KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium by Reinier A. Boon, Thomas A. Leyen, Ruud D. Fontijn, Joost O. Fledderus, Josefien M. C. Baggen, Oscar L. Volger, Geerten P. van Nieuw Amerongen, and Anton J. G. Horrevoets Blood Volume 115(12):2533-2542 March 25, 2010 ©2010 by American Society of Hematology

Time-course analysis after lentiviral KLF2 overexpression. Time-course analysis after lentiviral KLF2 overexpression. (A) HUVECs were transduced with mock (M) or KLF2 (K) lentivirus. After 24, 48, or 72 hours or 7 days, RNA was isolated, and microarray expression profiling was performed. A modified Venn analysis of the microarray expression data at 24, 48, 72, and 168 hours after lentiviral KLF2 transduction is displayed. Indicated are the numbers of genes per time point that meet the indicated criteria but do not meet the criteria for an earlier time point. Inflammatory genes, transforming growth factor-β (TGF-β) signaling genes and vasodilatation genes were previously published to be KLF2 dependent. HUVECs were transduced with mock or KLF2 lentivirus for 24, 48, and 72 hours, and levels of the indicated genes were measured by the use of real-time RT-PCR (n = 5; B) or Western blot and densitometric quantification (n = 3; C). α-Tubulin was used as a loading control. (D) Summary of levels of the different posttranslationally activated forms of FAK in KLF2-transduced compared with mock-transduced cells, as analyzed with the Kinex antibody microarray. The category axis shows antibody specificity for the indicated phosphorylated amino acid residue. Fold induction by KLF2 is indicated on the y-axis. *P < .05, **P < .01. Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

KLF2 induces early cytoskeleton changes, inhibits FAK S732 phosphorylation, and activates RhoA. KLF2 induces early cytoskeleton changes, inhibits FAK S732 phosphorylation, and activates RhoA. (A) HUVECs were cultured on glass coated with fibronectin and fixed after 24, 48, or 72 hours of lentiviral overexpression. Nuclei were stained with Hoechst 33342 (blue) and actin filaments with Phalloidin-TRITC (red), and fluorescent microscopy was performed. (B) Western blot analysis of levels of FAK phosphorylated on serine residue 732 in mock- (□) and KLF2-transduced HUVECs (■) at 24, 48, and 72 hours after transduction. α-Tubulin was used as a loading control. (Bottom) Densitometric quantification of the Western blot (n = 4). (C) Active RhoA was isolated with glutathione S-transferase–tagged Rhotekin, and a representative subsequent Western blot for RhoA is shown. Three independent experiments were quantified and corrected for α-tubulin. *P < .05, **P < .01. Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

KLF2 inhibits phosphorylation and nuclear localization of JNK in an actin-dependent manner. KLF2 inhibits phosphorylation and nuclear localization of JNK in an actin-dependent manner. (A) Fluorescent photomicrographs of mock- and KLF2-transduced cells treated with dimethyl sulfoxide (DMSO) or 200nmol/L Cytochalasin D (CytD) for 16 hours, demonstrating filamentous actin in red and nuclei in blue. (B) Western blot analysis of P-JNK1, 2, and 3 levels in mock- (□) and KLF2-transduced HUVECs (■) cultured in the presence of dimethyl sulfoxide (control) or cytochalasin D (200nmol/L) for 16 hours. α-Tubulin was used as a loading control. Quantifications are shown for 4 independent experiments. (C) Immunofluorescent photomicrographs of mock- and KLF2-transduced HUVECs, with phosphorylated JNK (P-JNK) shown in red and nuclei in blue. *P < .05. Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

KLF2 inhibits nuclear localization of P-c-Jun and P-ATF2 in an actin-dependent manner. KLF2 inhibits nuclear localization of P-c-Jun and P-ATF2 in an actin-dependent manner. Immunofluorescent photomicrographs and quantification thereof of mock- (A, E, I, M, Q, U, C, G, K, O, S, and W) and KLF2-transduced (B, F, J, N, R, V, D, H, L, P, T, and X) HUVECs treated with DMSO (A-B, E-F, I-J, M-N, Q-R, U-V) or cytochalasin D (200nM; C-D, G-H, K-L, O-P, S-T, W-X) for 16 hours. Phosphorylated c-Jun on serine residue 63 (A-H) or serine residue 73 (I-P) or phosphorylated ATF2 on threonine residue 71 (Q-X) are stained red and nuclei are stained blue with Hoechst 33 342 (E-H, M-P, and U-X). (Y) Average nuclear intensity was measured for the indicated fluorescent staining and condition (n = 5). For each staining, 1-way analysis of variance with Bonferroni correction for multiple comparisons was performed, comparing KLF2/DMSO vs Mock/DMSO and KLF2/CytD vs Mock/CytD. ***P < .001. N.S. indicates not significant (P > .05). Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

KLF2 induces distinct actin filament formation in a Rho-kinase–independent manner. KLF2 induces distinct actin filament formation in a Rho-kinase–independent manner. (A) Mock-transduced (left) and KLF2-transduced (right) HUVECs were fixed, and immunofluorescence microscopy was performed. Visualized in blue are nuclei (stained with Hoechst 33342), in red F-actin (phalloidin-tetramethyl rhodamine iso-thiocyanate [TRITC]), and in green focal adhesions (vinculin stained with Alexa647). The top images were taken at high-power magnification focused on the basal side of the cell, whereas the middle images were taken at approximately 4 μm higher magnification. The bottom images were also taken with focus on the basal side of the cell. Arrowheads indicate typical actin fibers for the given condition. (B) Mock- or KLF2-transduced HUVECs were stimulated for 24 hours with TNF-α (10 ng/mL, top left), Thrombin (1 U/mL, middle left), Y27632 (10μM, top right), or left unstimulated (bottom). Then, cells were fixed, and immunofluorescence microscopy was performed. Visualized in green are focal adhesions (vinculin), in red actin filaments (phalloidin-TRITC), and in blue nuclei (stained with Hoechst 33342; top 4 images) or P-MLC (bottom 2 images). (C) Mock- (left) and KLF2-transduced (right) HMVECs and HAECs were fixed, and immunofluorescence microscopy was performed. Visualized in blue are nuclei (stained with Hoechst 33342), in red F-actin (phalloidin-TRITC), and in green focal adhesions (vinculin stained with Alexa647). Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

KLF2 is essential for shear fiber formation, cell alignment, and inhibition of JNK signaling in vitro, and these shear fibers are very similar in endothelium in vivo. KLF2 is essential for shear fiber formation, cell alignment, and inhibition of JNK signaling in vitro, and these shear fibers are very similar in endothelium in vivo. (A) HUVECs transduced with either control siRNA lentivirus (filled bars) or KLF2-silencing lentivirus (open bars) were subjected to pulsatile shear stress and fixed. Differential interference contrast microscopy was performed, and approximately 100 cells per condition in 3 independent experiments were analyzed. Cell angle compared with the direction of the flow was measured and visualized in a bar graph. Differences in cell angles in the absence of KLF2 are not significant. (B) HUVECs were treated as in panel A, and immunofluorescence microscopy was performed as for Figure 5B. These images were acquired by merging the green channel (vinculin), the red channel (F-actin), which was acquired at 1 μm higher magnification than the green channel, and the blue channel (nuclei), which was taken at 4 μm higher magnification than the red channel. The flow direction was from left to right. The arrowheads indicate short basal actin filaments similar to those observed after KLF2 overexpression. (C) Intact rat renal arteries and saphenous veins were stained with phalloidin-TRITC (F-Actin, red) and Hoechst 33342 (Nuclei, blue) in situ and visualized by the use of immunofluorescence microscopy and computational deconvolution. The arrowheads show short basal actin filaments, similar to those observed after KLF2 overexpression. (D) Western blot analysis of P-JNK1 in HUVECs treated as in panels A and B. α-Tubulin was used as a loading control. Quantifications are shown for 4 independent experiments. *P < .05. Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology

Schematic representation of the proposed mechanism by which KLF2 inhibits JNK signaling and induces cell alignment through the actin cytoskeleton. Schematic representation of the proposed mechanism by which KLF2 inhibits JNK signaling and induces cell alignment through the actin cytoskeleton. Shear stress activates the MEK5/ERK5/MEF2 MAPK pathway that transcriptionally induces KLF2, which indirectly activates RhoA, which causes the formation of actin shear fibers. These fibers are both essential for the alignment in the direction of the flow and for inhibition of the FAK-JNK-c-Jun/ATF2 signaling pathway. Red arrows indicate activation; green blunted arrows indicate inhibition. Reinier A. Boon et al. Blood 2010;115:2533-2542 ©2010 by American Society of Hematology