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KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation.

SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, García-Cardeña G, Jain MK - J. Exp. Med. (2004)

Bottom Line: Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest.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.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA.

ABSTRACT
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-1beta 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.

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KLF2 inhibits NF-κB function. (A) KLF2 does not affect expression or nuclear translocation of various components of the NF-κB pathway. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2), stimulated with IL-1β for 30 or 60 min, and assessed for expression of the indicated factors by Western blot analysis. NE, nuclear extracts; Cyto, cytoplasmic extracts. (B) KLF2 does not affect NF-κB binding to DNA. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP (control) and Ad-KLF2 (KLF2) in the presence or absence of IL-1β. Induced NF-κB band is designated by the arrow. Specificity was verified by competition and supershift studies. (C) KLF2 inhibits TNFα-mediated induction of the VCAM-1 promoter and NF-κB concatemer. Transient transfection studies were performed in BAECs with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001. (D) KLF2 inhibits p65-mediated induction of the VCAM-1 and NF-κB concatemer. Transient transfection studies were performed in COS-7 cells with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001.
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fig4: KLF2 inhibits NF-κB function. (A) KLF2 does not affect expression or nuclear translocation of various components of the NF-κB pathway. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2), stimulated with IL-1β for 30 or 60 min, and assessed for expression of the indicated factors by Western blot analysis. NE, nuclear extracts; Cyto, cytoplasmic extracts. (B) KLF2 does not affect NF-κB binding to DNA. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP (control) and Ad-KLF2 (KLF2) in the presence or absence of IL-1β. Induced NF-κB band is designated by the arrow. Specificity was verified by competition and supershift studies. (C) KLF2 inhibits TNFα-mediated induction of the VCAM-1 promoter and NF-κB concatemer. Transient transfection studies were performed in BAECs with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001. (D) KLF2 inhibits p65-mediated induction of the VCAM-1 and NF-κB concatemer. Transient transfection studies were performed in COS-7 cells with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001.

Mentions: As a first step, we assessed the effect of adenovirally expressed KLF2 (versus control virus; Fig. 4 A, right, C) on p50, p65, IκB, and phosphorylated IκB levels at several time points after IL-1β treatment. As shown in Fig. 4 A (left), we did not observe any significant differences among the two groups. In addition, we did not observe any effect on nuclear accumulation of p65 after IL-1β treatment (Fig. 4 A, right). We next assessed if KLF2 can affect NF-κB DNA binding. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP and Ad-KLF2 in the presence or absence of IL-1β stimulation, and gel shift assays were performed using a NF-κB site derived from the VCAM-1 promoter (6). As expected, NF-κB binding was not detected in either Ad-GFP (Ctrl) or Ad-KLF2–infected cells in the absence of cytokine treatment (not depicted). However, as shown in Fig. 4 B (left) a DNA–protein complex is induced in Ad-GFP cells after treatment with IL-1β, and the presence of p50 and p65 in this complex was verified by competition and supershift studies. In the presence of KLF2 (right), a nearly identical binding pattern was observed. These data suggest that KLF2's inhibitory effect is not due to effects on NF-κB expression or DNA binding.


KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation.

SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, García-Cardeña G, Jain MK - J. Exp. Med. (2004)

KLF2 inhibits NF-κB function. (A) KLF2 does not affect expression or nuclear translocation of various components of the NF-κB pathway. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2), stimulated with IL-1β for 30 or 60 min, and assessed for expression of the indicated factors by Western blot analysis. NE, nuclear extracts; Cyto, cytoplasmic extracts. (B) KLF2 does not affect NF-κB binding to DNA. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP (control) and Ad-KLF2 (KLF2) in the presence or absence of IL-1β. Induced NF-κB band is designated by the arrow. Specificity was verified by competition and supershift studies. (C) KLF2 inhibits TNFα-mediated induction of the VCAM-1 promoter and NF-κB concatemer. Transient transfection studies were performed in BAECs with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001. (D) KLF2 inhibits p65-mediated induction of the VCAM-1 and NF-κB concatemer. Transient transfection studies were performed in COS-7 cells with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001.
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Related In: Results  -  Collection

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fig4: KLF2 inhibits NF-κB function. (A) KLF2 does not affect expression or nuclear translocation of various components of the NF-κB pathway. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2), stimulated with IL-1β for 30 or 60 min, and assessed for expression of the indicated factors by Western blot analysis. NE, nuclear extracts; Cyto, cytoplasmic extracts. (B) KLF2 does not affect NF-κB binding to DNA. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP (control) and Ad-KLF2 (KLF2) in the presence or absence of IL-1β. Induced NF-κB band is designated by the arrow. Specificity was verified by competition and supershift studies. (C) KLF2 inhibits TNFα-mediated induction of the VCAM-1 promoter and NF-κB concatemer. Transient transfection studies were performed in BAECs with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001. (D) KLF2 inhibits p65-mediated induction of the VCAM-1 and NF-κB concatemer. Transient transfection studies were performed in COS-7 cells with the indicated constructs. A similar degree of inhibition is seen with KLF2 and KLFΔZnF. n = 6–12 per group; *P < 0.00005; **P < 0.00001.
Mentions: As a first step, we assessed the effect of adenovirally expressed KLF2 (versus control virus; Fig. 4 A, right, C) on p50, p65, IκB, and phosphorylated IκB levels at several time points after IL-1β treatment. As shown in Fig. 4 A (left), we did not observe any significant differences among the two groups. In addition, we did not observe any effect on nuclear accumulation of p65 after IL-1β treatment (Fig. 4 A, right). We next assessed if KLF2 can affect NF-κB DNA binding. Nuclear extracts were harvested from HUVECs overexpressing Ad-GFP and Ad-KLF2 in the presence or absence of IL-1β stimulation, and gel shift assays were performed using a NF-κB site derived from the VCAM-1 promoter (6). As expected, NF-κB binding was not detected in either Ad-GFP (Ctrl) or Ad-KLF2–infected cells in the absence of cytokine treatment (not depicted). However, as shown in Fig. 4 B (left) a DNA–protein complex is induced in Ad-GFP cells after treatment with IL-1β, and the presence of p50 and p65 in this complex was verified by competition and supershift studies. In the presence of KLF2 (right), a nearly identical binding pattern was observed. These data suggest that KLF2's inhibitory effect is not due to effects on NF-κB expression or DNA binding.

Bottom Line: Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest.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.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA.

ABSTRACT
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-1beta 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.

Show MeSH
Related in: MedlinePlus