Cortactin Modulates Lung Endothelial Apoptosis Induced by Cigarette Smoke

Abstract

Cigarette smoke (CS) is the primary cause of Chronic Obstructive Pulmonary Disease (COPD), and an important pathophysiologic event in COPD is CS-induced apoptosis in lung endothelial cells (EC). Cortactin (CTTN) is a cytoskeletal actin-binding regulatory protein with modulation by Src-mediated tyrosine phosphorylation. Based upon data demonstrating reduced CTTN mRNA levels in the lungs of smokers compared to non-smokers, we hypothesized a functional role for CTTN in CS-induced mitochondrial ROS generation and apoptosis in lung EC. Exposure of cultured human lung EC to CS condensate (CSC) led to the rearrangement of the actin cytoskeleton and increased CTTN tyrosine phosphorylation (within hours). Exposure to CS significantly increased EC mitochondrial ROS generation and EC apoptosis. The functional role of CTTN in these CSC-induced EC responses was explored using cortactin siRNA to reduce its expression, and by using a blocking peptide for the CTTN SH3 domain, which is critical to cytoskeletal interactions. CTTN siRNA or blockade of its SH3 domain resulted in significantly increased EC mitochondrial ROS and apoptosis and augmented CSC-induced effects. Exposure of lung EC to e-cigarette condensate demonstrated similar results, with CTTN siRNA or SH3 domain blocking peptide increasing lung EC apoptosis. These data demonstrate a novel role for CTTN in modulating lung EC apoptosis induced by CS or e-cigarettes potentially providing new insights into COPD pathogenesis.

Keywords: COPD; cytoskeleton; e-cigarette; endothelium; lung injury; mitochondrial ROS.

Figure 1

Cortactin mRNA levels are decreased in human lung tissues from smokers compared to non-smokers. Cortactin (CTTN) mRNA levels were analyzed by qPCR in human lung tissues from current versus never/former smokers. The bar graph depicts fold-changes in CTTN mRNA expression normalized to the housekeeping gene, 18S rRNA. N = 3–4, * p < 0.05.

Figure 2

Cigarette smoke condensate induces cytoskeletal rearrangement in human lung endothelial cells. (A) HPAECs were treated with vehicle (DMSO) or CSC (40 µg/mL) for 6 h, fixed and subjected to immunofluorescence analysis. Confocal images were taken at 40× after staining with Alexa488 -CTTN (green), Alex 594- Phalloidin (F-actin staining, red), and DAPI (nucleus staining, blue). F-actin and total cortactin (T-CTTN) overlap was quantified. (B) HPAECs were treated with CSC (40 µg/mL, 24 h) and cell lysates were subjected to western blotting analysis for T-CTTN protein expression. Shown are representative blots and densitometry analysis. N = 3, * p < 0.05.

Figure 3

CSC induces CTTN tyrosine phosphorylation in human lung endothelial cells. HPAECs were treated with CSC (40 μg/mL) for 15–30 min. Tyrosine phosphorylation of CTTN was assessed at Y421/466/486 sites using phospho-specific antibodies for each site. (A) Representative blots of CTTN Y421/466/486 phosphorylation in cell lysates upon CSC challenge. (B) quantification of Y421/466/486 by densitometric analysis at 30 min for all 3 sites; data were normalized to total CTTN and pooled from 3 independent experiments. * p < 0.05.

Figure 4

Inhibition of CTTN expression or SH3 domain blockade augments CSC-induced MitoROS in lung EC. (A) HPAECs were transfected with siRNA (control or CTTN) for 48 h followed by CSC (40 µg/mL) for 2 h. (B) HPAECs were pre-treated with peptide control or CTTN blocking peptide (CBP) for 45 min followed by CSC challenge for 2 h. Mitochondrial superoxide production was assayed by the MitoSOX TM Red reagent. Shown are the representative confocal images of mitoROS staining (A,B, left images). Quantification of ROS intensity was performed in 30 cells from 5–8 different fields for three independent experiments. ** p < 0.01. (C) Representative western blot demonstrating reduced CTTN expression in lung EC after siRNA transfection.

Figure 5

CSC induces apoptosis in lung EC. HPAECs were treated with CSC (40 µg/mL) for 24 h. Apoptosis was assessed by cleavage of apoptotic marker PARP1 and by flow cytometry of Annexin-V/7-AAD double-positive cells (% late apoptotic cells) (A) protein expression of cleaved PARP1 in cell lysates upon CSC challenge. Shown is a representative blot from three independent experiments and the quantification of cleaved PARP1 by densitometry. Data were normalized to β-actin. (B) HPAECs challenged with CSC were analyzed by flow cytometry for apoptosis. Shown are representative dot plots of cells stained with Annexin V and 7-AAD (left) and bar graphs depicting normalized percentages of late apoptotic cells under each condition (right). ** p < 0.01.

Figure 6

CTTN expression regulates CSC-induced apoptosis in lung EC. HPAECs were transfected with siRNA (control or CTTN) before CSC challenge (40 μg/mL, 24 h). Apoptosis was assessed by flow cytometry. (A) Shown are representative dot plots of cells stained with Annexin V and 7-AAD, and (B) Bar graphs represent the percentage of double-positive cells (late apoptosis) pooled from 4 independent experiments. * p < 0.05, ** p < 0.01, ^# p = 0.053.

Figure 7

Blocking the SH3 domain of CTTN accentuates CSC-induced apoptosis in lung EC. HPAECs were pre-treated with peptide control or CTTN blocking peptide (CBP) for 45 min followed by CSC challenge (40 μg/mL, 24 h). Apoptosis was assessed by flow cytometry. (A) Shown are representative dot plots of cells stained with Annexin V and 7-AAD, and (B) Bar graphs represent the percentage of double-positive cells (late apoptosis) pooled from 4 independent experiments. * p < 0.05, ** p < 0.01.

Figure 8

CTTN expression regulates e-cigarette-induced apoptosis in lung EC. HPAECs were transfected with siRNA (control or CTTN) before e-cigarette (E-cig) challenge (50 μg/mL, 24 h). Apoptosis was assessed by flow cytometry. (A) Shown are representative dot plots of cells stained with Annexin V and 7-AAD, and (B) Bar graphs represent the normalized percentage of double-positive cells (late apoptosis) pooled from 3 independent experiments. ** p < 0.01, ^# p = 0.07.

Figure 9

Blocking the SH3 domain of CTTN accentuates e-cigarette-induced apoptosis in lung EC. HPAECs were pre-treated with peptide control or CTTN blocking peptide (CBP) for 45 min followed by e-cigarette (E-cig) challenge (50 μg/mL, 24 h). Apoptosis was assessed by flow cytometry (A) Shown are representative dot plots of cells stained with Annexin V and 7-AAD and (B) Bar graphs represent the normalized percentage of double-positive cells (late apoptosis) pooled from 3 independent experiments. ** p < 0.01, ^# p = 0.06.

Figure 10

Schema illustrating the mechanistic role of CTTN in mitoROS and lung endothelial apoptosis. CTTN contributes to cigarette smoke-induced lung injury by causing rearrangement of actin and activating Src-mediated phosphorylation at the major sites 421, 466, and 486. Cigarette smoke induces mitochondrial ROS production and lung endothelial apoptosis. In vitro studies showed altering CTTN expression or blocking its SH3 interaction domain enhanced mitochondrial ROS production and endothelial apoptosis. Here, we explore the undescribed mechanism of CTTN in mtROS induced apoptosis.