Bnip3 regulates airway smooth muscle cell focal adhesion and proliferation

Abstract

Increased airway smooth muscle (ASM) mass is a key contributor to airway narrowing and airway hyperresponsiveness in asthma. Besides conventional pathways and regulators of ASM proliferation, recent studies suggest that changes in mitochondrial morphology and function play a role in airway remodeling in asthma. In this study, we aimed at determining the role of mitochondrial Bcl-2 adenovirus E1B 19 kDa-interacting protein, Bnip3, in the regulation of ASM proliferation. Bnip3 is a member of the Bcl-2 family of proteins critical for mitochondrial health, mitophagy, and cell survival/death. We found that Bnip3 expression is upregulated in ASM cells from asthmatic donors compared with that in ASM cells from healthy donors and transient downregulation of Bnip3 expression in primary human ASM cells using an siRNA approach decreased cell adhesion, migration, and proliferation. Furthermore, Bnip3 downregulation altered the structure (electron density) and function (cellular ATP levels, membrane potential, and reacitve oxygen species generation) of mitochondria and decreased expression of cytoskeleton proteins vinculin, paxillin, and actinin. These findings suggest that Bnip3 via regulation of mitochondria functions and expression of adhesion proteins regulates ASM adhesion, migration, and proliferation. This study reveals a novel role for Bnip3 in ASM functions and establishes Bnip3 as a potential target in mitigating ASM remodeling in asthma.

Keywords: Bnip3; asthma; growth; remodeling.

Fig. 1.

Upregulation of Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) protein expression in airway smooth muscle (ASM) cells from asthmatic donors. Proteins were harvested from healthy (He) and asthmatic (As) human ASM cells. A: Bnip3 protein levels were determined by immunoblotting. GAPDH was used as an internal control for protein loading. He, healthy human ASM cells. B: quantification of immunoblots showing Bnip3 protein levels in He and As ASM cells (n = 5; *P < 0.05 He vs. As).

Fig. 2.

Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) downregulation impaired PDGF-induced human airway smooth muscle (ASM) proliferation and migration. Bnip3 expression was downregulated in human ASM cells by transient transfection of Bnip3 siRNA. Scrambled siRNA-transfected ASM cells were used as control. A: cells were stimulated with either 10 ng/mL of PDGF or vehicle for 24, 48, and 72 h in serum-free F-12 media and cell proliferation was measured by CyQuant assay (n = 12 measurements from 4 different ASM lines, #P < 0.05, significance relative to control siRNA with vehicle treatment condition. *P < 0.05, relative to control siRNA with 72-h PDGF treatment condition; ns: not significant). AU, arbitrary units. B: following transfection of siRNA for 24 h, cells were cultured in serum free F-12 media. Cells were scraped at the center of the dish and were treated with 10 ng/mL of PDGF or vehicle for 30 h. Light microscope images were taken before and after 30 h treatment. Bnip3 KD, Bnip3 knockdown. C: the wound area (between lines) was measured before and after PDGF treatment as percentage of control siRNA area without treatment (n = 24 measurements from 6 different ASM cell lines. *P < 0.05 Bnip3 siRNA vs. scrambled siRNA with 30 h of PDGF treatment). D: immunoblot showing efficiency of Bnip3 knockdown. C, control, scrambled siRNA; B, Bnip3 siRNA.

Fig. 3.

Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) downregulation negatively affects human airway smooth muscle (ASM) cell adhesion and spreading on tissue culture dish. Cell adhesion and spreading were assayed in human ASM cells 72 h after transient transfection of scrambled and Bnip3 siRNA. A–C: cell adhesion and spreading are expressed as cell index and data are represented as real-time (A), peak (B), and integrated (area under the curve, AUC) (C) cell index (*P < 0.05, Bnip3 vs. scrambled siRNA; n = 3 ASM lines from 3 different donors). D: representative confocal images of rhodamine-phalloidin staining showing cell adhesion and spreading 1 and 3 h after seeding cells were seeded on culture dish. Bar = 100 μm. E: immunoblot showing efficiency of Bnip3 knockdown. C, control, scrambled siRNA; B, Bnip3 siRNA.

Fig. 4.

F-actin and mitochondrial functions were compromised in Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) siRNA-transfected human airway smooth muscle (ASM) cells. A: human ASM cells were transfected with Bnip3 and scrambled siRNA for 72 h. Cells were stained with rhodamine-phalloidin and DAPI. Representative images of actin stress fiber were acquired by confocal microscopy at excitation of 540 nm. Bnip3 KD, Bnip3 knockdown. Bar = 100 μm. B: representative transmission electron microscopy images (×11,000) showing mitochondrial structure in control and Bnip3 knockdown ASM cells (red arrow). C: representative confocal live-cell image of mitochondria labeled by MitoTracker green. Bar = 20 μm. D: Western blot for determining protein levels of Bnip3, Mfn1, Mfn2, and DLP1 using β-actin as loading control. E–G: mitochondrial functions were assessed by determining cellular ATP levels (E) using a luciferase assay (n = 18 measurements from 6 different ASM cell lines, *P < 0.05, significance relative to scrambled siRNA), mitochondrial membrane potential (F) using tetramethyl rhodamine ester (TMRE) by confocal live-cell imaging (n = 29 cells from 3 separate experiments; *P < 0.05, significance relative to scrambled siRNA), and mitochondrial reactive oxygen species (G) by confocal live-cell imaging using MitoSox red (n = 43 cells from 3 different experiments; *P < 0.05, significance relative to scrambled siRNA). AU, arbitrary units.

Fig. 5.

Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) downregulation decreased human airway smooth muscle (ASM) cell adhesion and spreading on collagen I-coated culture plates and on hydrogels. Cell adhesion and spreading on collagen I and hydrogel matrixes were assessed in human ASM cells 72 h after transient transfection of scrambled and Bnip3 siRNA. A–C: real-time (A), peak (B), and integrated (area under the curve, AUC) (C) measurement of cell adhesion and spreading on collagen were performed using xCELLigence system (n = 3; *P < 0.05 significance relative to scrambled siRNA). D and E: representative confocal images of rhodamine-phalloidin staining showing cell adhesion and spreading 3 h after cells were seeded on hydrogel. DAPI was used as nuclear staining. Bar = 100 μm.

Fig. 6.

Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (Bnip3) downregulation in human airway smooth muscle (ASM) cells decreased paxillin, vinculin, and α-actinin protein levels. Expression of focal adhesion proteins and paxillin puncta in scrambled and Bnip3 siRNA-transfected human ASM cells. A: Western blot showing the protein levels of paxillin (pax), vinculin (vin), α-actinin (act), tensin 2 (ten), talin2 (tal), and Bnip3 using β-actin as loading control. B: quantified data with scrambled siRNA levels set at 1.0. *P < 0.05 significance relative to scrambled siRNA. C, scrambled siRNA; B, Bnip3 siRNA. C: representative confocal images of immunostaining for paxillin (green) in control (left panels) and Bnip3 knockdown (right panels; Bnip3 KD) human ASM cells on culture dish (top two panels; bar = 100 μm) and hydrogel (middle and bottom two panels; bar = 10 μm). Rhodamine (red) and DAPI (blue) were used as counterstaining for F-actin and nucleus, respectively.