KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation

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.

Figure 1.

KLF2 expression in endothelial cells in response to biochemcial and biomechanical stimuli. (A) Inhibition of KLF2 by IL-1β. HUVECs were treated with IL-1β (10 U/ml) for the indicated times, and expression was assessed by Taqman assay. *P < 0.05. (B) Induction of KLF2 by LSS. HUVEC monolayers were exposed to laminar flow (10 dynes/cm²) for 24 h, and the relative expression of KLF2 was assessed by Taqman assay. *P < 0.05.

Figure 2.

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.

Figure 2.

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.

Figure 3.

Effect of KLF2 on cytokine-mediated induction of adhesion molecules. (A) KLF2 inhibits VCAM-1 and E-selectin but not ICAM-1 mRNA. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2) at the indicated dose, stimulated with IL-1β for 4 h, and total RNA was assessed for adhesion molecule expression. In contrast to VCAM-1 and E-selectin, no effect is observed on ICAM-1 expression. Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 inhibits IL-1β–mediated induction of VCAM-1 and E-selectin protein levels. Experiments were performed as described in A except cells were harvested for total protein and Western blot analysis was performed. (C) KLF2 inhibits VCAM-1 and E-selectin in response to multiple cytokines. HUVECs infected with the adenovirus at the indicated dose, stimulated with cytokine for 4 h, and total RNA was assessed for adhesion molecule expression. (D) KLF2 inhibits IL-1β–mediated T cell attachment and rolling to endothelial cells. HUVECs infected with the indicated adenovirus (Ctrl, Ad-GFP), stimulated with IL-1β, and then perfused with T cells under flow conditions (0.75 dynes/cm²). *P < 0.05.

Figure 3.

Effect of KLF2 on cytokine-mediated induction of adhesion molecules. (A) KLF2 inhibits VCAM-1 and E-selectin but not ICAM-1 mRNA. HUVECs were infected with the adenovirus (C, Ad-GFP; K2, Ad-KLF2) at the indicated dose, stimulated with IL-1β for 4 h, and total RNA was assessed for adhesion molecule expression. In contrast to VCAM-1 and E-selectin, no effect is observed on ICAM-1 expression. Exo-KLF2 refers to exogenously expressed mouse KLF2. Endo-KLF2 refers to endogenous human KLF2. (B) KLF2 inhibits IL-1β–mediated induction of VCAM-1 and E-selectin protein levels. Experiments were performed as described in A except cells were harvested for total protein and Western blot analysis was performed. (C) KLF2 inhibits VCAM-1 and E-selectin in response to multiple cytokines. HUVECs infected with the adenovirus at the indicated dose, stimulated with cytokine for 4 h, and total RNA was assessed for adhesion molecule expression. (D) KLF2 inhibits IL-1β–mediated T cell attachment and rolling to endothelial cells. HUVECs infected with the indicated adenovirus (Ctrl, Ad-GFP), stimulated with IL-1β, and then perfused with T cells under flow conditions (0.75 dynes/cm²). *P < 0.05.

Figure 4.

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.

Figure 4.

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.

Figure 5.

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.

Figure 5.

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.