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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 interacts directly with p300. (A) p300 rescues KLF2-mediated inhibition. Transient transfection studies with the indicated plasmids were performed in COS-7 cells. Cotransfection studies demonstrate that p300 can rescue KLF2 and KLFΔZnF-mediated inhibition of the NF-κB concatemer. n = 6–12 per group. *P < 0.00001; **P < 0.00002. (B) KLF2 and p300 cooperate to induce the eNOS promoter. Cotransfection studies were performed in COS-7 cells. Cotransfection of KLF2 and p300 induces eNOS promoter activity greater than either factor alone. n = 6–12 per group; *P < 0.001; **P < 0.0001; #P < 0.05. (C) KLF2 and p300 interact. GST fusion proteins were generated for KLF2 and p300. Both KLF2 and KLFΔZnF can interact with p300 (top). Conversely, KLF2 interacts specifically with the NH2 terminus of p300. (D) KLF2 and p300 interact in cells. COS-7 cells were transfected with HA-KLF2 and Flag-p300. Immunoprecipitation was performed using the α-Flag antibody (or isotype control) followed by Western blot for KLF2 with α-HA antibody.
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fig5: KLF2 interacts directly with p300. (A) p300 rescues KLF2-mediated inhibition. Transient transfection studies with the indicated plasmids were performed in COS-7 cells. Cotransfection studies demonstrate that p300 can rescue KLF2 and KLFΔZnF-mediated inhibition of the NF-κB concatemer. n = 6–12 per group. *P < 0.00001; **P < 0.00002. (B) KLF2 and p300 cooperate to induce the eNOS promoter. Cotransfection studies were performed in COS-7 cells. Cotransfection of KLF2 and p300 induces eNOS promoter activity greater than either factor alone. n = 6–12 per group; *P < 0.001; **P < 0.0001; #P < 0.05. (C) KLF2 and p300 interact. GST fusion proteins were generated for KLF2 and p300. Both KLF2 and KLFΔZnF can interact with p300 (top). Conversely, KLF2 interacts specifically with the NH2 terminus of p300. (D) KLF2 and p300 interact in cells. COS-7 cells were transfected with HA-KLF2 and Flag-p300. Immunoprecipitation was performed using the α-Flag antibody (or isotype control) followed by Western blot for KLF2 with α-HA antibody.

Mentions: The data presented above demonstrate that KLF2 can induce eNOS and inhibit cytokine-mediated induction of endothelial adhesion molecules. The induction of eNOS requires DNA binding, whereas the inhibition of VCAM-1 does not. Furthermore, the inhibitory effect occurs in the absence of any effect on p50/p65 expression or DNA binding. To reconcile these observations, we considered the possibility that KLF2 may recruit away from NF-κB, a critical cofactor that is required for its function. Previous studies have demonstrated that NF-κB activity is critically modulated by several cofactors such as p300/CBP, PCAF-1, and SRC-1, (47, 48, 8). We chose to focus on p300/CBP, since it is essential for optimal NF-κB–mediated transcriptional activity in endothelial cells (8). In addition, previous studies demonstrate that the interaction of other KLF family members with p300 is important for the ability of these factors to transactivate reporter genes (49–51). Consistent with this hypothesis, cotransfection of CBP/p300 rescued both the KLF2 and KLF2ΔZnF-mediated repression of the NF-κB concatemer (Fig. 5 A). Furthermore, cotransfection of KLF2 (but not ZnF) and CBP/p300 augmented eNOS promoter activity to a greater degree than either factor alone (Fig. 5 B). These data suggest that KLF2 and p300 can work in a cooperative fashion.


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 interacts directly with p300. (A) p300 rescues KLF2-mediated inhibition. Transient transfection studies with the indicated plasmids were performed in COS-7 cells. Cotransfection studies demonstrate that p300 can rescue KLF2 and KLFΔZnF-mediated inhibition of the NF-κB concatemer. n = 6–12 per group. *P < 0.00001; **P < 0.00002. (B) KLF2 and p300 cooperate to induce the eNOS promoter. Cotransfection studies were performed in COS-7 cells. Cotransfection of KLF2 and p300 induces eNOS promoter activity greater than either factor alone. n = 6–12 per group; *P < 0.001; **P < 0.0001; #P < 0.05. (C) KLF2 and p300 interact. GST fusion proteins were generated for KLF2 and p300. Both KLF2 and KLFΔZnF can interact with p300 (top). Conversely, KLF2 interacts specifically with the NH2 terminus of p300. (D) KLF2 and p300 interact in cells. COS-7 cells were transfected with HA-KLF2 and Flag-p300. Immunoprecipitation was performed using the α-Flag antibody (or isotype control) followed by Western blot for KLF2 with α-HA antibody.
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Related In: Results  -  Collection

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fig5: KLF2 interacts directly with p300. (A) p300 rescues KLF2-mediated inhibition. Transient transfection studies with the indicated plasmids were performed in COS-7 cells. Cotransfection studies demonstrate that p300 can rescue KLF2 and KLFΔZnF-mediated inhibition of the NF-κB concatemer. n = 6–12 per group. *P < 0.00001; **P < 0.00002. (B) KLF2 and p300 cooperate to induce the eNOS promoter. Cotransfection studies were performed in COS-7 cells. Cotransfection of KLF2 and p300 induces eNOS promoter activity greater than either factor alone. n = 6–12 per group; *P < 0.001; **P < 0.0001; #P < 0.05. (C) KLF2 and p300 interact. GST fusion proteins were generated for KLF2 and p300. Both KLF2 and KLFΔZnF can interact with p300 (top). Conversely, KLF2 interacts specifically with the NH2 terminus of p300. (D) KLF2 and p300 interact in cells. COS-7 cells were transfected with HA-KLF2 and Flag-p300. Immunoprecipitation was performed using the α-Flag antibody (or isotype control) followed by Western blot for KLF2 with α-HA antibody.
Mentions: The data presented above demonstrate that KLF2 can induce eNOS and inhibit cytokine-mediated induction of endothelial adhesion molecules. The induction of eNOS requires DNA binding, whereas the inhibition of VCAM-1 does not. Furthermore, the inhibitory effect occurs in the absence of any effect on p50/p65 expression or DNA binding. To reconcile these observations, we considered the possibility that KLF2 may recruit away from NF-κB, a critical cofactor that is required for its function. Previous studies have demonstrated that NF-κB activity is critically modulated by several cofactors such as p300/CBP, PCAF-1, and SRC-1, (47, 48, 8). We chose to focus on p300/CBP, since it is essential for optimal NF-κB–mediated transcriptional activity in endothelial cells (8). In addition, previous studies demonstrate that the interaction of other KLF family members with p300 is important for the ability of these factors to transactivate reporter genes (49–51). Consistent with this hypothesis, cotransfection of CBP/p300 rescued both the KLF2 and KLF2ΔZnF-mediated repression of the NF-κB concatemer (Fig. 5 A). Furthermore, cotransfection of KLF2 (but not ZnF) and CBP/p300 augmented eNOS promoter activity to a greater degree than either factor alone (Fig. 5 B). These data suggest that KLF2 and p300 can work in a cooperative fashion.

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