Tensin1 expression and function in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies have shown significant associations between airflow obstruction or COPD with a non-synonymous SNP in the TNS1 gene, which encodes tensin1. However, the expression, cellular distribution and function of tensin1 in human airway tissue and cells are unknown. We therefore examined these characteristics in tissue and cells from controls and people with COPD or asthma. Airway tissue was immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression. Tensin1 expression was increased in the airway smooth muscle and lamina propria in COPD tissue, but not asthma, when compared to controls. Tensin1 was expressed in HASMCs and upregulated by TGFβ1. TGFβ1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised, and tensin1 depletion in HASMCs attenuated both αSMA expression and contraction of collagen gels. In summary, tensin1 expression is increased in COPD airways, and may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs.


Subjects
For the study of human airway smooth muscle cells (HASMCs) in culture, healthy control subjects and subjects with COPD or asthma were recruited from respiratory clinics and underwent bronchoscopy.
COPD subjects were all ex or current smokers with spirometric evidence of airflow obstruction according to the Global Obstructive Lung Disease (GOLD) criteria for COPD 1 . Subjects with a history of asthma had a <10 pack year smoking history and met a diagnosis for asthma as described previously 2 . Control subjects included those with and without a smoking history without airflow obstruction.
To study tissue by immunohistochemistry, we used two sources of tissue; i) airway tissue collected at the time of lung resection from lung cancer patients who also met the GOLD criteria for COPD. Control tissue was also collected from subjects undergoing lung resection without evidence of COPD; ii) bronchial biopsies from healthy volunteers and asthmatic patients as described for HASMC cell culture 2,3 . All subjects gave written informed consent, and the collection of tissue was approved by the National Research Ethics Service (reference numbers: 07/MRE08/42, 04/Q2502/74, 08/H0406/189). All methods were performed in accordance with the relevant guidelines and regulations.

Human airway smooth muscle cell (HASMC) isolation and culture
Subjects underwent bronchoscopy, and mucosal biopsies were collected for HASMC culture as described previously 3 . Pure human airway smooth muscle (ASM) bundles in airways isolated from lung resection and bronchial biopsy tissue were dissected free of surrounding tissue. The ASM bundles were cultured in DMEM supplemented with 10% FCS, 4 mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin, and 0.25μg/ml amphotericin. HASMC characteristics were determined by immunofluorescence with antibodies to α-smooth muscle actin (αSMA) (FITC directly conjugated) and myosin indirectly labelled with FITC 3 .

Immunohistochemistry
Bronchial tissue remote from the cancer was dissected from the lung resection material and embedded in glycol-methacrylate (GMA) and stored at −20°C as described previously 4 . Bronchial biopsies taken at bronchoscopy were also embedded in GMA as described previously 4 . GMA sections of 2 μm thickness were immunostained for Tensin1 (SAB4200283, 2.5 μg/ml, Sigma-Aldrich and sc-28542, 1 μg/ml, Santa-Cruz) or the appropriate isotype control at the same concentration as the primary antibody (X0936, 2.5 µg/ml, Dako) using the Dako EnVsion FLEX+ staining technique.
Sections were counter-stained using Mayer's haematoxylin and visualized using a light microscope.
The tensin1 antibodies were validated further using immunoprecipitation and siRNA downregulation (see below).
Images were collected and tensin1 immunostaining was quantified using Cell F software version 5.0 (Olympus). The thresholding technique was used to quantify tensin1 immunostaining based on the hue saturation and intensity (HSI) value 2 . Qiagen). PCR products were run on an agarose gel to confirm the product amplified was the correct size, and were also sequenced. Tensin1 and αSMA mRNA expression was quantified using the ΔCT method 5 .

Quantitative Real Time PCR
Cells were stimulated with 10 ng/ml transforming growth factor-β1 (TGFβ1) (R&D systems), cells were grown to confluence and then serum-starved for 24 hours prior to stimulation for a further

Western blotting
Tensin1 expression in primary HASMCs was analysed by western blotting as described previously 6 .

Immunofluorescence
HASMCs were seeded into 8-well chamber slides, grown to confluence, and immunostained using mouse monoclonal αSMA (0.7 µg/ml, Dako) and isotype control FITC-conjugated mouse IgG2a (X0933, 10 µg/ml, Dako, Ely, UK), anti-Tensin1 antibody (4.5µg/ml, Sigma-Aldrich) and isotype control rabbit IgG (4.5µg/ml, Dako). Secondary antibodies labelled with AlexaFluor594 (A-11012, ThermoFisher) or FITC (FO382, Sigma-Aldrich) were applied and the cells were counterstained with 4', 6-diamidino-2-phenylindole (DAPI) (32670, Sigma-Aldrich). To study the effect of the extracellular matrix protein fibronectin and TGFβ1 on tensin1 expression, slides were coated with human recombinant fibronectin (F0895, Sigma-Aldrich) for 1 hour prior seeding and stimulated with TGFβ1 (10ng/ml) for 24 hours. When 50% confluent, cells were stained as above and mounted with fluorescent mounting medium. Original images were captured on a confocal immunofluorescence microscope (Leica TCS SP5, UK) and staining was quantified using Cell F imaging software (Olympus UK Ltd). Matched exposures were used for isotype controls. Quantification of the co-localisation of tensin1 and αSMA proteins was performed using an Image J plugin, JaCoP, in which Mander's overlap coefficient and Pearson's correlation were calculated (National Institutes of Health; http://rsbweb.nih.gov/ij/). Quantification of fibrillar adhesion length was measured by Cell F imaging software (Olympus UK Ltd).

Human airway smooth muscle cell transfection with siRNA
HASMCs were transfected with siRNA smartpools directed against tensin1 (M-009976-00, Dharmacon) and a non-targeting siCONTROL (D-001206-14, Dharmacon). HASMCs were plated in culture media in the absence of antibiotics in a 6 well plate and incubated at 37°C, 5% CO2 overnight. 5 μl of Lipofectamine 2000 (11668, Invitrogen) was diluted in 105 μl serum and antibiotic-free media and incubated for 5 minutes before adding to siRNA diluted in 105 μl of serum and antibiotic-free media. The mixture was incubated for 20 minutes and added to cells with an additional 1.78 ml culture media. The cells were incubated with the complexes for 5 hours. After 5 hours, medium was replaced with antibiotic-free media for 48 hours 7 .

Survival/Proliferation assay
The MTS assay (G3582, Promega) was used to assess survival and proliferation of the cells after tensin1 silencing. 48 hours after transfection, cells were collected and 2 x 10 3 cells were plated into 96-well plates in culture media in the absence of antibiotics overnight. Cells were stimulated with TGFβ1 for 24 hours. 20 μl of MTS solution was added to each well. Plates were then incubated at 37°C for 4 hours. The optical density (OD) at 490nm was determined with a spectrophotometer. Each experimental condition was run in triplicate.

TGFβ1 ELISA
Tensin1 knockdown was performed as described above and supernatants were collected. A commercial ELISA was used to measure TGFβ1 in tensin1 knockdown sample supernatants according to the manufacturer's protocol (DY240, R&D systems). The lower limit of TGFβ1 detection was 0.031 pg/ml and the upper limit was 2 pg/ml. Assay samples were diluted and activated before ELISA. Each experimental condition was run in triplicate.

Collagen gel contraction assay
Tensin1 knockdown was performed as above and then cells were detached and embedded in collagen gels as described previously 8 . Bradykinin was then added to appropriate wells to a final concentration of 1nM (B3259, Sigma-Aldrich). Photographs were taken at 0, 4, 18, 24 and 48 hours.