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Am J Physiol Heart Circ Physiol 292: H1443-H1451, 2007. First published November 10, 2006; doi:10.1152/ajpheart.01056.2006
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Inhibition of PPAR-{alpha} activity in mice with cardiac-restricted expression of tumor necrosis factor: potential role of TGF-beta/Smad3

Kenichi Sekiguchi, Qi Tian, Masakuni Ishiyama, Jana Burchfield, Feng Gao, Douglas L. Mann, and Philip M. Barger

Winters Center for Heart Failure Research, Department of Medicine, Baylor College of Medicine, Houston, Texas

Submitted 26 September 2006 ; accepted in final form 6 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A shift in energy substrate utilization from fatty acids to glucose has been reported in failing hearts, resulting in improved oxygen efficiency yet perhaps also contributing to a state of energy deficiency. Peroxisome proliferator-activated receptor (PPAR)-{alpha}, the principal transcriptional regulator of cardiac fatty acid beta-oxidation (FAO) genes, is downregulated in heart failure, and this may contribute to reduced fatty acid utilization. Cardiomyopathic states are also accompanied by elevated levels of circulating cytokines, such as tumor necrosis factor (TNF), as well as increased local production of cytokines and profibrotic factors, such as transforming growth factor (TGF)-beta. However, whether these molecular pathways directly modulate cardiac energy metabolism and PPAR-{alpha} activity is not known. Therefore, FAO capacity and FAO gene expression were determined in mice with cardiac-restricted overexpression of TNF (MHCsTNF3). MHCsTNF3 hearts had significantly lower FAO capacity and decreased expression of PPAR-{alpha} and FAO target genes compared with control hearts. Surprisingly, TNF had little effect on PPAR-{alpha} activity and FAO rates in cultured ventricular myocytes, suggesting that TNF acts indirectly on myocyte FAO in vivo. We found that TGF-beta expression was upregulated in MHCsTNF3 hearts and that treatment of cultured myocytes with TGF-beta significantly suppressed FAO rates and directly impaired PPAR-{alpha} activity, a result reproduced by Smad3 overexpression. This work demonstrates that TGF-beta signaling pathways directly suppress PPAR-{alpha} activity and reduce FAO in cardiac myocytes, perhaps in response to locally elevated TNF. Although speculative, TGF-beta-driven repair mechanisms may also include the additional benefit of limiting FAO in injured myocardium.

metabolism; inflammation; genes; signal transduction

PRODUCTION OF ATP in the mammalian heart is a dynamic process, regulated by energy demand and substrate supply within the confines of normal cardiac physiology. When the heart becomes diseased, energy metabolic homeostasis is perturbed at many levels, including changes in the identity and quantity of energy substrate uptake and utilization, altered activity of metabolic enzymes, and abnormal expression of genes encoding the various components of the energy metabolic machinery. It remains controversial as to whether these events are simply another consequence of the pathophysiological remodeling that occurs with cardiac disease or whether altered energy production is an early event, contributing in a primary way to disease progression through such postulated mechanisms as lipo- or glucotoxicity (29, 32). Although the evidence for alterations in energy metabolic pathways with cardiac disease is clear, the molecular mechanisms and responsible signaling pathways remain poorly defined (4).

One metabolic pathway that has received significant attention in recent years is the peroxisome proliferator-activated receptor (PPAR)-{alpha} transcriptional pathway. Like other members of the nuclear hormone receptor superfamily, PPAR-{alpha} is a ligand-activated transcription factor and can be activated either by natural ligands, such as long-chain fatty acids, or synthetic compounds, such as the fibric acid class of lipid-lowering agents. The PPAR-{alpha} transcriptional complex serves as a master regulator of cardiac myocyte lipid flux via coordinate control of the expression of genes that encode enzymes and fatty acid transporters at all steps involved in myocellular fatty acid utilization, from uptake to mitochondrial fatty acid beta-oxidation (FAO) (reviewed in Ref. 4).

Various models of cardiac hypertrophy and left ventricular (LV) dysfunction have been used to investigate disease-related consequences with respect to cardiac energy metabolism and the PPAR-{alpha} signaling pathway. These studies have demonstrated that disruption of PPAR signaling occurs at multiple levels during pathological stress, including reduction in both PPAR-{alpha} RNA and protein levels in hypertrophied hearts induced by pressure overload (2, 27). In addition, in cultured ventricular myocytes, G protein-coupled receptor activation by phenylephrine (PE) induces ERK MAPK to target the PPAR-{alpha} complex, leading to both impaired activity of PPAR-{alpha} and repressed FAO capacity (2). Many other signaling pathways have been implicated in various pathological phenotypes in the heart, although whether these are also linked to direct effects on energy metabolism or PPAR-{alpha} activity is unknown.

Over the past decade, evidence has mounted that elevated levels of circulating cytokines accompany many cardiomyopathic states. Indeed, diseased myocardium produces cytokines locally, including tumor necrosis factor (TNF), with the potential for both autocrine and paracrine effects (5, 23, 31). TNF activity in the heart contributes to an enhanced inflammatory state and decreased contractile performance (5, 7, 12, 19, 20, 28, 36). The use of transgenic mouse lines with cardiac-restricted expression of various forms of TNF has led to a greater understanding of the role of TNF in altering the biology of the cardiac myocyte (7, 12, 20, 28). Little is known about whether TNF signaling directly modulates cardiac energy metabolism. This study was undertaken to determine whether TNF modulates FAO in the heart and, if so, to investigate the molecular mechanisms. We found that although transgenic mice with cardiac-restricted overexpression of TNF did indeed have a reduced capacity to perform FAO, TNF had only a modest effect on FAO rates in ventricular myocytes in vitro. Instead, we found that transforming growth factor (TGF)-beta, shown to be upregulated by TNF in vivo, not only led to reduced FAO in vitro but also led to a direct repression of PPAR-{alpha} activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal studies. Creation of transgenic mice with targeted cardiac overexpression of wild-type (WT) TNF (MHCsTNF3, C57BL/6 x ICR hybrid background) or TNF with the TNF-{alpha} cleavage enzyme (TACE) site removed (MHCmTNF, FVB background) has been described previously (9, 28). TNF levels in myocardial extracts and serum were determined by ELISA (9, 28), and LV function was assessed with a 1.4-Fr microtipped Millar catheter (Millar Instruments, Houston, TX) as previously described (26). For fasting studies, singly housed male littermate mice were fasted for 24 h while control mice were allowed ad libitum access to standard laboratory chow. All animal experimental protocols were reviewed and approved by the Animal Care Committee of Baylor College of Medicine.

FAO capacity in heart tissue. FAO capacity was measured by the method of Watanabe et al. (34). Hearts were homogenized in 0.25 M sucrose containing 1 mM EDTA. Five hundred micrograms of homogenate was incubated with the assay medium in 0.2 ml of 150 mM potassium chloride, 10 mM HEPES, pH 7.2, 0.1 mM EDTA, 1 mM potassium phosphate buffer, pH 7.2, 5 mM Tris malonate, 10 mM magnesium chloride, 1 mM carnitine, 0.15% bovine serum albumin, 5 mM ATP, and 50 µM [1-14C]palmitic acid (5.0 x 104 cpm of radioactive substrate). [1-14C]palmitic acid (54 mCi/mmol) was purchased from Perkin Elmer (Wellesley, MA). After 30 min at 25°C, the reaction was stopped by the addition of 0.2 ml of 0.6 N perchloric acid. After centrifugation at 2,000g for 10 min, the unreacted fatty acid was extracted with n-hexane. Radioactive degradation products in the water phase were counted, and FAO activity was expressed as counts per minute per milligram of heart tissue.

Neonatal rat ventricular myocyte culture. Cultures of 1- to 2-day-old neonatal rat ventricular myocytes were prepared as described previously (2). For gene expression studies, TNF (1,000 U/ml) or TGF-beta (20 ng/ml) (R&D Systems, Minneapolis, MN) was added 24 h after plating. Cells were harvested for RNA extraction at 48 h after agonist addition. Transient transfection assays were performed with Lipofectamine (Invitrogen, Carlsbad, CA) and the PPAR reporter plasmid MCPT.Luc.781 (Ref. 6; kindly provided by Dr. Daniel P. Kelly, Washington University School of Medicine). Twenty-four hours after transfection, cells were stimulated with TNF or TGF-beta. Forty-eight hours later, myocytes were harvested and luciferase activity was measured with the Promega Luciferase assay system and a Dynex MLX microtiter plate luminometer according to manufacturer's instructions.

Gene expression analysis. Total RNA was extracted from mouse hearts or rat neonatal cardiac myocytes. Twenty micrograms of total RNA was analyzed by ribonuclease protection assay (RPA). For making RPA templates, total RNA was extracted from mouse hearts or rat neonatal cardiac myocytes and reverse-transcribed with a 1st Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany). cDNA fragments were amplified with PCR primers as shown in Table 2 and then inserted into pCR 2.1 TOPO cloning vector (Invitrogen, Carlsbad, CA), followed by linearization for use as RPA templates. TGF-beta1 expression was determined with the mCK-3b RiboQuant MultiProbe template (Pharmingen, San Diego, CA). RPA was performed according to manufacturers' instructions (Pharmingen). [{gamma}-32P]UTP was purchased from Amersham Biosciences (Little Chalfont, UK).


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  		Table 2. PCR primers used to create RPA templates

 
FAO studies in vitro. Measurements of cellular FAO rates were performed as described previously (2). In brief, cardiac myocytes were prepared as above and then plated in equivalent numbers in T25 flasks. TNF, TGF-beta, or vehicle control exposure began at the switch to serum-free medium. Forty-eight hours later, the cells were given fresh TNF, TGF-beta, or vehicle-containing medium with [1-14C]palmitate, and no. 1 Whatman filter paper was suspended within each flask. The flasks were sealed, and 6 h later palmitate oxidation was terminated with 6 N hydrochloric acid. 14CO2 was collected overnight on the Whatman paper alkalinized with 2 N sodium hydroxide and quantified by scintillation counting.

Transient transfection assays in CV-1 cells. CV-1 cells were transfected by the calcium phosphate precipitation method as described previously (11). CDMPPAR{alpha} (Ref. 16; provided by Dr. Daniel P. Kelly); CDMRXR{alpha} (1) and UASTKLUC (3) (both provided by David Moore, Baylor College of Medicine); and GAL4PPAR{alpha} (3) have been described previously. Jae Lee (Baylor College of Medicine) provided the Smad3 expression vector.

Statistical analysis. Values are expressed as means ± SE. One-way analysis of variance was used to evaluate differences between groups. Where appropriate, post hoc testing with a Tukey test was performed to evaluate differences between the control and experimental groups. A P value <0.05 was accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Capacity for mitochondrial FAO. To determine whether cytokine-induced cardiomyopathy is accompanied by alterations in normal cardiac energy metabolism, we measured the capacity for mitochondrial FAO in cardiac homogenates from transgenic mice that overexpress TNF in a cardiac-restricted manner. The characteristics of 8-wk-old MHCsTNF3 mice are presented in Table 1. At this age, MHCsTNF3 animals exhibit concentric cardiac hypertrophy with normal systolic function and evidence of a relaxation abnormality based on increased {tau}. With the use of radiolabeled palmitic acid as an exogenous substrate, FAO capacity was measured in homogenates of hearts from MHCsTNF3 and WT littermate controls and showed that mean palmitate oxidation capacity was 53.9% lower in MHCsTNF3 than in WT hearts (P < 0.01; Fig. 1A).


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  		Table 1. Characterization of wild-type and MHCsTNF3 mice

 

Figure 1
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  		Fig. 1. Cardiac fatty acid beta-oxidation (FAO) capacity and FAO gene expression in MHCsTNF3 (sTNF) mice. A: capacity of cardiac homogenates to oxidize [1-14C]palmitic acid expressed as % of control wild-type (WT) hearts and normalized for cardiac mass. Data are presented as means ± SE; n ≥ 6. *P < 0.05 by unpaired Student's t-test compared with WT littermate controls. B: representative ribonuclease protection assay for cardiac expression of genes encoding transcriptional regulators and enzymes involved in cardiac energy metabolism. Large ribosomal protein L32 (L32) is used as an internal control for loading. TNF, tumor necrosis factor; PGC1{alpha}, peroxisome proliferator-activated receptor (PPAR)-{gamma} coactivator 1{alpha}; FAT, fatty acid translocase; VLCAD, very long-chain acyl-CoA dehydrogenase; MCPT-1, muscle-type carnitine palmitoyltransferase-1; ACO, acyl-CoA oxidase; UCP2, uncoupling protein 2; GLUT4, glucose transporter 4. C: collective gene expression data normalized to L32 as % of WT control. Data are presented as means ± SE; n ≥ 6. *P < 0.05 by unpaired Student's t-test compared with WT littermate controls.

 
FAO gene expression in MHCsTNF3 mouse hearts. To determine whether the reduced capacity for FAO was associated with altered FAO gene expression as has been seen in other models of cardiac hypertrophy, RPAs were performed on MHCsTNF3 and WT hearts. The expression of nuclear genes encoding the mitochondrial FAO enzymes (defined in Table 2) muscle-type carnitine palmitoyltransferase (MCPT)-1 and very long-chain acyl-CoA dehydrogenase (VLCAD), the peroxisomal enzyme acyl-CoA oxidase (ACO), and the fatty acid transporter fatty acid translocase (FAT) were all significantly reduced in MHCsTNF3 hearts (Fig. 1, B and C). In addition, the mRNA expression of PPAR-{alpha}, a known transcriptional regulator of these genes, and its coactivator, PPAR-{gamma} coactivator 1{alpha} (PGC1{alpha}), was also reduced in MHCsTNF3 hearts compared with WT controls (P < 0.05; representative autograph shown in Fig. 1B). The gene encoding another mitochondrial protein, uncoupling protein 2 (UCP2), was not altered in these mice. Interestingly, the expression of the gene encoding insulin-responsive glucose transporter (GLUT)4 was also significantly reduced in MHCsTNF3 hearts, suggesting that the expression of genes encoding proteins involved in both major energy production pathways is impaired in this model of cytokine-induced hypertrophy.

Response to metabolic stress. Given the reduced expression of PPAR-{alpha} and PPAR-{alpha} target genes in MHCsTNF3 hearts, we sought to determine whether these animals might be susceptible to metabolic stress as has been reported in PPAR-{alpha}–/– mice (22). We therefore subjected MHCsTNF3 mice and WT littermate controls to a 24-h fast, previously reported to cause acute upregulation of FAO capacity in WT mice via PPAR-{alpha}-mediated induction of FAO gene expression (22). RPA for representative FAO genes indicated that MHCsTNF3 transgenic animals not only have a lower basal expression of VLCAD and FAT but also exhibit a blunted transcriptional response to fasting (Fig. 2). Given the previously described role of PPAR-{alpha} in the normal fasting response in the heart (22), the question is raised as to whether TNF signaling alters PPAR-{alpha} activity in MHCsTNF3 hearts.


Figure 2
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  		Fig. 2. Effect of fasting on cardiac FAO gene expression in MHCsTNF3 mice. A: representative ribonuclease protection assay for cardiac expression of FAT and VLCAD in MHCsTNF3 and WT littermate controls subjected to 24 h of fasting (F). B: collective gene expression data normalized to L32 as % of WT control; n ≥ 6. Statistical analysis performed post hoc by Student-Newman-Keuls test. *P < 0.05. n.s. Not significant.

 
Myocyte response to TNF in vitro. To determine whether acute administration of TNF alters FAO and/or PPAR-{alpha} activity by a direct mechanism, FAO rates and FAO gene expression were assessed in cultured neonatal rat ventricular myocytes. For these experiments, myocytes were exposed to TNF or vehicle for 48 h in serum-free medium before addition of [1-14C]palmitate. Although FAO rates in TNF-treated cells were significantly lower than in control cells (P < 0.01; Fig. 3A), the absolute reduction was fairly modest compared with the FAO reduction seen in MHCsTNF3 hearts in vivo. We next examined whether short-term exposure of cultured myocytes to TNF would also lead to downregulation of gene expression for the PPAR-{alpha} target MCPT-1. Unexpectedly, MCPT-1 mRNA levels were not altered in myocytes exposed to TNF compared with vehicle-treated cells, either at baseline or in the presence of the PPAR-{alpha} agonist oleic acid, a known positive regulator of MCPT-1 (Fig. 3B). This suggests that endogenous PPAR-{alpha} activity is not impaired by acute administration of TNF and thus this mechanism does not account for the modest degree of reduction in FAO rates mediated by TNF exposure in vitro. This is in contrast to previously published reports with another hypertrophic stimulus, the {alpha}1-adrenergic agonist PE (2), which leads to a greater reduction in FAO in cultured myocytes than that produced by TNF in the present study. Also in contrast to TNF treatment, PE reduces both basal and fatty acid-induced MCPT-1 gene expression in hypertrophied myocytes (2).


Figure 3
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  		Fig. 3. FAO capacity, MCPT-1 gene expression, and MCPT-1 promoter activity in neonatal rat ventricular myocytes (NRVM) treated with TNF. A: capacity of TNF-treated NRVM in culture to oxidize [1-14C]palmitic acid expressed as % of control cells; n ≥ 6. *P < 0.05 by unpaired Student's t-test compared with vehicle-treated control cells. B: representative ribonuclease protection assay for MCPT-1 gene expression in NRVM treated with TNF, the PPAR-{alpha} agonist oleic acid (oleate), and/or vehicle control. C: transient transfection assay performed in NRVM utilizing a MCPT-1 gene promoter construct driving luciferase expression (MCPT.Luc.781). Cells were treated with TNF, oleic acid (oleate), and/or vehicle control. Bars represent mean ± SE luciferase activity [in relative luciferase units (RLU)] normalized (= 1) to the activity of MCPT.Luc.781 under basal conditions. Data represent means of at least 3 independent experiments, each performed in triplicate. *P < 0.05 by unpaired Student's t-test.

 
These observations were further confirmed by transient transfection assays using a reporter construct containing the PPAR-{alpha}-responsive human MCPT-1 gene promoter fused to a luciferase cDNA (MCPT.Luc.781; Fig. 3C). As previously published and consistent with the RPA study above, addition of oleic acid significantly increases the activity of the MCPT-1 promoter, a process dependent on PPAR (6). Addition of TNF to the cultured myocytes did not alter the activity of MCPT1.Luc.781 in the presence or absence of PPAR-{alpha} agonist, thus confirming that TNF does not alter PPAR-{alpha} activity or PPAR-{alpha} target gene expression when applied to neonatal myocytes in culture under these conditions.

Secreted vs. membrane-bound TNF transgenic animals. These results suggest that the reduction in FAO and expression of PPAR-{alpha} target genes in MHCsTNF3 hearts occurs through an indirect mechanism, i.e., one that is not mediated by the TNF signal transduction cascade that is activated by TNF binding to surface receptors on cardiac myocytes. To examine the possibility that TNF does not alter the cardiac myocyte metabolic phenotype through direct TNF signaling on the myocyte, we took advantage of another transgenic mouse line that expresses a modified TNF in the heart that cannot be secreted. MHCmTNF transgenic mice express TNF that lacks the TACE site and therefore can only signal in the myocyte population because the TNF remains membrane bound yet still activates canonical TNF signaling pathways (9). The characterization of MHCmTNF mouse hearts has been previously reported (9), and these mice show concentric cardiac hypertrophy with preserved systolic function. Given that direct application of TNF to myocytes in culture failed to significantly alter metabolic gene expression or FAO, the prediction was that FAO gene expression would be preserved in MHCmTNF mouse hearts. As can be seen in Fig. 4, A and B, the expression of MCPT-1 is unchanged in MHCmTNF hearts, even though the degree of cardiac hypertrophy in these animals is greater than that seen in the MHCsTNF3 animals (Fig. 4C). This suggests that, with respect to cardiac energy metabolism, TNF signals differentially in MHCmTNF vs. MHCsTNF3 animals and supports the concept that TNF alters myocyte FAO in MHCsTNF3 hearts via an indirect mechanism.


Figure 4
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  		Fig. 4. Comparison of MCPT-1 gene expression in the hearts of transgenic mice with cardiac-restricted expression of TNF, as either a membrane-bound or a secreted form. A: representative ribonuclease protection assay of MCPT-1 gene expression in hearts of MHCmTNF (mTNF), MHCsTNF3, or WT littermate control mice. B: collective MCPT-1 gene expression data from hearts of MHCmTNF or MHCsTNF3 mice normalized to L32 as % of WT control. Data are presented as means ± SE; n = 6. *P < 0.05 by unpaired Student's t-test compared with WT. C: heart weight (HW)-to-body weight (BW) ratio (mg/g) of the animals used in Fig. 5B. *P < 0.05 by unpaired Student's t-test.

 
TGF-beta expression in MHCsTHF3 hearts and in vitro myocyte response to TGF-beta. Given that direct effects of TNF on the cardiac myocyte could not account for the in vivo observations, we chose to examine potential involvement of secondary mediators known to lie downstream of TNF. A previously characterized separate line of MHCsTNF mice showed increased cardiac expression of TGF-beta (28) as well as angiotensin II (ANG II) (14), which itself has been shown to increase the expression and production of TGF-beta in nonmyocyte cardiac cells (13, 15, 21). RPA of TGF-beta expression in MHCsTNF3 hearts showed a 15-fold increase in mRNA expression in transgenic vs. control animals (Fig. 5A). To examine whether TGF-beta alters cardiac FAO or PPAR target gene expression in vitro, studies were repeated on cultured ventricular myocytes, substituting TGF-beta for TNF. TGF-beta-treated cells showed significantly lower FAO rates than control cells (Fig. 5B) and a greater reduction than that produced by TNF. Also in contrast to the in vitro TNF experiments, RPA demonstrated significant downregulation of MCPT-1 gene expression at baseline and an impairment of induction when the PPAR-{alpha} agonist oleic acid was added, similar to the observations from the in vivo fasting experiments (Fig. 5, C and D).


Figure 5
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  		Fig. 5. Transforming growth factor (TGF)-beta gene expression in hearts of MHCsTNF3 mice; FAO capacity and MCPT-1 gene expression in TGF-beta-treated NRVM. A: collective TGF-beta gene expression data in hearts from MHCsTNF3 mice normalized to L32 as fold vs. WT control. Data are presented as means ± SE; n ≥ 3. *P < 0.05 by unpaired Student's t-test compared with WT littermate controls. Inset: representative ribonuclease protection assay for TGF-beta gene expression. B: capacity of TGF-beta-treated NRVM to oxidize [1-14C]palmitic acid expressed as % of control cells; n ≥ 6. *P < 0.05 by unpaired Student's t-test compared with vehicle-treated controls. C: representative ribonuclease protection assay for MCPT-1 gene expression in NRVM treated with TGF-beta, oleic acid, and/or vehicle control. D: collective MCPT-1 gene expression data from NRVM treated with TGF-beta, oleic acid, and/or vehicle control normalized to L32 as % of controls. Data are presented as means ± SE; n = 6. *P < 0.05 by unpaired Student's t-test compared with vehicle-treated control cells.

 
To determine whether TGF-beta signaling directly alters PPAR-{alpha} transcriptional function, we performed transient transfection experiments initially in cultured cardiac myocytes. Consistent with the in vitro gene expression studies, TGF-beta suppressed both basal and oleate-induced transcriptional activation of the MCPT-1 promoter (Fig. 6A). To further determine whether TGF-beta directly alters PPAR-{alpha} activity, transient transfection assays were performed in CV-1 cells, which are null for PPAR-{alpha}. In this case, PPAR-{alpha} and its obligate heterodimeric partner retinoid X receptor (RXR)-{alpha} must be cotransfected with the reporter construct. Figure 6B demonstrates that TGF-beta markedly reduces both basal and oleic acid activation of PPAR-{alpha}/RXR-{alpha} heterodimers driving expression of MCPT1.Luc.781. To determine whether the effects were directed specifically at PPAR-{alpha}, a mammalian one-hybrid transfection assay was performed, using an expression vector encoding a fusion protein of full-length PPAR-{alpha} and the DNA-binding domain of the yeast transcription factor Gal4. Figure 6C shows that, similar to the PPAR/RXR heterodimer, Gal4-PPAR-{alpha} activity is repressed under basal and ligand-activated conditions by TGF-beta. This effectively confirms that TGF-beta signaling directly interferes with PPAR-{alpha} activity.


Figure 6
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  		Fig. 6. Effect of TGF-beta or Smad3 overexpression on MCPT-1 gene promoter and/or PPAR-{alpha} transcriptional complex activity. A: transient transfection assay performed in NRVM in culture utilizing MCPT.Luc.781. Cells were treated with TGF-beta, oleic acid, and/or vehicle control. Bars represent mean ± SE luciferase activity (in RLU) normalized (= 1) to the activity of MCPT.Luc.781 under basal conditions. B: transient transfection assay performed in CV-1 cells utilizing MCPT.Luc.781 and cotransfection of expression plasmids for PPAR-{alpha} and RXR-{alpha} in all conditions. Cells were treated with TGF-beta, oleic acid, and/or vehicle control. Bars represent mean ± SE luciferase activity (in RLU) normalized (= 1) to the activity of MCPT.Luc.781 + PPAR-{alpha} and RXR-{alpha} under basal conditions. C: transient transfection assay performed in CV-1 cells utilizing a Gal4-responsive reporter (UASTKLuc) and cotransfection of a construct expressing a Gal4-PPAR{alpha} fusion protein. Cells were treated with TGF-beta, oleic acid, and/or vehicle control. Bars represent mean ± SE luciferase activity (in RLU) normalized (= 1) to the activity of UASTKLuc + Gal4PPAR{alpha} under basal conditions. D: transient transfection assay performed in CV-1 cells utilizing MCPT.Luc.781 and cotransfection of expression plasmids for PPAR-{alpha}, RXR-{alpha}, and/or Smad3 as indicated. Cells were treated with oleic acid (oleate) or bovine serum albumin (BSA) vehicle control. Bars represent mean ± SE luciferase activity (in RLU) normalized (= 1) to the activity of MCPT.Luc.781 under basal conditions. E: transient transfection assay performed in CV-1 cells utilizing UASTKLuc and cotransfection of expression plasmids for Gal4-PPAR{alpha} and Smad3. Cells were treated with oleic acid or BSA vehicle control. Bars represent mean ± SE luciferase activity (in RLU) normalized (= 1) to the activity of UASTKLuc + Gal4-PPAR{alpha} under basal conditions. All data represent means of at least 3 independent experiments, each performed in triplicate. *P < 0.05 by unpaired Student's t-test.

 
To determine whether the canonical downstream effectors of TGF-beta, members of the Smad family of transcription factors, were responsible for the observed repression of PPAR-{alpha} activity, transient transfection experiments were repeated, utilizing cotransfection of a Smad3 expression vector in place of TGF-beta treatment. As can be seen in Fig. 6, D and E, cotransfection of Smad3 inhibited PPAR-{alpha}/RXR-{alpha} heterodimer activity on the MCPT-1 promoter to a similar degree as TGF-beta (Fig. 6D). This Smad3 inhibition likely occurs through direct interference with PPAR-{alpha} activity, as demonstrated by the reduction in ligand-mediated activation of Gal4-PPAR-{alpha} with coexpression of Smad3. Taken together, these data show that TGF-beta/Smad3 interferes with PPAR-{alpha} activation in cultured myocytes, leading to reduced PPAR-{alpha} target gene expression and lower rates of FAO.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The capacity of the heart to maintain normal levels of FAO is frequently impaired in animal models of cardiac hypertrophy and cardiomyopathy, such as pressure overload-induced cardiac hypertrophy secondary to aortic banding (37) or hypertension (8). The present study demonstrates that functional FAO capacity is suppressed in parallel with reduced expression of genes encoding mitochondrial FAO cycle enzymes in TNF-induced hypertrophy in vivo. We further show that this effect is apparently not through direct TNF action on ventricular myocytes. Our data suggest that a signal is generated downstream of TNF in a paracrine or endocrine fashion. One excellent candidate for this is TGF-beta, which we show is upregulated in MHCsTNF3 transgenic mouse hearts. Indeed, we show for the first time that TGF-beta potently suppresses FAO in cultured myocytes and, along with its downstream effector Smad3, directly impairs both endogenous and overexpressed PPAR-{alpha} activity.

Smads are downstream effectors of the TGF-beta family of signaling molecules and have previously been suggested to physically interact with PPAR-{alpha} (18). In that scenario, however, PPAR-{alpha} activation antagonized TGF-beta-induced beta5-integrin transcription in vascular smooth muscle cells. PPAR-{alpha} ligands inhibited TGF-beta-stimulated Sp1-Smad4 nuclear complex formation with a parallel induction of PPAR-{alpha}-Smad4 complexes. In a wound healing model using keratinocytes, TGF-beta-Smad3 has also been shown to antagonize TNF induction of PPAR-beta/{delta} expression via interference with AP-1 binding to the PPAR-beta/{delta} promoter, although no interaction between Smad3 and PPAR-beta/{delta} was reported (30). In addition, PPAR-{alpha} ligands have been demonstrated to inhibit induction of TGF-beta gene expression in several models, including in response to H2O2 (25) and ANG II (10), and in Dahl salt-sensitive rats (17). These observations suggest that PPAR-{alpha} may antagonize other transcriptional regulators that are involved in producing a TGF-beta response, underscoring the complex interrelationship and cross-regulation that occur between PPARs and inflammatory pathways. Although our data also indicate a reduction in PPAR-{alpha} mRNA expression in MHCsTNF3 transgenic mouse hearts, the mechanism for that observation is unknown and could not account for the repression seen in the transient transfection assays where PPAR-{alpha} was overexpressed. The present study is the first to demonstrate direct Smad interference with PPAR-{alpha} activity.

In the canonical TGF-beta signal transduction pathway, binding of TGF-beta ligand to a membrane receptor induces phosphorylation of a subset of receptor-regulated Smads, including Smad3, which then results in dimerization with the ubiquitous partner Smad4 and markedly enhances nuclear relocalization from the cytoplasm. In the unstimulated state, nonphosphorylated Smads undergo continuous shuttling between nucleus and cytoplasm (25). Thus, in our Smad overexpression studies, Smad3 would be expected to spend some time in the nuclear compartment, where it would be available for interfering with PPAR-{alpha} function. This also suggests that phosphorylation of Smad3 may not be required for this effect. Nevertheless, TGF-beta stimulation of cells in culture also led to the inhibition of PPAR-{alpha} activity and would be accompanied by a significant increase in Smad phosphorylation, thus implying that phosphorylated Smad can also antagonize PPAR-{alpha}. Sorting out these possibilities will require the use of phosphoresistant mutant Smads. Likewise, loss-of-function models will be necessary to determine whether the other receptor-regulated Smads also inhibit PPAR-{alpha} function and/or which endogenous Smad(s) may mediate the metabolic effects of TGF-beta in vivo. In addition, the specific molecular mechanism by which Smad3 interferes with PPAR-{alpha} activity remains to be determined. Possibilities include physical interaction with PPAR-{alpha} and active recruitment of a corepressor to the PPAR transcriptional complex or steric interference with PPAR ligand-mediated recruitment of the coactivator complex.

Although both TNF and TGF-beta are known to be produced by cardiac fibroblasts (24) and TGF-beta expression is upregulated in MHCsTNF3 transgenic mouse hearts, we have not identified the cellular source of TGF-beta expression in these animals. Furthermore, it is not known whether TNF induces cardiac fibroblasts to produce TGF-beta, although the predominance of currently available evidence suggests that TGF-beta production in the heart originates in the nonmyocyte population. TNF is known to stimulate TGF-beta expression in mouse lung fibroblasts (33), and this pathway has been suggested to play a role in interstitial pulmonary fibrosis, similar to the proposed role for TGF-beta in modulating cardiac fibrosis. ANG II has been demonstrated to be a potent inducer of both TGF-beta and endothelin-1 in cardiac fibroblasts (13, 15, 21), and coculture experiments indicate that ANG II-mediated stimulation of myocyte hypertrophy is significantly augmented by the presence of fibroblasts (15). Although relatively understudied, this latter observation highlights the role that cardiac fibroblasts play in paracrine signaling to the myocyte population in the heart. While our present studies do not directly link fibroblast-produced TGF-beta to altered cardiac myocyte energy metabolism or PPAR-{alpha} activity, it seems quite plausible that this pathway is active in vivo when TNF or ANG II signaling is upregulated in the heart.

The role of TGF-beta in the cardiac repair process following injury is mainly adaptive to counteract inflammation and promote wound healing. Although speculative, a TGF-beta-driven reduction in FAO and consequent upregulation of glucose utilization in the hypertrophied or injured heart would also be beneficial, as this results in lower oxygen requirements during the repair process. However, after healing and subsequent reduction in TGF-beta activity, a return to reliance on FAO would allow for greater production of ATP from this high-energy substrate. Thus the TGF-beta signaling cascade could potentially link repair mechanisms and energy metabolism in a coordinated adaptive response to injury.

In summary, we provide evidence that the cardiomyopathy that accompanies TNF overexpression includes an alteration in normal myocellular energy production that is mediated, at least in part, at the transcriptional level through antagonism of PPAR-{alpha} activity. We show that a potential mediator of this effect is TGF-beta, shown to be upregulated in MHCsTNF3 hearts and demonstrated here to directly impair PPAR-{alpha} function, an effect that is reproduced by overexpression of Smad3. This is the first report to link TGF-beta signaling to alterations in cardiac FAO and presents an alternative pathway to previously described G protein-coupled receptor-dependent pathways (2) known to remodel cardiac myocyte energy production capacity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded in part by National Heart, Lung, and Blood Institute Grant 5-R01-HL-071911 to P. M. Barger.


   ACKNOWLEDGMENTS
 
Present addresses: K. Sekiguchi, Dept. of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma, Japan 371-8511; M. Ishiyama, Heart Institute of Japan, Dept. of Cardiovascular Surgery, Tokyo Women's Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, Japan 162-8666; J. Burchfield, Molecular Cardiology, University of Frankfurt, Theodor Stern Kai 7, Haus 25 Floor 4, Room 414, 60590 Frankfurt am Main, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Barger, Winters Center for Heart Failure Research, Baylor College of Medicine, 6621 Fannin St., FC0450.01, Houston, TX 77030 (e-mail: pbarger{at}bcm.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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