Nicotine-mediated induction of E-selectin in aortic endothelial cells requires Src kinase and E2F1 transcriptional activity

Abstract

Smoking is highly correlated with enhanced likelihood of atherosclerosis by inducing endothelial dysfunction. In endothelial cells, various cell-adhesion molecules including E-selectin, are shown to be upregulated upon exposure to nicotine, the addictive component of tobacco smoke; however, the molecular mechanisms underlying this induction are poorly understood. Here we demonstrate that nicotine-induced E-selectin transcription in human aortic endothelial cells (HAECs) could be significantly blocked by α7-nAChR subunit inhibitor, α-BT, Src-kinase inhibitor, PP2, or siRNAs against Src or β-Arrestin-1 (β-Arr1). Further, chromatin immunoprecipitations show that E-selectin is an E2F1 responsive gene and nicotine stimulation results in increased recruitment of E2F1 on E-selectin promoter. Inhibiting E2F1 activity using RRD-251, a disruptor of the Rb-Raf-1 kinase interaction, could significantly inhibit the nicotine-induced recruitment of E2F1 to the E-selectin promoter as well as E-selectin expression. Interestingly, stimulation of HAECs with nicotine results in increased adhesion of U937 monocytic cells to HAECs and could be inhibited by pre-treatment with RRD-251. Similarly, depletion of E2F1 or Src using RNAi blocked the increased adhesion of monocytes to nicotine-stimulated HAECs. These results suggest that nicotine-stimulated adhesion of monocytes to endothelial cells is dependent on the activation of α7-nAChRs, β-Arr1 and cSrc regulated increase in E2F1-mediated transcription of E-selectin gene. Therefore, agents such as RRD-251 that can target activity of E2F1 may have potential therapeutic benefit against cigarette smoke induced atherosclerosis.

Fig. 1. Signal transduction dependent induction of E-selectin transcription in response to nicotine stimulation

(A) Nicotine induced E-selectin transcription was inhibited by of α7-nAChR inhibitor α-BT or Src inhibitor PP2. Data represent the mean±SD (p < 0.05). (B) Nicotine induced E-selectin transcription was suppressed by β-Arr1 or Src siRNA. (C and D) Suppression of Src expression by Src siRNA (C) and suppression of β-Arr1 expression by β-Arr1 siRNA was evaluated by RT-PCR analysis.

Fig. 2. E-selectin promoter is E2F1 responsive

(A) Three putative E2F binding sites are shown in relation to TSS. (B) E-selectin was significantly upregulated at transcriptional level upon transient overexpression of E2F1 in HAECs (p<0.05). (C) ChIP assay showing the binding of endogenous E2F1 on E-selectin promoter in serum stimulated HAECs.

Fig. 3. RRD-251 inhibits nicotine mediated induction of E-selectin and monocyte binding

(A) Nicotine stimulated E2F1 binding on E-selectin promoter was suppressed by RRD-251 as seen in a ChIP assay. (B) Nicotine stimulated expression of E-selectin was significantly suppressed by RRD-251 in dose dependent manner. (C) U937 monocytic cells adhered on nicotine stimulated HAECs, which was significantly blocked by RRD-251 (p < 0.05).

Fig. 4. Adhesion of Monocytic cells on HAECs is dependent on Src and E2F1 activity

(A and B) siRNA mediated depletion of Src or E2F1 results in suppressed adhesion of U937 cells on nicotine stimulated HAECs. The image is a composite of fluorescent U937 cells adhered on a confluent monolayer of endothelial cells. (B) Bar graph representing the average number of U937 adhered on the HAECs. (C) Schematic of nicotine induced monocyte adhesion on endothelial cells. Nicotine stimulation results in α7-nAChR/β-Arr-1/Src/Raf-1-Rb/E2F1 mediated transcriptional upregulation of E-selectin in HAECs. Targeting β-Arr1, Src or E2F1 suppressed the expression of E-selectin which results in decreased monocyte adhesion.