KLF2 suppresses TGF-beta signaling in endothelium through induction of Smad7 and inhibition of AP-1 (original) (raw)
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KLF2 suppresses TGF-β signaling in endothelium through induction of Smad7 and inhibition of AP-1
2007
Objective-The flow-responsive Kruppel-like factor 2 (KLF2) is crucial for maintaining endothelial cell quiescence. Here, we describe its detailed effects on transforming growth factor- (TGF-) signaling, which normally has proatherogenic effects on endothelium. Methods and Results-In-depth analysis of genome-wide expression data shows that prolonged lentiviral-mediated overexpression of KLF2 in human umbilical vein endothelial cells (HUVECs) diminishes the expression of a large panel of established TGF--inducible genes. Both baseline and TGF--induced expression levels of plasminogen activator inhibitor 1 (PAI-1) and thrombospondin-1 are greatly diminished by KLF2. Using a combination of ectopic expression, small interfering RNA-mediated knockdown, and promoter activity assays, we show that KLF2 partly inhibits the phosphorylation and subsequent nuclear accumulation of Smad2, thereby suppressing the TGF--induced Smad4mediated transcriptional activity. This is achieved through TGF--independent induction of inhibitory Smad7. Additionally, a full inhibition of TGF- signaling is functionally achieved through a simultaneous suppression of activator protein 1 (AP-1), which is an essential cofactor for TGF--dependent transcription of many genes.
Blood, 2006
The flow-responsive transcription factor KLF2 is acquiring a leading role in the regulation of endothelial cell gene expression. A genome-wide microarray expression profiling is described employing lentivirus-mediated, 7-day overexpression of human KLF2 at levels observed under prolonged flow. KLF2 is not involved in lineage typing, as 42 endothelial-specific markers were unaffected. Rather, KLF2 generates a gene transcription profile (> 1000 genes) affecting key functional pathways such as cell migration, vasomotor function, inflammation, and hemostasis and induces a morphology change typical for shear exposure including stress fiber formation. Protein levels for thrombomodulin, endothelial nitric oxide synthase, and plasminogen activator inhibitor type-1 are altered to atheroprotective levels, even in the presence of the inflammatory cytokine TNF-α. KLF2 attenuates cell migration by affecting multiple genes including VEGFR2 and the potent antimigratory SEMA3F. The distribution ...
TGF-β induces proangiogenic and antiangiogenic factorsvia parallel but distinct Smad pathways1
Kidney International, 2004
TGF-b induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways. Background. Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-b1 (TGF-b1), but the mechanism remains unclear. Methods. We examined the role of TGF-b1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-b1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-b1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation. Results. Induction of VEGF-A and TSP-1 by TGF-b1 in NRK52E cells was associated with activation of pathwayrestricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-b1. Conditioned media from NRK52E, which was stimulated by TGF-b1 for 24 hours, did not induce endothelial cell 1 See Editorial by Woolf, p. 862.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2006
Objective-The purpose of this study is to investigate the expression and regulation of type-2 tissue factor pathway inhibitor (TFPI-2) in endothelial cells, as well as the regulation of human endothelial cell (EC) function by TFPI-2. Methods and Results-Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that vascular endothelial growth factor (VEGF) induced both time-and dose-dependent increase in TFPI-2 mRNA and protein expression in endothelial cells. TFPI-2 mRNA expression was also significantly upregulated by IL-1, and modestly increased by both tumor necrosis factor (TNF)-␣ and fibroblast growth factor (FGF)-2, but not placental growth factor (PlGF). VEGF upregulation of TFPI-2 was dramatically reduced by inhibition of the MEK pathway. Administration of TFPI-2 protein suppressed both VEGF and FGF-2 stimulation of EC proliferation in a dose-dependent manner. A recombinant preparation of the first Kunitz-type domain of TFPI-2 (KD1) did not suppress growth factor stimulation of EC proliferation, suggesting a mechanism distinct from the proteinase inhibitory activity of TFPI-2. Exogenously added TFPI-2 protein suppressed VEGF-induced EC migration in 2 different assays. Recombinant wt-KD1 or the R24K mutant of KD1, but not the R24Q mutant, dramatically suppressed VEGF-induced EC migration. TFPI-2 protein, but not recombinant KD1, blocked VEGF-induced activation of both Akt and ERK1/2 in ECs. At higher doses, TFPI-2 protein blocked VEGFR2 activation. Conclusion-Our data suggest that VEGF-upregulation of TFPI-2 expression in endothelial cells may represent a mechanism for negative feedback regulation and modulation of its pro-angiogenic action on endothelial cells. TFPI-2, or derivatives of TFPI-2, may be novel therapeutics for treatment of angiogenic disease processes.
Journal of Biological …, 2001
Vascular endothelial growth factor (VEGF) and tumor necrosis factor-␣ (TNF-␣) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-␣, wild-type VEGF-or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-␣ with wild-type VEGF-or KDR-selective variants resulted in an approximate 250fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of proteintyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF. Vascular endothelial growth factor (VEGF) 1 is a potent endothelial cell (EC)-specific mitogen that promotes the proliferation and migration of EC, remodeling of the extracellular matrix, formation of capillary tubules, and vascular leakage (1-6). As a key angiogenic growth factor, VEGF plays a critical role in the development of the fetal cardiovascular system, as well as a significant role in the physiological and pathological angiogenesis (7-10). Although it is well accepted that Flt-1 and KDR are both high affinity receptors for VEGF, it is not clear which receptor activates the downstream signaling pathways responsible for the diverse biological responses of VEGF. Transgenic knockout studies in mice showed that both receptors are essential for animal survival, because mouse embryos null for KDR or Flt-1 die in utero (11-12). Experiments using receptor-specific binding variants or receptor-specific inhibitors have associated KDR receptor activity with EC proliferation, migration, vascular permeability, cell survival, and angiogenesis (13-17). The role of Flt-1 receptor signaling in VEGF biology is less clear. Studies indicate that Flt-1 may mediate, at least in part, chemotaxis and procoagulatant activity in macrophages and up-regulation of matrix metalloproteinases in vascular smooth muscle cells (18-19). Upon VEGF stimulation, KDR receptor is strongly tyrosine-phosphorylated, but little or no autophosphorylation of Flt-1 occurs (19-21). Rahimi et al. (19) recently proposed that KDR activation plays a dominant role in angiogenesis by promoting EC proliferation, whereas Flt-1 binding plays a stationary role by antagonizing the interaction of VEGF with KDR. A novel VEGF receptor with a sequence identical to that of neuropilin was recently identified by Soker et al. (22). This receptor binds to VEGF via an interaction with C-terminal heparin binding domain. The role of neuropilin in VEGF signaling is currently under active investigation (22-24). VEGF has previously been demonstrated to regulate tissue factor (TF) expression in monocytes and ECs (25-27). Although VEGF alone induced only a moderate increase in TF expression, it significantly enhanced TNF-␣-induced TF up-regulation through a synergistic mechanism in EC (26-27). The role that the two TNF receptors, TNFR60 and TNFR80, play in the synergy between VEGF and TNF has been studied. Clauss et al. (27) reported that stimulation of the 60-kDa TNF receptor by a mutant of TNF specific for TNFR60 induced a TF upregulation comparable with wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutant did not enhance TF expression. Thus, TNFR60 is the principle receptor involved in the synergistic up-regulation of TF induced by TNF and VEGF. The role that the VEGF receptors, KDR and Flt-1, play in this process is unknown. Therefore, in the present study we have investigated the role of KDR and Flt-1 receptor signaling in TF up-regulation and in its synergy with TNF by using VEGF receptor-selective variants. VEGF and TNF work in concert to synergistically up-regulate TF expression in human umbilical vein endothelial (HUVEC) cells. Here we demonstrate that KDR receptor signaling is necessary and sufficient for the potentiation of TNF-induced TF up-regulation.
KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation
2004
The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1  and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.
Fibroblast Growth Factor-2 Induces Lef/Tcf-dependent Transcription in Human Endothelial Cells
Journal of Biological Chemistry, 2002
Lef/Tcf proteins belong to a family of architectural transcription factors that control developmental processes and play an important role in oncogenesis. Classical activators of Lef/Tcf-dependent transcription comprise the Wnt family of proteins, which translocate -catenin into the nucleus and allow the formation of transactivation-competent Lef/Tcf--catenin complexes.
Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2
Journal of Clinical …, 2006
In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway. Overexpression and silencing of KLF2 in the context of flow, combined with findings from genome-wide analyses of gene expression, demonstrate that the induction of KLF2 results in the orchestrated regulation of endothelial transcriptional programs controlling inflammation, thrombosis/hemostasis, vascular tone, and blood vessel development. Our data also indicate that KLF2 expression globally modulates IL-1b-mediated endothelial activation. KLF2 therefore serves as a mechano-activated transcription factor important in the integration of multiple endothelial functions associated with regions of the arterial vasculature that are relatively resistant to atherogenesis. Nonstandard abbreviations used: Ang, angiopoietin; ASS, argininosuccinate synthetase; ChIP, chromatin immunoprecipitation; CNP, C-type natriuretic peptide; ET-1, endothelin-1; HUVEC, human umbilical vein endothelial cell; KLF2, Kruppel-like factor 2; MEF2, myocyte enhancer factor-2; MEK5, MAPK kinase 5; PTGDS, prostaglandin D2 synthase; sih, silent heart; siRNA, small interfering RNA; TF, tissue factor; TM, thrombomodulin.
Molecular Regulation of Placental Growth Factor (PlGF) Expression in Endothelial Cells
2011
Placental growth factor (PlGF) is a pro-angiogenic and inflammatory mediator that promotes many pathological conditions including, diabetes, atherosclerosis and cancer. In mouse models, the loss of PlGF or inhibition of vascular endothelial growth factor receptor-1 (VEGFR-1) activity suppresses these disorders. Hyperglycaemia plays a fundamental role in the pathogenesis of type-2 diabetes and associated conditions, resulting in a loss of PI3 kinase (PI3K) signalling and dysfunction in endothelial cells. Using pharmacological inhibitors, siRNA, and adenoviral constructs to modulate the PI3K/Akt signalling activity, I found that the induction of PlGF expression in human umbilical vein endothelial cells (HUVEC) by hyperglycaemia is PI3K/Akt-dependent. Using similar approaches, the FOXO1 transcription factor was identified as the downstream target of Akt involved in the regulation of both PlGF and VEGFR-1 expression. FOXO1 was found to interact directly with the VEGFR-1 gene promoter in vitro, and over-expression of constitutively-active FOXO1 promotes PlGF expression in vivo. Although VEGF activates PI3K/Akt, it stimulates robust PlGF release in endothelial cells. Here I show that this effect is both VEGFR-2 and PKC-dependent, but independent of PI3K/Akt. The PI3K/Akt/FOXO1 axis is an important regulator of vascular homeostasis and stress responses and the identification of its involvement in PlGF expression may provide new therapeutic targets for disorders characterised by endothelial dysfunction.