Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

Abstract

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-␤1-induced PAI-1 expression in the cardiovascular system and adipose tissue. Taken Key Words: plasminogen activator inhibitor-1 promoter Ⅲ insulin response element Ⅲ TGF-␤ Ⅲ response site Ⅲ forkhead transcription factor I nsulin 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. [3][4][5] 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 acids 11 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 la...

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“…6 These findings are consistent with the notion that FOXO1 competes with Foxc2 via the insulin response element (IRE) on the PAI-1 promoter and that FOXO1 antagonizes the synergistic interaction of Foxc2 with smad proteins in the induction of PAI-1 in endothelial cells. Matsuzaki et al 11 reported that insulininduced phosphorylation of Foxo1 results in exclusion from the nucleus and subsequent ubiquitin-mediated proteasomal degradation.…”

“…Rather, they argue that our knowledge of molecular pathways and factors that influence them remains inadequate, as was proposed in relation to the lack of an effect of diet on breast cancer. 5 It is in this context that the report of Fujita et al 6 in this issue of Circulation Research is of significant interest. The data presented here link winged helix family transcription factor Foxc2 to the inflammatory cytokine, TGF-␤1, and to expression of plasminogen activator inhibitor type-1 (PAI-1), a key regulator of intravascular thrombosis.…”

“…The polymorphisms studies are of great potential interest, 9 yet we know little on the relative activity of polymorphisms in transactivation of important genes. The findings of Fujita et al 6 suggest additional levels of complexity for the field, but do, at the end of the day, tie obesity and insulin resistance to coagulation at the molecular level-an important finding. Their data convincingly demonstrate that Foxc2 is a transcriptional activator for both insulin and TGF-␤1-induced PAI-1 expression in vitro, and that Foxc2 ϩ/Ϫ mice have lowered expression of PAI-1 in response to TGF-␤1 treatment.…”

“…However, the connection between obesity, inflammation, and atherosclerosis potentially provided by Fujita et al 6 suggests that in addition to conventional measures, it may be important to measure molecular pathways impacted by various obesity treatments.…”

Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration

“…2A). Consistently, a constitutively active form of Foxc2 (caFoxc2) [24], which only includes the N-terminal activation domain and the DNA binding domain, further transactivated the CXCR4 promoter by 10-fold. Since there are several putative Foxc-binding sites within the promoter region (data not shown), we next generated a series of deletion constructs with different regions of the CXCR4 promoter (Fig.…”