Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expr
http://www.100md.com
《循环研究杂志》
the Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tenn.
Abstract
Elevated plasma levels of plasminogen activator inhibitor type I (PAI-1), a significant risk factor of ischemic heart disease, are associated with insulin resistance in which insulin and transforming growth factor (TGF)- play a pivotal role in regulating PAI-1 production. Forkhead transcription factor FOXC2 is an important regulator of insulin resistance. However, the underlying molecular mechanisms to link FOXC2 to PAI-1 levels in insulin resistance remain to be elucidated. Here, we demonstrate that Foxc2 is a common transcriptional activator of insulin and TGF- signaling to directly regulate PAI-1 expression via 2 distinct target sites, an insulin response element (IRE) and a novel forkhead-binding element (FBE), adjacent to a Smad-binding site. We found that in adipocytes and endothelial cells Foxc2 mediates insulin action competing with another Forkhead protein, FOXO1, via the insulin response element, and simultaneously cooperate with the TGF-/Smad pathway to transactivate PAI-1. Importantly, Foxc2 haploinsufficiency in mice significantly attenuates TGF-1eCinduced PAI-1 expression in the cardiovascular system and adipose tissue. Taken together, we propose that Foxc2 is a key molecule to regulate PAI-1 gene expression.
Key Words: plasminogen activator inhibitor-1 promoter insulin response element TGF- response site forkhead transcription factor
Introduction
Insulin resistance, defined as an attenuated responsiveness to insulin, is widely recognized as a key component of prediabetic state and obesity. Clinical studies have identified a strong link between insulin resistance and ischemic heart disease.1,2 Nevertheless, little is known about the underlying mechanisms to elucidate their precise interaction.
Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation, and animal studies have identified PAI-1 as a mediator of ischemic heart disease in obesity.3 PAI-1 is synthesized in many tissues, including vascular endothelium and adipose tissue.3eC5 Elevated plasma levels of PAI-1 are strongly associated with increased risk of ischemic heart disease.2,6 Plasma levels of PAI-1 are determined by genetic, hormonal,7,8 circadian,9,10 and metabolic factors, such as fatty acids11 and glucose.12
In the context of insulin resistance, as well as obesity and type 2 diabetes mellitus, several signaling pathways are known to play important roles in regulating PAI-1 gene expression. Insulin per se induces PAI-1 even in insulin-resistant adipocytes and insulin-resistant mice.13 Transforming growth factor (TGF)- levels are elevated in adipose tissues and TGF- administration increases PAI-1 in genetically obese (ob/ob) mice.14 Elevated levels of tumor necrosis factor also increase PAI-1 levels in adipose tissues of obese mice.15 Importantly, inflammatory process has recently emerged as a critical biological mechanism underlying obesity-related insulin resistance.16,17 However, the mechanisms underlying transcriptional regulation of PAI-1 in insulin resistance are not well understood.
Forkhead box (Fox) proteins constitute a large family of transcription factors that share an evolutionarily conserved DNA-binding domain with highly divergent flanking regions, leading to pleiotropic functions, such as the control of metabolism.18 Based on the homology of DNA-binding domains, they are classified into 17 subfamilies (subclasses A through Q), where the abbreviation contains all uppercase letters for human (eg, FOXC2), only the first letter capitalized for mouse (eg, Foxc2), and the first and subclass letters capitalized for all the chordates (eg, FoxC2). There is extensive evidence that Fox transcription factors are components of several signaling pathways, such as insulin.18 FoxO acts downstream of insulin signaling, and Akt-dependent phosphorylation of FoxO results in its exclusion from the nucleus.18eC21 Interestingly, FOXO1 was previously shown to bind to the insulin response element (IRE) of the PAI-1 promoter, although it had little effect on insulin-driven PAI-1 expression.22,23 This suggests that another Fox protein(s) plays a role in insulin-mediated induction of the PAI-1 gene.
FOXC2 is shown to be upregulated by insulin signaling in adipocytes and functions as a key regulator in adipose tissues in counteracting insulin resistance in adult mice as well as human subjects of insulin resistance or obesity.24,25 FOXC2 upregulates the transcription of the RI subunit of protein kinase A (PKA) to augment the -adrenergiceCcAMPeCPKA response, leading to increased sensitivity to insulin.26 We have previously shown that Foxc2, in cooperation with a closely related Foxc, Foxc1, plays pivotal roles in the development of cardiovascular, ocular, and genitourinary systems.27eC31
In the present study, we demonstrate that Foxc2 directly transactivates the PAI-1 promoter via 2 Fox-binding sites, IRE and a novel forkhead-binding element (FBE), which are associated with the insulin and TGF- signaling pathways, respectively. These studies led us to propose that Foxc2 mediates insulin and TGF- signaling to regulate PAI-1 gene expression. This mechanistic model provides new insights into the potential molecular pathways of cardiovascular disease in patients with insulin resistance.
Materials and Methods
Cell Culture
Bovine arterial endothelial cells (BAECs) were isolated and maintained as described previously.10 Human microvascular endothelial cells (HMEC-1)32 were cultured in Endothelial Cell Basal Medium-2 (Clonetics). 3T3-L1 (American Type Culture Collection) and COS-7 cells as well as mouse embryonic endothelial cells (MEECs) were maintained in DMEM supplemented with 10% FBS.
Plasmid Constructs
We amplified the full-length and truncated forms of Foxc1 and Foxc2 as well as full-length FOXO1 by PCR using Pfu Turbo (Stratagene) and primers (supplemental Table IA in the online data supplement available at http://circres.ahajournals.org) and cloned them into either the GAL4-fusion expression vector pSG42433 or pcDNA3.0 (Invitrogen). Mutated reporters for pGLuc884 were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and primers (supplemental Table IB). Expression vectors for Smad proteins and PAI-1eCpromotereCluciferase reporter vectors (pGLuc884, pGLuc107, and pGLuc85) have been described previously.34,35
Transfection and Luciferase Assay
BAECs or 3T3-L1 cells were plated at a density of 5x104 to 1x105 cells/mL into 12-well tissue culture plates and were subjected to transfection as described previously.10 Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) at 48 hours after transfection. For insulin treatment, BAECs or 3T3-L1 cells were cultured in DMEM with or without 10 e/mL human recombinant insulin (R&D) at 24 hours after transfection. Each transfection was performed in triplicate and replicated at least 3 times.
TGF-1 Treatment for Mice
Foxc2+/eC mice were maintained and genotyped as previously described.27,36 We injected age- and sex-matched wild-type (n=10) or Foxc2+/eC (n=10) mice with 25 e/kg human recombinant TGF-1 (R&D) (n=5 for each genotype) or saline (n=5 for each genotype) as a negative control through tail vein. Four hours after injection, organs and tissues were snap-frozen in liquid nitrogen, as described previously.37
Real-Time Quantitative RT-PCR
We extracted total RNA from tissues using the RNeasy Kit (Qiagen) and 1 e of mRNAs were reverse transcribed using random hexamers with Universal R.T. Reagents (ABI). We used SYBR Green PCR Master Mix (ABI) and primers (supplemental Table IE) on an i-Cycler instrument (Bio-Rad), as described previously.37 The 2eCCT method was used for analysis.38 Primers used are shown in supplemental Table IE.
An expanded Materials and Methods section can be found in the online data supplement.
Results
Foxc2 Activates the PAI-1 Promoter In Vitro
To determine whether Foxc1 or Foxc2 can induce PAI-1 expression directly, we first examined the transcriptional activity of Foxc proteins using the human PAI-1 promoter in BAECs (Figure 1A and 1B). We found that Foxc2 could transactivate the eC884-bp promoter in a dose-dependent manner, whereas Foxc1 failed to show significant activation of the PAI-1 promoter (Figure 1A). As shown in Figure 1B, we systematically examined Foxc2-mediated luciferase activity using a series of deletion constructs of the human PAI-1 promoter.34 All constructs contain the IRE, which is shown to be a FOXO1-binding site,23 although pGLuc884 also has TGF-eCresponsive sequences (TRSs).39 Foxc2-responsiveness was evident in pGLuc884 and pGLuc107 and was still retained in the shortest construct (pGLuc85) containing the IRE. Based on analysis of functional domains in Foxc (supplemental Figure IA through ID), we generated constitutively active forms of Foxc1 and Foxc2 (caFoxc1 and caFoxc2, respectively), which consist of the activation domain 1 (AD1) and forkhead DNA-binding domain (FHD) (supplemental Figure IE). Significantly, transfection of caFoxc2 resulted in considerably enhanced activation of the PAI-1 promoter in BAECs (Figure 1B), although similar induction by caFoxc1 was observed (data not shown). It should be noted that PAI-1 promoter activity was more than 85-fold using caFoxc2 in pGLuc884, whereas it remained approximately 70-fold with pGLuc107 and pGLuc85, suggesting that there are multiple Foxc2-responsive sites in the PAI-1 promoter (see below).
We further tested the effects of Foxc on the activation of the PAI-1 promoter in murine preadipocytic 3T3-L1 cell line (Figure 1C). In contrast to BAECs, caFoxc2 showed only 6.2-fold induction in 3T3-L1 cells, whereas basal luciferase activity of the PAI-1 promoter was 25-fold higher in 3T3-L1 cells than in COS-7 cells. We found that the FoxC2 protein is highly expressed in 3T3-L1 cells compared with other cell lines, such as BAECs and COS-7 (supplemental Figure II), suggesting that PAI-1 expression is constitutively upregulated by endogenous Foxc2 in 3T3-L1 cells. As shown in Figure 1C, this observation was supported by the finding that basal luciferase activity was reduced in a dose-dependent manner by a dominant-negative form of Foxc2 (dnFoxc2), which consists of the FHD (supplemental Figure IE).
Foxc2 Regulates the PAI-1 Promoter via Two Fox-Binding Sites
In addition to the previously reported IRE,23 we found another Fox-binding element (FBE) located at eC723 bp. This site is adjacent to 1 of the TRSs (Figure 2A) and has recently been shown to be a putative FoxD1-binding site.40 Because FoxC1, FoxC2, and FoxO1 are coexpressed in endothelial and preadipocytic cell lines (supplemental Figure II), we examined whether these Fox proteins directly bind to the Fox-binding sites on the PAI-1 promoter (Figure 2B). Specific complexes were formed between IRE or FBE probe and recombinant Fox proteins. The specificity of these complexes was confirmed by demonstrating their disappearance in the presence of unlabeled corresponding probes. In addition, no complexes were detected between the Foxc proteins and either of the 2 mutated oligonucleotides (IREmut and FBEmut). The intensity of the bands was the strongest using Foxc2, suggesting that Foxc2 can bind both FBE and IRE more efficiently than Foxc1 or FOXO1. To further confirm the significance of the FBE and IRE, we tested luciferase activity of mutant reporter constructs in which the FBE and/or IRE are disrupted (Figure 2C). Although pGLuc884IREmut or pGLuc884FBEmut was transactivated by Foxc2 significantly less than pGLuc884 in BAECs, pGLuc884BOTHmut showed substantially low transactivation by Foxc2. These results indicate that both FBE and IRE are responsible for Foxc2-mediated transactivation of the PAI-1 promoter.
FOXO1 Antagonizes Foxc2 to Regulate the PAI-1 Promoter
Although FOXO1 binds to the IRE, it has little effect on transcription of PAI-1 in response to insulin.23 Because Foxc2 can transactivate the PAI-1 promoter via this site (Figure 2B and 2C), it is possible that FOXO1 competes with Foxc2 to suppress the activation of the PAI-1 promoter. Indeed, transactivation of the shortest promoter by Foxc2 was significantly attenuated when FOXO1 was coexpressed in BAECs (Figure 3A).
Because FOXO1 is a downstream component of insulin signaling and is excluded from the nucleus in response to insulin,19 we tested the functional interaction between Foxc2 and FOXO1 in the insulin-mediated induction of PAI-1 using BAECs and 3T3-L1 cells that respond to insulin (Figure 3B and 3C). The attenuation of Foxc2-mediated PAI-1 induction by FOXO1 was significantly abolished in the presence of insulin. These results indicate that activation of insulin signaling prevents FOXO1 from competing with Foxc2, resulting in PAI-1 induction in endothelial and preadipocytic cells. Moreover, we tested the effects of mutations of the IRE and FBE on insulin action in BAECs. The results using pGLuc884FBEmut were similar to those of pGLuc85 (Figure 3B and 3D), indicating that the FBE is dispensable for insulin action on the PAI-1 promoter. In contrast, neither pGLuc884IREmut nor pGLuc884BOTHmut exhibited significant changes in transactivation by addition of FOXO1 or insulin. These results together demonstrate that FOXO1 functionally competes with Foxc2 via the IRE on the PAI-1 promoter.
Foxc2 Is Associated With the TGF-/Smad Pathway
Because Smad3 and Smad4 mediate TGF- signaling through direct binding to the TRSs in the PAI-1 promoter,22,39,41 which are adjacent to the FBE (Figure 2A), we investigated whether Foxc proteins activate the PAI-1 promoter in cooperation with Smad proteins. Cotransfection of Foxc2 and Smad3 expression vectors in BAECs resulted in additive induction of PAI-1 (4.2-fold) (Figure 4A). Significantly, overexpression of Foxc2 and Smad4 resulted in synergistic induction (7.3-fold), which is more than a summation of individual induction by Foxc2 and Smad4. Moreover, cotransfection of Foxc2 together with Smad3 and Smad4 activated the PAI-1 promoter by 13.6-fold, whereas Smad3 and Smad4 by themselves were significantly less potent (4-fold), demonstrating that Foxc2 functionally cooperates with Smad3/Smad4 for synergistic activation of the PAI-1 promoter. These data also suggest that Smad4 is more responsible for functional synergism with Foxc2 than Smad3.
To determine whether Foxc can physically interact with Smad proteins, we performed a GST pull-down experiment (Figure 4B). GST-Smad3 and GST-Smad4 proteins physically bind to Foxc1, whereas Foxc2 interacts with GST-Smad2, GST-Smad3, and GST-Smad4. Therefore, these findings support the observation that Foxc2 function with Smad proteins to transactivate the PAI-1 promoter in a cooperative manner. By contrast, although Foxc1 can physically interact with Smad3 and Smad4, no synergistic effect was observed between Foxc1 and Smad proteins in PAI-1 induction (Figure 4A).
We further examined whether FOXO1 has an inhibitory effect on the functional synergism between Foxc2 and Smad proteins in BAECs (Figure 4C). FOXO1 alone did not show any effect on the activation of the PAI-1 promoter by Smad3 and Smad4. However, the extent of luciferase activity by Smad3, Smad4, and Foxc2 was significantly attenuated when FOXO1 was cotransfected, suggesting that FOXO1 functionally antagonizes Foxc2 in the context of Smad-mediated PAI-1 induction.
Foxc2 Directly Regulates PAI-1 Transcription In Vivo
To detect direct binding of Foxc2 to the human PAI-1 promoter in chromatin in response to insulin and TGF-, we next performed chromatin immunoprecipitation (ChIP) assays in HMEC-1 (Figure 5A). DNA fragments containing the FBE or IRE were immunoprecipitated by anti-Smad4, anti-FOXC2, or anti-FOXO1 antibody from untreated HMEC-1. In insulin treatment, a smaller amount of DNA was detected in FOXO1-bound chromatin complex, consistent with the current understanding that FOXO1 is excluded from the nucleus in response to insulin. Importantly, more DNA fragments were detected in FOXC2-bound chromatin in both insulin- and TGF-eCtreated HMEC-1. It should be noted that in this assay, sonicated genomic DNA (<1000 bp) was used for subsequent PCR analysis and that the FBE and IRE are located within 600 bp, so it is possible that the 2 sites were present in some DNA fragments. Nonetheless, although the precise mechanisms still remain to be elucidated, these data suggest that under the activation of insulin or TGF- signaling, Foxc2 binds to both FBE and IRE more efficiently than FOXO1.
To examine the transcriptional impact of Foxc2 on endogenous PAI-1 expression in response to insulin and TGF- signaling, we performed semiquantitative RT-PCR analysis using mouse embryonic endothelial cells (MEECs)42 (Figure 5B). Although upregulation of PA1eC1 expression was observed in Foxc2-transfected cells, transcripts of PAI-1 were further increased in these cells when treated with insulin or TGF-. Consistent with our findings described above, these results demonstrate that Foxc2 is an important regulator for PAI-1 gene expression in insulin and TGF- signaling.
Foxc2 Regulates TGF-eCMediated PAI-1 Expression in Adult Tissues
As a first step to investigate the functional role of Foxc2 in PAI-1 regulation in postnatal life, we examined expression levels of FoxC2 in murine and human adult tissues (supplemental Figure IIIA and IIIB). Transcripts as well as proteins of FoxC2 were detected in a variety of adult tissues involved in insulin responsiveness. Consistently, analysis of real-time quantitative RT-PCR38 revealed that murine Foxc2 was abundantly expressed in aorta, heart, kidney, pancreas, white adipose tissue (WAT), and brown adipose tissue (BAT), whereas Foxc2 expression were detected at much lower levels in liver and spleen (supplemental Figure IIIC and data not shown).
To address the functional significance of Foxc2 in regulating PAI-1 gene expression in vivo, we used a TGF-1 induction model established previously37 using wild-type and Foxc2 heterozygous mutant mice. We performed quantitative analysis of Foxc2 and PAI-1 mRNA in kidney, heart, and WAT using real-time RT-PCR based on the treatment (with vehicle or TGF-) and genotype (wild-type or Foxc2+/eC) (Figure 6A and 6B). The basal levels of Foxc2 mRNA in vehicle-treated Foxc2+/eC mice were lower in the 3 tissues compared with those of the vehicle-treated wild-type mice (Figure 6A). This is likely attributable to Foxc2 haploinsufficiency. In both wild-type and Foxc2+/eC mice, TGF-1 upregulated Foxc2 expression in kidney but not in heart and WAT. To examine the relative mRNA abundance among 4 groups, 2-factor ANOVA analysis was performed. Foxc2 expression levels were significantly decreased by genotype in heart (P=0.044), whereas the differences in kidney and white adipose tissue were marginal (P=0.076).
Next, we analyzed the relative abundance of PAI-1 mRNA under the same conditions (Figure 6B). Two-factor ANOVA analysis revealed that genotype significantly differentiates these 4 groups with regard to PAI-1 expression levels in heart (P=0.040) and WAT (P=0.001). No statistical significance was observed in kidney. The relative induction of PAI-1 mRNA in response to TGF-1 treatment were 9.29±10.2, 1.86±2.04, and 7.77±12.3 in kidney, heart, and WAT, respectively, in wild-type animals, and 4.21±3.01, 0.179±0.215, and 1.46±1.59, respectively, in Foxc2+/eC mice. These results demonstrate that TGF-1eCinduced PAI-1 expression is significantly impaired in heart and WAT of Foxc2+/eC mice, indicating a physiological role for Foxc2 in regulating PAI-1 production in vivo.
Discussion
The human FOXC2 has attracted increasing attention because it is reported to serve as a key regulator of genes associated with insulin sensitivity.24,43 Our data indicate that Foxc2 directly induces PAI-1 expression competing with FOXO1 under the control of insulin signaling. Furthermore, Foxc2 are associated with Smad-mediated PAI-1 gene expression. Lastly, reduction of Foxc2 expression levels dramatically affect PAI-1 production in response to TGF- in postnatal heart and adipose tissue. This novel regulatory pathway mediated by Foxc2 provides new insights into the pathophysiological mechanisms underlying cardiovascular risk.
We demonstrate that Foxc2 has the ability to induce PAI-1 expression by directly binding to 2 distinct sites, IRE and FBE. Interestingly, the inhibitory effects of FOXO1 on Foxc2-mediated PAI-1 induction in insulin signaling through the IRE (Figure 3B through 3D) are consistent with the previous report that FOXO1 acts not only as a transcriptional activator but also as a repressor, depending on target genes in tumor suppression.44 TGF- signaling has been recognized as an important player that maintains elevated plasma PAI-1 levels and consequently increases the morbidity of obesity.45 We identified a novel Fox-binding element, FBE, adjacent to 1 of the Smad-binding sequences (TRSs) in the PAI-1 promoter. Others have recently identified the same site as a putative binding element of FoxD1,40 although binding of FoxD1 to this site still remains to be elucidated. One of the most important findings is that Foxc2 is a key binding partner of Smad proteins in PAI-1 gene regulation. It has been reported that the physical and functional interaction between FoxA and Smad3 takes place in TGF-eCmediated repression of the pulmonary surfactant protein B gene46 and that FoxO and FoxG proteins interact with Smad proteins to regulate the p21Cip1 gene in neuroepithelial and glioblastoma cell proliferation.47 Taken together with these studies, our findings lead to the underlying hypothesis that Fox proteins are common binding partners of Smad proteins.
PAI-1 gene expression is physiologically regulated by a variety of transcription factors. Importantly, Sp1 mediates hyperglycemia-induced PAI-1 expression through 2 Sp1-binding sites adjacent to the IRE34,48 and interacts with Smad3 and Smad4 in response to TGF- signaling.22 Based on these findings, we speculate that Foxc2 might also associate with Sp1 or Sp1-like proteins/KLF49 in the context of mediating glucose, insulin, and TGF- signaling pathways.
We show that Foxc2 is highly expressed in heart and adipose tissues as well as in kidney during postnatal life (Figure 6A and supplemental Figure III). This is in contrast to a previous report that FoxC2 is expressed exclusively in adipose tissue in adult humans and mice,24 although human FOXC2 is reported to be expressed in other adult tissues, such as skeletal muscle.50 The reason for this discrepancy is unclear at this time. Of note, we demonstrate in this article that TGF-1eCinduced PAI-1 transcription is impaired in heart and WAT of Foxc2 heterozygous mice. Taken together with our in vitro results, these data strongly suggest a physiological role of Foxc2 in regulation of PAI-1 induction in vivo.
FOXC2 counteracts insulin resistance when it is forcibly expressed in adipose tissue in mice.24 Conversely, it has been reported that insulin-resistant human subjects had 3-fold higher levels of FOXC2 mRNA in adipose tissues than insulin-sensitive subjects.43 Thus, the exact physiological role for FOXC2 in insulin resistance still remains to be elucidated.51 Given evidence that FOXC2 sensitizes the -adrenergiceCcAMPeCPKA pathway in adipocytes26 and that PAI-1 expression is induced in response to sympathetic (epinephrine) stimulation (eg, physiological stress),52,53 PAI-1 gene regulation by Foxc2 in adipocytes reinforces the linkage of FoxC2 to sympathetic efference on this cell type. Taken together, our results suggest that Foxc2 protein plays a pivotal role in insulin and TGF-eCinduced transcriptional regulation of the PAI-1 gene and may significantly contribute to a better understanding of the cellular and molecular basis of ischemic heart disease associated with insulin resistance.
Acknowledgments
This work was supported by grants from the NIH (HL65192 to D.E.V.; HL67105, DK68547, and HL74121 to T.K.), a grant for cardiovascular research from Banyu Pharmaceuticals (Tokyo, Japan), and Merck Co. H.F. is the recipient of a fellowship from Banyu Pharmaceuticals and Merck Co. We thank Corrie Ann Painter for technical assistance.
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Abstract
Elevated plasma levels of plasminogen activator inhibitor type I (PAI-1), a significant risk factor of ischemic heart disease, are associated with insulin resistance in which insulin and transforming growth factor (TGF)- play a pivotal role in regulating PAI-1 production. Forkhead transcription factor FOXC2 is an important regulator of insulin resistance. However, the underlying molecular mechanisms to link FOXC2 to PAI-1 levels in insulin resistance remain to be elucidated. Here, we demonstrate that Foxc2 is a common transcriptional activator of insulin and TGF- signaling to directly regulate PAI-1 expression via 2 distinct target sites, an insulin response element (IRE) and a novel forkhead-binding element (FBE), adjacent to a Smad-binding site. We found that in adipocytes and endothelial cells Foxc2 mediates insulin action competing with another Forkhead protein, FOXO1, via the insulin response element, and simultaneously cooperate with the TGF-/Smad pathway to transactivate PAI-1. Importantly, Foxc2 haploinsufficiency in mice significantly attenuates TGF-1eCinduced PAI-1 expression in the cardiovascular system and adipose tissue. Taken together, we propose that Foxc2 is a key molecule to regulate PAI-1 gene expression.
Key Words: plasminogen activator inhibitor-1 promoter insulin response element TGF- response site forkhead transcription factor
Introduction
Insulin resistance, defined as an attenuated responsiveness to insulin, is widely recognized as a key component of prediabetic state and obesity. Clinical studies have identified a strong link between insulin resistance and ischemic heart disease.1,2 Nevertheless, little is known about the underlying mechanisms to elucidate their precise interaction.
Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation, and animal studies have identified PAI-1 as a mediator of ischemic heart disease in obesity.3 PAI-1 is synthesized in many tissues, including vascular endothelium and adipose tissue.3eC5 Elevated plasma levels of PAI-1 are strongly associated with increased risk of ischemic heart disease.2,6 Plasma levels of PAI-1 are determined by genetic, hormonal,7,8 circadian,9,10 and metabolic factors, such as fatty acids11 and glucose.12
In the context of insulin resistance, as well as obesity and type 2 diabetes mellitus, several signaling pathways are known to play important roles in regulating PAI-1 gene expression. Insulin per se induces PAI-1 even in insulin-resistant adipocytes and insulin-resistant mice.13 Transforming growth factor (TGF)- levels are elevated in adipose tissues and TGF- administration increases PAI-1 in genetically obese (ob/ob) mice.14 Elevated levels of tumor necrosis factor also increase PAI-1 levels in adipose tissues of obese mice.15 Importantly, inflammatory process has recently emerged as a critical biological mechanism underlying obesity-related insulin resistance.16,17 However, the mechanisms underlying transcriptional regulation of PAI-1 in insulin resistance are not well understood.
Forkhead box (Fox) proteins constitute a large family of transcription factors that share an evolutionarily conserved DNA-binding domain with highly divergent flanking regions, leading to pleiotropic functions, such as the control of metabolism.18 Based on the homology of DNA-binding domains, they are classified into 17 subfamilies (subclasses A through Q), where the abbreviation contains all uppercase letters for human (eg, FOXC2), only the first letter capitalized for mouse (eg, Foxc2), and the first and subclass letters capitalized for all the chordates (eg, FoxC2). There is extensive evidence that Fox transcription factors are components of several signaling pathways, such as insulin.18 FoxO acts downstream of insulin signaling, and Akt-dependent phosphorylation of FoxO results in its exclusion from the nucleus.18eC21 Interestingly, FOXO1 was previously shown to bind to the insulin response element (IRE) of the PAI-1 promoter, although it had little effect on insulin-driven PAI-1 expression.22,23 This suggests that another Fox protein(s) plays a role in insulin-mediated induction of the PAI-1 gene.
FOXC2 is shown to be upregulated by insulin signaling in adipocytes and functions as a key regulator in adipose tissues in counteracting insulin resistance in adult mice as well as human subjects of insulin resistance or obesity.24,25 FOXC2 upregulates the transcription of the RI subunit of protein kinase A (PKA) to augment the -adrenergiceCcAMPeCPKA response, leading to increased sensitivity to insulin.26 We have previously shown that Foxc2, in cooperation with a closely related Foxc, Foxc1, plays pivotal roles in the development of cardiovascular, ocular, and genitourinary systems.27eC31
In the present study, we demonstrate that Foxc2 directly transactivates the PAI-1 promoter via 2 Fox-binding sites, IRE and a novel forkhead-binding element (FBE), which are associated with the insulin and TGF- signaling pathways, respectively. These studies led us to propose that Foxc2 mediates insulin and TGF- signaling to regulate PAI-1 gene expression. This mechanistic model provides new insights into the potential molecular pathways of cardiovascular disease in patients with insulin resistance.
Materials and Methods
Cell Culture
Bovine arterial endothelial cells (BAECs) were isolated and maintained as described previously.10 Human microvascular endothelial cells (HMEC-1)32 were cultured in Endothelial Cell Basal Medium-2 (Clonetics). 3T3-L1 (American Type Culture Collection) and COS-7 cells as well as mouse embryonic endothelial cells (MEECs) were maintained in DMEM supplemented with 10% FBS.
Plasmid Constructs
We amplified the full-length and truncated forms of Foxc1 and Foxc2 as well as full-length FOXO1 by PCR using Pfu Turbo (Stratagene) and primers (supplemental Table IA in the online data supplement available at http://circres.ahajournals.org) and cloned them into either the GAL4-fusion expression vector pSG42433 or pcDNA3.0 (Invitrogen). Mutated reporters for pGLuc884 were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and primers (supplemental Table IB). Expression vectors for Smad proteins and PAI-1eCpromotereCluciferase reporter vectors (pGLuc884, pGLuc107, and pGLuc85) have been described previously.34,35
Transfection and Luciferase Assay
BAECs or 3T3-L1 cells were plated at a density of 5x104 to 1x105 cells/mL into 12-well tissue culture plates and were subjected to transfection as described previously.10 Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) at 48 hours after transfection. For insulin treatment, BAECs or 3T3-L1 cells were cultured in DMEM with or without 10 e/mL human recombinant insulin (R&D) at 24 hours after transfection. Each transfection was performed in triplicate and replicated at least 3 times.
TGF-1 Treatment for Mice
Foxc2+/eC mice were maintained and genotyped as previously described.27,36 We injected age- and sex-matched wild-type (n=10) or Foxc2+/eC (n=10) mice with 25 e/kg human recombinant TGF-1 (R&D) (n=5 for each genotype) or saline (n=5 for each genotype) as a negative control through tail vein. Four hours after injection, organs and tissues were snap-frozen in liquid nitrogen, as described previously.37
Real-Time Quantitative RT-PCR
We extracted total RNA from tissues using the RNeasy Kit (Qiagen) and 1 e of mRNAs were reverse transcribed using random hexamers with Universal R.T. Reagents (ABI). We used SYBR Green PCR Master Mix (ABI) and primers (supplemental Table IE) on an i-Cycler instrument (Bio-Rad), as described previously.37 The 2eCCT method was used for analysis.38 Primers used are shown in supplemental Table IE.
An expanded Materials and Methods section can be found in the online data supplement.
Results
Foxc2 Activates the PAI-1 Promoter In Vitro
To determine whether Foxc1 or Foxc2 can induce PAI-1 expression directly, we first examined the transcriptional activity of Foxc proteins using the human PAI-1 promoter in BAECs (Figure 1A and 1B). We found that Foxc2 could transactivate the eC884-bp promoter in a dose-dependent manner, whereas Foxc1 failed to show significant activation of the PAI-1 promoter (Figure 1A). As shown in Figure 1B, we systematically examined Foxc2-mediated luciferase activity using a series of deletion constructs of the human PAI-1 promoter.34 All constructs contain the IRE, which is shown to be a FOXO1-binding site,23 although pGLuc884 also has TGF-eCresponsive sequences (TRSs).39 Foxc2-responsiveness was evident in pGLuc884 and pGLuc107 and was still retained in the shortest construct (pGLuc85) containing the IRE. Based on analysis of functional domains in Foxc (supplemental Figure IA through ID), we generated constitutively active forms of Foxc1 and Foxc2 (caFoxc1 and caFoxc2, respectively), which consist of the activation domain 1 (AD1) and forkhead DNA-binding domain (FHD) (supplemental Figure IE). Significantly, transfection of caFoxc2 resulted in considerably enhanced activation of the PAI-1 promoter in BAECs (Figure 1B), although similar induction by caFoxc1 was observed (data not shown). It should be noted that PAI-1 promoter activity was more than 85-fold using caFoxc2 in pGLuc884, whereas it remained approximately 70-fold with pGLuc107 and pGLuc85, suggesting that there are multiple Foxc2-responsive sites in the PAI-1 promoter (see below).
We further tested the effects of Foxc on the activation of the PAI-1 promoter in murine preadipocytic 3T3-L1 cell line (Figure 1C). In contrast to BAECs, caFoxc2 showed only 6.2-fold induction in 3T3-L1 cells, whereas basal luciferase activity of the PAI-1 promoter was 25-fold higher in 3T3-L1 cells than in COS-7 cells. We found that the FoxC2 protein is highly expressed in 3T3-L1 cells compared with other cell lines, such as BAECs and COS-7 (supplemental Figure II), suggesting that PAI-1 expression is constitutively upregulated by endogenous Foxc2 in 3T3-L1 cells. As shown in Figure 1C, this observation was supported by the finding that basal luciferase activity was reduced in a dose-dependent manner by a dominant-negative form of Foxc2 (dnFoxc2), which consists of the FHD (supplemental Figure IE).
Foxc2 Regulates the PAI-1 Promoter via Two Fox-Binding Sites
In addition to the previously reported IRE,23 we found another Fox-binding element (FBE) located at eC723 bp. This site is adjacent to 1 of the TRSs (Figure 2A) and has recently been shown to be a putative FoxD1-binding site.40 Because FoxC1, FoxC2, and FoxO1 are coexpressed in endothelial and preadipocytic cell lines (supplemental Figure II), we examined whether these Fox proteins directly bind to the Fox-binding sites on the PAI-1 promoter (Figure 2B). Specific complexes were formed between IRE or FBE probe and recombinant Fox proteins. The specificity of these complexes was confirmed by demonstrating their disappearance in the presence of unlabeled corresponding probes. In addition, no complexes were detected between the Foxc proteins and either of the 2 mutated oligonucleotides (IREmut and FBEmut). The intensity of the bands was the strongest using Foxc2, suggesting that Foxc2 can bind both FBE and IRE more efficiently than Foxc1 or FOXO1. To further confirm the significance of the FBE and IRE, we tested luciferase activity of mutant reporter constructs in which the FBE and/or IRE are disrupted (Figure 2C). Although pGLuc884IREmut or pGLuc884FBEmut was transactivated by Foxc2 significantly less than pGLuc884 in BAECs, pGLuc884BOTHmut showed substantially low transactivation by Foxc2. These results indicate that both FBE and IRE are responsible for Foxc2-mediated transactivation of the PAI-1 promoter.
FOXO1 Antagonizes Foxc2 to Regulate the PAI-1 Promoter
Although FOXO1 binds to the IRE, it has little effect on transcription of PAI-1 in response to insulin.23 Because Foxc2 can transactivate the PAI-1 promoter via this site (Figure 2B and 2C), it is possible that FOXO1 competes with Foxc2 to suppress the activation of the PAI-1 promoter. Indeed, transactivation of the shortest promoter by Foxc2 was significantly attenuated when FOXO1 was coexpressed in BAECs (Figure 3A).
Because FOXO1 is a downstream component of insulin signaling and is excluded from the nucleus in response to insulin,19 we tested the functional interaction between Foxc2 and FOXO1 in the insulin-mediated induction of PAI-1 using BAECs and 3T3-L1 cells that respond to insulin (Figure 3B and 3C). The attenuation of Foxc2-mediated PAI-1 induction by FOXO1 was significantly abolished in the presence of insulin. These results indicate that activation of insulin signaling prevents FOXO1 from competing with Foxc2, resulting in PAI-1 induction in endothelial and preadipocytic cells. Moreover, we tested the effects of mutations of the IRE and FBE on insulin action in BAECs. The results using pGLuc884FBEmut were similar to those of pGLuc85 (Figure 3B and 3D), indicating that the FBE is dispensable for insulin action on the PAI-1 promoter. In contrast, neither pGLuc884IREmut nor pGLuc884BOTHmut exhibited significant changes in transactivation by addition of FOXO1 or insulin. These results together demonstrate that FOXO1 functionally competes with Foxc2 via the IRE on the PAI-1 promoter.
Foxc2 Is Associated With the TGF-/Smad Pathway
Because Smad3 and Smad4 mediate TGF- signaling through direct binding to the TRSs in the PAI-1 promoter,22,39,41 which are adjacent to the FBE (Figure 2A), we investigated whether Foxc proteins activate the PAI-1 promoter in cooperation with Smad proteins. Cotransfection of Foxc2 and Smad3 expression vectors in BAECs resulted in additive induction of PAI-1 (4.2-fold) (Figure 4A). Significantly, overexpression of Foxc2 and Smad4 resulted in synergistic induction (7.3-fold), which is more than a summation of individual induction by Foxc2 and Smad4. Moreover, cotransfection of Foxc2 together with Smad3 and Smad4 activated the PAI-1 promoter by 13.6-fold, whereas Smad3 and Smad4 by themselves were significantly less potent (4-fold), demonstrating that Foxc2 functionally cooperates with Smad3/Smad4 for synergistic activation of the PAI-1 promoter. These data also suggest that Smad4 is more responsible for functional synergism with Foxc2 than Smad3.
To determine whether Foxc can physically interact with Smad proteins, we performed a GST pull-down experiment (Figure 4B). GST-Smad3 and GST-Smad4 proteins physically bind to Foxc1, whereas Foxc2 interacts with GST-Smad2, GST-Smad3, and GST-Smad4. Therefore, these findings support the observation that Foxc2 function with Smad proteins to transactivate the PAI-1 promoter in a cooperative manner. By contrast, although Foxc1 can physically interact with Smad3 and Smad4, no synergistic effect was observed between Foxc1 and Smad proteins in PAI-1 induction (Figure 4A).
We further examined whether FOXO1 has an inhibitory effect on the functional synergism between Foxc2 and Smad proteins in BAECs (Figure 4C). FOXO1 alone did not show any effect on the activation of the PAI-1 promoter by Smad3 and Smad4. However, the extent of luciferase activity by Smad3, Smad4, and Foxc2 was significantly attenuated when FOXO1 was cotransfected, suggesting that FOXO1 functionally antagonizes Foxc2 in the context of Smad-mediated PAI-1 induction.
Foxc2 Directly Regulates PAI-1 Transcription In Vivo
To detect direct binding of Foxc2 to the human PAI-1 promoter in chromatin in response to insulin and TGF-, we next performed chromatin immunoprecipitation (ChIP) assays in HMEC-1 (Figure 5A). DNA fragments containing the FBE or IRE were immunoprecipitated by anti-Smad4, anti-FOXC2, or anti-FOXO1 antibody from untreated HMEC-1. In insulin treatment, a smaller amount of DNA was detected in FOXO1-bound chromatin complex, consistent with the current understanding that FOXO1 is excluded from the nucleus in response to insulin. Importantly, more DNA fragments were detected in FOXC2-bound chromatin in both insulin- and TGF-eCtreated HMEC-1. It should be noted that in this assay, sonicated genomic DNA (<1000 bp) was used for subsequent PCR analysis and that the FBE and IRE are located within 600 bp, so it is possible that the 2 sites were present in some DNA fragments. Nonetheless, although the precise mechanisms still remain to be elucidated, these data suggest that under the activation of insulin or TGF- signaling, Foxc2 binds to both FBE and IRE more efficiently than FOXO1.
To examine the transcriptional impact of Foxc2 on endogenous PAI-1 expression in response to insulin and TGF- signaling, we performed semiquantitative RT-PCR analysis using mouse embryonic endothelial cells (MEECs)42 (Figure 5B). Although upregulation of PA1eC1 expression was observed in Foxc2-transfected cells, transcripts of PAI-1 were further increased in these cells when treated with insulin or TGF-. Consistent with our findings described above, these results demonstrate that Foxc2 is an important regulator for PAI-1 gene expression in insulin and TGF- signaling.
Foxc2 Regulates TGF-eCMediated PAI-1 Expression in Adult Tissues
As a first step to investigate the functional role of Foxc2 in PAI-1 regulation in postnatal life, we examined expression levels of FoxC2 in murine and human adult tissues (supplemental Figure IIIA and IIIB). Transcripts as well as proteins of FoxC2 were detected in a variety of adult tissues involved in insulin responsiveness. Consistently, analysis of real-time quantitative RT-PCR38 revealed that murine Foxc2 was abundantly expressed in aorta, heart, kidney, pancreas, white adipose tissue (WAT), and brown adipose tissue (BAT), whereas Foxc2 expression were detected at much lower levels in liver and spleen (supplemental Figure IIIC and data not shown).
To address the functional significance of Foxc2 in regulating PAI-1 gene expression in vivo, we used a TGF-1 induction model established previously37 using wild-type and Foxc2 heterozygous mutant mice. We performed quantitative analysis of Foxc2 and PAI-1 mRNA in kidney, heart, and WAT using real-time RT-PCR based on the treatment (with vehicle or TGF-) and genotype (wild-type or Foxc2+/eC) (Figure 6A and 6B). The basal levels of Foxc2 mRNA in vehicle-treated Foxc2+/eC mice were lower in the 3 tissues compared with those of the vehicle-treated wild-type mice (Figure 6A). This is likely attributable to Foxc2 haploinsufficiency. In both wild-type and Foxc2+/eC mice, TGF-1 upregulated Foxc2 expression in kidney but not in heart and WAT. To examine the relative mRNA abundance among 4 groups, 2-factor ANOVA analysis was performed. Foxc2 expression levels were significantly decreased by genotype in heart (P=0.044), whereas the differences in kidney and white adipose tissue were marginal (P=0.076).
Next, we analyzed the relative abundance of PAI-1 mRNA under the same conditions (Figure 6B). Two-factor ANOVA analysis revealed that genotype significantly differentiates these 4 groups with regard to PAI-1 expression levels in heart (P=0.040) and WAT (P=0.001). No statistical significance was observed in kidney. The relative induction of PAI-1 mRNA in response to TGF-1 treatment were 9.29±10.2, 1.86±2.04, and 7.77±12.3 in kidney, heart, and WAT, respectively, in wild-type animals, and 4.21±3.01, 0.179±0.215, and 1.46±1.59, respectively, in Foxc2+/eC mice. These results demonstrate that TGF-1eCinduced PAI-1 expression is significantly impaired in heart and WAT of Foxc2+/eC mice, indicating a physiological role for Foxc2 in regulating PAI-1 production in vivo.
Discussion
The human FOXC2 has attracted increasing attention because it is reported to serve as a key regulator of genes associated with insulin sensitivity.24,43 Our data indicate that Foxc2 directly induces PAI-1 expression competing with FOXO1 under the control of insulin signaling. Furthermore, Foxc2 are associated with Smad-mediated PAI-1 gene expression. Lastly, reduction of Foxc2 expression levels dramatically affect PAI-1 production in response to TGF- in postnatal heart and adipose tissue. This novel regulatory pathway mediated by Foxc2 provides new insights into the pathophysiological mechanisms underlying cardiovascular risk.
We demonstrate that Foxc2 has the ability to induce PAI-1 expression by directly binding to 2 distinct sites, IRE and FBE. Interestingly, the inhibitory effects of FOXO1 on Foxc2-mediated PAI-1 induction in insulin signaling through the IRE (Figure 3B through 3D) are consistent with the previous report that FOXO1 acts not only as a transcriptional activator but also as a repressor, depending on target genes in tumor suppression.44 TGF- signaling has been recognized as an important player that maintains elevated plasma PAI-1 levels and consequently increases the morbidity of obesity.45 We identified a novel Fox-binding element, FBE, adjacent to 1 of the Smad-binding sequences (TRSs) in the PAI-1 promoter. Others have recently identified the same site as a putative binding element of FoxD1,40 although binding of FoxD1 to this site still remains to be elucidated. One of the most important findings is that Foxc2 is a key binding partner of Smad proteins in PAI-1 gene regulation. It has been reported that the physical and functional interaction between FoxA and Smad3 takes place in TGF-eCmediated repression of the pulmonary surfactant protein B gene46 and that FoxO and FoxG proteins interact with Smad proteins to regulate the p21Cip1 gene in neuroepithelial and glioblastoma cell proliferation.47 Taken together with these studies, our findings lead to the underlying hypothesis that Fox proteins are common binding partners of Smad proteins.
PAI-1 gene expression is physiologically regulated by a variety of transcription factors. Importantly, Sp1 mediates hyperglycemia-induced PAI-1 expression through 2 Sp1-binding sites adjacent to the IRE34,48 and interacts with Smad3 and Smad4 in response to TGF- signaling.22 Based on these findings, we speculate that Foxc2 might also associate with Sp1 or Sp1-like proteins/KLF49 in the context of mediating glucose, insulin, and TGF- signaling pathways.
We show that Foxc2 is highly expressed in heart and adipose tissues as well as in kidney during postnatal life (Figure 6A and supplemental Figure III). This is in contrast to a previous report that FoxC2 is expressed exclusively in adipose tissue in adult humans and mice,24 although human FOXC2 is reported to be expressed in other adult tissues, such as skeletal muscle.50 The reason for this discrepancy is unclear at this time. Of note, we demonstrate in this article that TGF-1eCinduced PAI-1 transcription is impaired in heart and WAT of Foxc2 heterozygous mice. Taken together with our in vitro results, these data strongly suggest a physiological role of Foxc2 in regulation of PAI-1 induction in vivo.
FOXC2 counteracts insulin resistance when it is forcibly expressed in adipose tissue in mice.24 Conversely, it has been reported that insulin-resistant human subjects had 3-fold higher levels of FOXC2 mRNA in adipose tissues than insulin-sensitive subjects.43 Thus, the exact physiological role for FOXC2 in insulin resistance still remains to be elucidated.51 Given evidence that FOXC2 sensitizes the -adrenergiceCcAMPeCPKA pathway in adipocytes26 and that PAI-1 expression is induced in response to sympathetic (epinephrine) stimulation (eg, physiological stress),52,53 PAI-1 gene regulation by Foxc2 in adipocytes reinforces the linkage of FoxC2 to sympathetic efference on this cell type. Taken together, our results suggest that Foxc2 protein plays a pivotal role in insulin and TGF-eCinduced transcriptional regulation of the PAI-1 gene and may significantly contribute to a better understanding of the cellular and molecular basis of ischemic heart disease associated with insulin resistance.
Acknowledgments
This work was supported by grants from the NIH (HL65192 to D.E.V.; HL67105, DK68547, and HL74121 to T.K.), a grant for cardiovascular research from Banyu Pharmaceuticals (Tokyo, Japan), and Merck Co. H.F. is the recipient of a fellowship from Banyu Pharmaceuticals and Merck Co. We thank Corrie Ann Painter for technical assistance.
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