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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 regulates eNOS expression and promoter activity. (A) KLF2 induces eNOS expression. HUVECs were infected with control (Ad-GFP; control) and KLF2 adenovirus (KLF2) at 10 MOI for 24 h and assessed for eNOS mRNA (Northern) and protein expression (Western). Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 induces eNOS enzymatic activity. HUVECs were infected with indicated adenoviral constructs, and eNOS activity was assessed by measuring the conversion of L-[3H] arginine to L-[3H] citrulline. *P < 0.0001. (C) Deletion and mutational analyses of eNOS promoter. Transient transfection and mutational studies identify the KLF site between −644 and −652 as critical for KLF2-mediated induction of the eNOS promoter activity. Top graph indicate studies in COS-7, whereas bottom graph is in BAECs. n = 6–12 per group. *P < 0.00005; **P < 0.00001; ‡P < 0.001. (D) KLF2 induction of the eNOS promoter is dependent on DNA binding. Transient transfection studies performed in BAECs and COS-7 cells demonstrate that KLF2 but not mutant constructs (DBD–DNA binding domain; ZnF–DNA binding domain alone; KLF2ΔZnF–non-DNA binding domain) can induce the eNOS promoter. n = 6–12 per group. *P < 0.0001. (E) KLF2 binds the eNOS promoter. Probe is derived from the sequence between −644 and −652 in the eNOS promoter. Gel shift studies were performed using GST–KLF2 fusion protein (left) and nuclear extracts from adenovirally overexpressed GFP (Ctrl) or KLF2 (right). Arrow denotes major retarded band, and arrowhead identifies the supershifted band using α-KLF2 antibody.
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fig2: KLF2 regulates eNOS expression and promoter activity. (A) KLF2 induces eNOS expression. HUVECs were infected with control (Ad-GFP; control) and KLF2 adenovirus (KLF2) at 10 MOI for 24 h and assessed for eNOS mRNA (Northern) and protein expression (Western). Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 induces eNOS enzymatic activity. HUVECs were infected with indicated adenoviral constructs, and eNOS activity was assessed by measuring the conversion of L-[3H] arginine to L-[3H] citrulline. *P < 0.0001. (C) Deletion and mutational analyses of eNOS promoter. Transient transfection and mutational studies identify the KLF site between −644 and −652 as critical for KLF2-mediated induction of the eNOS promoter activity. Top graph indicate studies in COS-7, whereas bottom graph is in BAECs. n = 6–12 per group. *P < 0.00005; **P < 0.00001; ‡P < 0.001. (D) KLF2 induction of the eNOS promoter is dependent on DNA binding. Transient transfection studies performed in BAECs and COS-7 cells demonstrate that KLF2 but not mutant constructs (DBD–DNA binding domain; ZnF–DNA binding domain alone; KLF2ΔZnF–non-DNA binding domain) can induce the eNOS promoter. n = 6–12 per group. *P < 0.0001. (E) KLF2 binds the eNOS promoter. Probe is derived from the sequence between −644 and −652 in the eNOS promoter. Gel shift studies were performed using GST–KLF2 fusion protein (left) and nuclear extracts from adenovirally overexpressed GFP (Ctrl) or KLF2 (right). Arrow denotes major retarded band, and arrowhead identifies the supershifted band using α-KLF2 antibody.

Mentions: Because KLF2 was induced by LSS, we considered the possibility that this transcription factor may regulate the expression of certain LSS-inducible genes. Previous studies have identified several genes that are induced by LSS such as eNOS, a central regulator of endothelial cell function (10). As shown in Fig. 2 A, adenoviral overexpression of KLF2 resulted in a marked induction of eNOS mRNA and protein (Fig. 2 A). This effect was specific, since no induction was observed on the expression of another LSS-inducible gene COX-2 (not depicted). Furthermore, we consistently noted that exogenous KLF2 (Fig. 2 A, exo-KLF2) inhibited the expression of endogenous KLF2 (Fig. 2 A, endo-KLF2), perhaps via a negative feedback mechanism.


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 regulates eNOS expression and promoter activity. (A) KLF2 induces eNOS expression. HUVECs were infected with control (Ad-GFP; control) and KLF2 adenovirus (KLF2) at 10 MOI for 24 h and assessed for eNOS mRNA (Northern) and protein expression (Western). Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 induces eNOS enzymatic activity. HUVECs were infected with indicated adenoviral constructs, and eNOS activity was assessed by measuring the conversion of L-[3H] arginine to L-[3H] citrulline. *P < 0.0001. (C) Deletion and mutational analyses of eNOS promoter. Transient transfection and mutational studies identify the KLF site between −644 and −652 as critical for KLF2-mediated induction of the eNOS promoter activity. Top graph indicate studies in COS-7, whereas bottom graph is in BAECs. n = 6–12 per group. *P < 0.00005; **P < 0.00001; ‡P < 0.001. (D) KLF2 induction of the eNOS promoter is dependent on DNA binding. Transient transfection studies performed in BAECs and COS-7 cells demonstrate that KLF2 but not mutant constructs (DBD–DNA binding domain; ZnF–DNA binding domain alone; KLF2ΔZnF–non-DNA binding domain) can induce the eNOS promoter. n = 6–12 per group. *P < 0.0001. (E) KLF2 binds the eNOS promoter. Probe is derived from the sequence between −644 and −652 in the eNOS promoter. Gel shift studies were performed using GST–KLF2 fusion protein (left) and nuclear extracts from adenovirally overexpressed GFP (Ctrl) or KLF2 (right). Arrow denotes major retarded band, and arrowhead identifies the supershifted band using α-KLF2 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211816&req=5

fig2: KLF2 regulates eNOS expression and promoter activity. (A) KLF2 induces eNOS expression. HUVECs were infected with control (Ad-GFP; control) and KLF2 adenovirus (KLF2) at 10 MOI for 24 h and assessed for eNOS mRNA (Northern) and protein expression (Western). Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 induces eNOS enzymatic activity. HUVECs were infected with indicated adenoviral constructs, and eNOS activity was assessed by measuring the conversion of L-[3H] arginine to L-[3H] citrulline. *P < 0.0001. (C) Deletion and mutational analyses of eNOS promoter. Transient transfection and mutational studies identify the KLF site between −644 and −652 as critical for KLF2-mediated induction of the eNOS promoter activity. Top graph indicate studies in COS-7, whereas bottom graph is in BAECs. n = 6–12 per group. *P < 0.00005; **P < 0.00001; ‡P < 0.001. (D) KLF2 induction of the eNOS promoter is dependent on DNA binding. Transient transfection studies performed in BAECs and COS-7 cells demonstrate that KLF2 but not mutant constructs (DBD–DNA binding domain; ZnF–DNA binding domain alone; KLF2ΔZnF–non-DNA binding domain) can induce the eNOS promoter. n = 6–12 per group. *P < 0.0001. (E) KLF2 binds the eNOS promoter. Probe is derived from the sequence between −644 and −652 in the eNOS promoter. Gel shift studies were performed using GST–KLF2 fusion protein (left) and nuclear extracts from adenovirally overexpressed GFP (Ctrl) or KLF2 (right). Arrow denotes major retarded band, and arrowhead identifies the supershifted band using α-KLF2 antibody.
Mentions: Because KLF2 was induced by LSS, we considered the possibility that this transcription factor may regulate the expression of certain LSS-inducible genes. Previous studies have identified several genes that are induced by LSS such as eNOS, a central regulator of endothelial cell function (10). As shown in Fig. 2 A, adenoviral overexpression of KLF2 resulted in a marked induction of eNOS mRNA and protein (Fig. 2 A). This effect was specific, since no induction was observed on the expression of another LSS-inducible gene COX-2 (not depicted). Furthermore, we consistently noted that exogenous KLF2 (Fig. 2 A, exo-KLF2) inhibited the expression of endogenous KLF2 (Fig. 2 A, endo-KLF2), perhaps via a negative feedback mechanism.

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