional functions of Sp3 have shown that Sp3 can act as an activator or a repressor. Whether Sp3 functions as an activator or inhibitor of gene expression depends partly on posttranslational modifications of the protein (2), the number of GC-box binding sites (8, 28) within a particular promoter, and the cell type within which Sp3 is expressed (25). It has been reported that promoters containing a single binding site are activated, whereas promoters containing multiple binding sites often are not activated or respond weakly to Sp3 (7, 10, 11). This model is in contradistinction to cTnT promoter responsiveness to the Sp family of factors, because the cTnT promoter contains a single GC-box element and is inhibited by Sp3 coexpression. The inhibitory effect was dose dependent. Higher concentrations of Sp3 resulted in greater degrees of inhibition of Sp1-mediated promoter activation, presumably by competing for Sp1 binding sites (17). The cell-specific expression of Sp3 within the heart is consistent with its transcriptional effects on –268cTnT promoter constructs. Endogenous Sp3 was not detected in embryonic cardiomyocytes by immunocytochemical staining, but endogenous Sp3 expression was detected in the fibroblast, smooth muscle cells, endothelial cells, and epicardial-epithelial cells of the avian embryonic heart.
GC-box DNA elements and Sp transcription factors have also been implicated in the hypertrophic response in cardiomyocytes stimulated by 1-adrenergic agonists (24). Fetal sarcomeric and fetal metabolic gene programs are activated by -agonists and seem to require Sp elements combined with increased binding of their respective factors Sp1 and Sp3. Sp factors have also been implicated in skeletal muscle hypertrophy and inactivity (30). In adult skeletal muscle, -myosin heavy chain (-MHC) gene expression is primarily restricted to slow-twitch type I fibers; however, its expression is downregulated in response to muscle inactivity. Tsika et al. (30) showed that, under non-weight-bearing conditions, downregulation of -MHC gene expression is associated with increased binding of Sp3 to GC-rich elements. Conversely, binding of Sp3 to these elements decreased, whereas Sp1 binding increased, with nuclear extracts from plantaris muscle exposed to mechanical overload, a stimulus that increases -MHC gene expression. Sp3 proteins acted as competitive inhibitors of Sp1-mediated -MHC reporter gene trans-activation in Drosophila SL-2 and mouse C2C12 myotubes. Our cell culture studies indicate that Sp3 acts as a repressor of cardiac promoters. Because 1) Sp3 downregulation is associated with mechanical overload in skeletal muscle, 2) Sp3 represses cardiac promoter function in cultured cells, and 3) Sp elements are implicated in the cardiac hypertrophic program, we postulated that Sp factors have a role in myocardial hypertrophy in vivo.
Placement of a large aortopulmonary shunt in lambs produces significant left-to-right shunting, increases in pulmonary blood flow, volume loading of the left ventricle, pulmonary hypertension, and pressure loading of the right ventricle. The model used in this study is clinically relevant. Volume loading of the left ventricle is common with valvular heart disease, such as aortic and mitral insufficiency. Right ventricular pressure overload is also commonly seen clinically, as in pulmonary hypertension, tetralogy of Fallot, and pulmonary stenosis. Patent ductus arteriosus, large ventricular septal defects, atrial septal defects, and aortopulmonary windows produce significant left-to-right shunts and pathophysiology similar to that in the shunted lamb model. The right and left ventricles undergo concentric and eccentric hypertrophy, respectively. Both chambers are enlarged and thickened (right more than left). Surgically created aortopulmonary shunts are also common for the management of single ventricle anomalies with inadequate pulmonary blood flow. The resultant pathophysiology includes volume loading of the ventricle.
Sp3 Is Expressed in Ovine Cardiomyocytes and Is Downregulated During Pathological Cardiac Hypertrophy
In the lamb model of aortopulmonary shunt, the heart is enlarged and hypertrophied after 2 wk of left-to-right shunting, with volume loading of the left ventricle and pressure loading of the right ventricle. Western blotting shows increased myosin and troponin T expression in both ventricles, concomitant with significant chamber enlargement and wall thickening. The hypertrophic response, in vivo, in a clinically relevant model of ventricular hypertrophy includes an increase in Sp1 expression and a simultaneous decrease in Sp3 expression. This is consistent with the findings that Sp3 inhibits, whereas Sp1 activates, the cTnT promoter; however, these data do not provide direct evidence that Sp1 upregulation and Sp3 downregulation, in vivo, are mechanistically responsible, in part, for the changes in sarcomeric protein expression during pathological cardiac hypertrophy in the ovine model. In sheep, Sp3 localizes to the myocyte population of the heart under normal and shunted conditions (data not shown). The mechanism by which biomechanical forces produce differential expression and/or activity of Sp factors remains to be elucidated. Numerous kinases are implicated in the transduction of a variety of hypertrophic stimuli, ultimately leading to the hypertrophic phenotype and implicating common transcriptional regulatory mechanisms. In the hypertrophied ventricle of a shunted lamb, increased levels of TEF-1, Nkx2.5, and GATA factors were found, implicating combinatorial interactions to produce the hypertrophic response (data not shown). Histone and transcription factor acetylation is also implicated in regulating gene expression during myocardial hypertrophy and may play a role in modulating activities of Sp factors (23, 32).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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1-adrenergic induction of the skeletal -actin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth. J Biol Chem 270: 410–417, 1995.-actin promoter by helix-loop-helix proteins. Proc Natl Acad Sci USA 89: 4047–4051, 1992.-myosin heavy chain gene expression during skeletal muscle inactivity. Mol Cell Biol 24: 10777–10791, 2004.1 transcription by hyperoxia is mediated by SP1/SP3 binding. J Biol Chem 275: 41396–41404, 2000.1 subunit gene transcription by low external potassium in cardiac myocytes. Role of Sp1 and Sp3. J Biol Chem 275: 24173–24184, 2000.This article has been cited by other articles:
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  T. Hucl, J. R. Brody, E. Gallmeier, C. A. Iacobuzio-Donahue, I. K. Farrance, and S. E. Kern High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor Dependent MCAT Motif Cancer Res., October 1, 2007; 67(19): 9055 - 9065. [Abstract] [Full Text] [PDF]
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 W. K. Steagall, J.-P. Lin, and J. Moss The C/A(-18) polymorphism in the surfactant protein B gene influences transcription and protein levels of surfactant protein B Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L448 - L453. [Abstract] [Full Text] [PDF]
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 A. Azakie, J. R. Fineman, and Y. He Myocardial transcription factors are modulated during pathologic cardiac hypertrophy in vivo J. Thorac. Cardiovasc. Surg., December 1, 2006; 132(6): 1262 - 1271. [Abstract] [Full Text] [PDF]
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