Enhanced Hsp70 expression protects against acute lung injury by modulating apoptotic pathways

Abstract

The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury. Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFα. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co-immunoprecipitation and a caspase-3 activity assay. TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes. We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery.

Figure 1. AdHSP reduces apoptosis in 2CLP induced ARDS.

Representative TUNEL-stained fixed tissue section. Positive staining depicted in green. T0 - Untreated controls, 2CLPPBS - septic animals treated with PBS, 2CLPAdHSP - septic animals treated with AdHSP, 2CLPAdGFP – septic animals treated with AdGFP. Nuclei stained with DAPI. Upper panel: 20× magnification, lower panel: 40× magnification. Positive control - DNAse treatment. Graph – Graphic representation of fluorescent nuclei/low powered field. Quantification performed on 20 lung fields/slide at 20× magnification, five slides per animal and at least 3 animals for each intervention. Quantification of TUNEL staining was performed using Nikon software. 20 fields per slide were randomly manually selected by a single observer who was blinded to the intervention (AdHSP, AdGFP, PBS). Data are mean +/− standard deviation * = significantly different from T0 and AdHSP.

Figure 2. AdHSP reduces Myeloperoxidase (MPO) in 2CLP-induced lung injury.

A. Myeloperoxidase (MPO) immunostaining. 20× Magnification. MPO containing cells stain brown. Left panel: 2CLPAdHSP, Right panel: 2CLPPBS. TUNEL and MPO double staining. 40× Magnification. Left panel: Myeloperoxidase (MPO) Immunostaining, MPO containing cells stain brown. Right panel: TUNEL fluorescent staining. TUNEL positive cells are green. White arrows depict apoptotic neutrophils – brown staining for MPO and green for TUNEL. Yellow arrows depict apoptotic cells that are negative for MPO but positive for TUNEL. Red arrows depict nuclei stained for dapi –showing the orientation of the slide. Black arrows depict MPO-positive cells that were not apoptotic. TUNEL and Aquaporin 5 (AQP5) double staining. AQP5 – a specific marker for alveolar type I cells. 40× magnification. Left panel: AQP5 immunostaining, AQP5 containing cell stain brown. Right panel: TUNEL fluorescent staining. TUNEL positive cells are green. Red arrows indicate positive staining of alveolar type I cells (both AQP5 and TUNEL). Yellow arrows indicate alveolar type II cells that are negatively stained for AQP5 and positively stained for TUNEL.

Figure 3. AdHSP reduces the abundance of caspases 8 and 9 in 2CLP-induced lung injury.

A. Immunoblotting of whole cytosolic extracts for Caspase 8 and 9. Abbreviations as in Fig. 1. Upper panel: Representative autoradiogram of SDS-PAGE, 30 µg of cytosolic extract/lane. Primary rabbit polyclonal antibody to caspase 8. Secondary goat anti rabbit IgG. .Graphic representation of relative density of cytosolic caspase 8. Middle panel: Representative autoradiogram of SDS-PAGE, 30 µg of cytosolic extracts. Primary rabbit polyclonal antibody to caspase 9, secondary goat anti rabbit IgG. Graph - Graphic representation of relative density (mean +/− standard deviation) of cytosolic caspase 9. * = significantly different from 2CLPPBS and 2CLPAdGFP. Lower panels: Representative autoradiogram of SDS-PAGE, 30 µg of mitochondrial extract/lane. Primary mouse monoclonal antibody to Bcl2, secondary goat anti mouse IgG and primary mouse monoclonal antibody to COX IV, secondary goat anti mouse IgG. COX IV serves as mitochondrial loading control. B. Hsp70 in vivo interaction with apopotosomal Apaf-1. Representative autoradiograms. Samples were immunoprecipitated with a rabbit polyclonal antibody to Apaf-1 and subjected to SDS-PAGE. Upper panels: Immunoblotting with a primary rabbit polyclonal antibody to pro-caspase 9, secondary goat anti rabbit IgG. Middle panels: Immunoblotting with primary mouse monoclonal antibody to Hsp70, secondary goat anti mouse IgG. Lower panels: IgG detection IgG serves as loading control. 250 µg of cytosolic extracts obtained from TO, 2CLPPBS, 2CLPAdHSP or 2CLPAdGFP treated animals sacrificed 48 hrs after the induction of sepsis. C. Hsp70 in vitro, MLE-12 cells, interaction with apopotosomal Apaf-1. Representative autoradiograms. 250 µg of cytosolic extracts obtained from non treated MLE-12 cells (controls), stimulated with tumor necrosis factor (TNF) and treated with AdHSP or AdGFP. Samples were immunoprecipitated with a rabbit polyclonal antibody to Apaf-1 and subjected to SDS-PAGE. Upper panels: Immunoblotting with a primary rabbit polyclonal antibody to pro-caspase 9, secondary goat anti rabbit IgG. Middle panels: Immunoblotting with primary mouse monoclonal antibody to Hsp70, secondary goat anti mouse IgG. Lower panels: IgG serves as loading control. D. Apaf-1 – CARD dissociates from Pro-caspase-9 in the presence of Hsp70. Representative autoradiograms. 100 µg of cytosolic extracts obtained from 2CLPPBS and 2CLAdHSP treated animals, were immunoprecipitated with Pro-caspase-9 and further incubated with GST-Apaf-1 – CARD obtained from BL-21 cells, together with 5 mM ATP and 5 µg/ml human Cytochrome C for 5, 10, 20 and 30 minutes. Samples were subjected to SDS-PAGE, immunoblotted and the membranes were incubated with primary rabbit polyclonal antibody to Cleaved Caspase-9, secondary to goat anti rabbit IgG.

Figure 4. AdHSP prevents nuclear translocation of activated Caspase-3.

A. Representative autoradiograms for pro-caspase 3 and activated (cleaved) Caspase 3. 30 µg of cytosolic (upper panels) and nuclear (lower panel) extracts were subjected to SDS-PAGE. Immunoblotting performed with primary rabbit polyclonal antibody to pro- caspase 3 and secondary goat anti rabbit IgG, primary goat antibody to β-actin and secondary donkey anti goat IgG, primary rabbit polyclonal antibody to active (cleaved) caspase-3 and secondary goat anti rabbit IgG, primary mouse monoclonal antibody to histone (H1) and secondary goat anti mouse IgG.. β-actin and histone serve as loading controls. B. Representative stained fixed tissue section depicting intra-nuclear staining for Caspase 3. Sections obtained from T0 control, 2CLPPBS and 2CLPHSP rats. Tissue isolated 48 hrs after the induction of sepsis. Upper panel: 40× magnifications. Black arrows indicate active caspase 3 stained nuclei. Lower panel: 100× magnification of upper panel. C. Caspase 3 Activity Assay. Graphic representation of relative caspase-3 enzymatic activity (mean +/− standard deviation) * = significantly different from Control and AdHSP+TNF.

Figure 5. AdHSP alters interactions between Caspase 8, 9, 3 and Apaf-1.

A and B: Representative Autoradiogram Demonstrating AdHSP treatment disrupts interaction between caspase 8 and caspase 9. Representative autoradiograms. 250 µg of cytosolic extracts were immunoprecipitated with rabbit polyclonal antibody to caspase 8 or caspase-9 and subjected to SDS-PAGE. Immunoblotting performed with either primary rabbit polyclonal antibodies to caspase 9 or caspase-8, secondary goat anti rabbit IgG. Lower panels: IgG detection. IgG serves as loading control. Abbreviations as in Figure 1. C: Hsp70 disrupts caspases 3, 8 9 and Apaf-1 complexes. 250 µg of cytosolic extracts from lung tissue fractionated via column chromatography, eluted by molecular weight, immunoprecipitated with an antibody to caspase-9 and subjected to 9% SDS-PAGE. Molecular weight of each fraction (kDa) indicated at the top of the figure. Detecting antibodies (anti-caspase 9, anti-caspase 8, anti-pro-caspase 3, anti-Hsp70 and anti-Apaf-1,) noted to the left of the panels. Lower panel: IgG detection. IgG serves as loading control. Abbreviations as in Figure 1.