Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS by Weiye Wang, Chang Hoon Ha, Bong Sook Jhun, Chelsea Wong, Mukesh K. Jain, and Zheng-Gen Jin Blood Volume 115(14):2971-2979 April 8, 2010 ©2010 by American Society of Hematology

Fluid shear stress stimulated HDAC5 phosphorylation through a Ca2+/calmodulin–dependent pathway in endothelial cells. Fluid shear stress stimulated HDAC5 phosphorylation through a Ca2+/calmodulin–dependent pathway in endothelial cells. (A) HUVECs were exposed to flow for 0, 15, 30, 60, 120, and 240 minutes. (B-C) HUVECs were exposed to either flow or static condition for 1 hour with the pretreatment of dimethyl sulfoxide (DMSO), KN-62 (30μM), CaM inhibitory peptide (60nM), W-13 (100μM), and BAPTA/AM (30μM) + EGTA (2μM) for 30 minutes. (D) HUVECs were infected with Ad-PKD-KN or Ad-LacZ for 24 hours before the 1-hour flow or static treatment. HDAC5 phosphorylation (Ser259 and Ser498) and HDAC5 were probed by phospho-specific HDAC5 antibody and HDAC5 antibody. *P < .05 compared with control. #P < .05 compared with cells treated with flow + DMSO/Ad-LacZ. Error bars represent ±SEM. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

Fluid shear stress induced phosphorylation-dependent HDAC5 nuclear export. Fluid shear stress induced phosphorylation-dependent HDAC5 nuclear export. (A-B) HUVECs were infected with Ad-GFP-HDAC5 or Ad-GFP-HDAC5-S/A. Twenty-four hours later, cells were conditioned with flow (left lane) or static (right lane) for the time indicated in each image. Images were captured at a magnification of ×60, using a fluorescence microscope. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

HDAC5 down-regulated flow-mediated KLF2 expression. HDAC5 down-regulated flow-mediated KLF2 expression. (A-B) HUVECs were exposed to 1-hour flow or to static conditions after 24-hour infection with Ad-HDAC5-S/A or Ad-LacZ (control). KLF2 mRNA levels were assayed by real-time reverse-transcription (RT)–PCR (A) and protein levels were detected by KLF2 antibody (B). (C-D) HUVECs were exposed to flow or to static conditions after pretreatment with DMSO (control), W-13 (100μM), or PD 98059 (another control; 30μM) for 30 minutes. KLF2 mRNA level were assayed by RT-PCR. Vertical lines have been inserted to indicate repositioned gel lanes. *P < .05 compared with control. #P < .05 compared with that treated with flow + Ad-LacZ or DMSO. Error bars represent ±SEM Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

HDAC5 negatively regulated MEF2 transcriptional activation and KFL2 expression. HDAC5 negatively regulated MEF2 transcriptional activation and KFL2 expression. (A) HUVECs were infected with Ad-Flag-HDAC5. Twenty-four hours later, cells were conditioned under static conditions (first lane) or flow (second lane) for 1 hour. Immunoprecipitation was performed with Flag antibody–conjugated agarose. MEF2C and Flag-HDAC5 were detected by probing with anti-MEF2C and anti-Flag antibodies. (B-C) HUVECs were transfected with 3xMEF2-luciferase or −221/+1 KLF2 promoter or −221/+1 MEF2 binding site–deleted KLF2 promoter reporter gene together with HDAC5-S/A or pcDNA as control. Twenty-four hours later, cells were conditioned with static or flow for 8 hours. Luciferase activities were assayed by Promega Luciferase assay kit. *P < .05 compared with control (static). #P < .05 compared with that treated with flow + pcDNA. Error bars represent ±SEM. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

HDAC5 down-regulated flow-mediated eNOS expression. HDAC5 down-regulated flow-mediated eNOS expression. (A-B) HUVECs were exposed to 8 or 24 hours of flow or to static conditions 24 hours after infection with Ad-HDAC5-S/A or Ad-LacZ (control). eNOS mRNA levels were assayed by real-time RT-PCR (A) and protein levels were detected by eNOS antibody (B). Vertical lines have been inserted to indicate repositioned gel lanes. *P < .05 compared with control (static). #P < .05 compared with that treated with flow + Ad-LacZ. Error bars represent ±SEM. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

HDAC5-dependent pathway modulated flow anti-inflammation in endothelial cells. HDAC5-dependent pathway modulated flow anti-inflammation in endothelial cells. (A-B) Overexpression of Ad-HDAC5-S/A attenuated flow anti-inflammation. HUVECs were infected with Ad-HDAC5-S/A or Ad-LacZ as control. Twenty-four hours later, cells were subjected to flow or to static conditions for 24 hours followed by the treatment of TNFα or vehicle for 6 hours. U937 monocytes were added to HUVECs and incubated for 30 minutes. HUVECs were washed 3 times. The adherent monocytes were counted in 5 randomly optical fields in each dish. *P < .05, flow + TNFα + Ad-HDAC5-S/A vs flow + TNFα + Ad-LacZ. Error bars represent ±SEM. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology

Model for HDAC5 modulated flow-induced KLF2 and eNOS expression. Model for HDAC5 modulated flow-induced KLF2 and eNOS expression. Fluid shear stress stimulates HDAC5 phosphorylation and nuclear export through a Ca2+/calmodulin-dependent pathway, freeing MEF2 to activate KLF2 expression and its downstream gene expression. Weiye Wang et al. Blood 2010;115:2971-2979 ©2010 by American Society of Hematology