Journal: The European Respiratory Journal

Article Title: Pharmacological inhibition of Epac1 protects against pulmonary fibrosis by blocking FoxO3a neddylation

doi: 10.1183/13993003.02250-2024

Figure Lengend Snippet: Molecular responses to AM-001 and neddylation inhibition in lung fibroblasts (FBs). a) The transcript levels of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-activating enzyme E1 ( NAE1 ), ubiquitin conjugating enzyme E2 M ( UBE2M ) and NEDD8 in lung tissue samples from healthy donors (n=7) and idiopathic pulmonary fibrosis (IPF) patients (n=7) were assessed by quantitative reverse-transcriptase PCR (qRT-PCR). b) Co-immunostaining of NEDD8 (red) and α-smooth muscle actin (α-SMA) (green) in lung tissue sections from both healthy donors and IPF patients. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. c) Forkhead box protein O3 ( FoxO3a ) mRNA levels (n=8). d) Representative images of co-immunostaining of FoxO3a (red) with α-SMA (green) in lung tissue sections from healthy donors and IPF patients. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. e) Representative immunoblot analysis of FoxO3a, UBE2M and NEDD8 in normal human lung (NHL) FBs and IPF-FBs (left panel). Right panels show the ratio quantification using GAPDH as the loading control (n=3). f) Transcript levels of NAE1 , NEDD8 and UBE2M in NHL-FBs and IPF-FBs after 48 h of treatment with either vehicle or AM-001 (20 μM) for 48 h (n=3). g) FoxO3a mRNA expression in NHL-FBs and IPF-FBs after 48 h of treatment with either vehicle or AM-001 (20 μM) for 48 h (n=3). h) Representative immunoblot analysis showing the levels of phosphorylated FoxO3a at Thr32 (p-FoxO3a Thr32 ), total FoxO3a and NEDD8 under the indicated conditions. GAPDH was used as a loading control. i) mRNA expression levels of fibrosis markers transforming growth factor β1 ( TGF-β1 ), collagen type 1 ɑ1 ( COL1A1 ), collagen type 3 ɑ1 ( COL3A1 ) and connective tissue growth factor ( CTGF ) were measured by qRT-PCR in NHL-FBs and IPF-FBs treated with MLN4924 (5 µM) alone, a small molecule inhibitor of NAE, or in combination with AM-001 (20 μM) for 48 h (n=3). j) Representative immunoblot analysis of p-FoxO3a Thr32 , total FoxO3a and NEDD8 protein levels in NHL-FBs or IPF-FBs treated with vehicle, MLN4924 alone or in combination with AM-001 for 48 h. k) Immunoprecipitation of haemagglutinin (HA)-tagged NEDD8 (HA-NEDD8) followed by immunoblot analysis of NEDD8 and total FoxO3a in IPF-FBs transfected with HA-NEDD8, in IPF-FBs pretreated with proteasome inhibitor MG132 (10 µM) to inhibit protein degradation, and under the indicated conditions. Data are presented as mean± sem . 8-CPT: Sp-8-(4-chlorophenylthio)-2′-O-methyl-cAMP; IB: immunoblot; IgG: immunoglobulin G; ns : nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Article Snippet: Pevonedistat (MLN4924) is a potent and selective inhibitor of the NEDD8-activating enzyme (NAE) with a half-maximal inhibitory concentration of 4.7 nM, obtained from MedChemExpress (Monmouth Junction, NJ, USA; catalogue number: HY-70062).

Techniques: Inhibition, Ubiquitin Proteomics, Reverse Transcription, Quantitative RT-PCR, Immunostaining, Western Blot, Control, Expressing, Immunoprecipitation, Transfection

Journal: The European Respiratory Journal

Article Title: Pharmacological inhibition of Epac1 protects against pulmonary fibrosis by blocking FoxO3a neddylation

doi: 10.1183/13993003.02250-2024

Figure Lengend Snippet: Pharmacological inhibition of exchange factor directly activated by cAMP 1 (Epac1) by AM-001 attenuates bleomycin (BLM)-induced pulmonary fibrosis (PF). a) Schematic outlining the experimental design. C57BL/6 mice received a single intratracheal (IT) delivery of BLM (4 U·kg −1 ) for 28 days (∼4 weeks). AM-001 was then administered via intraperitoneal (IP) injection every other day at a dose of 10 mg·kg −1 for an additional 2 weeks. b) Representative images of Masson's trichrome staining (left panel) of lung sections from control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001. The right panel shows the quantification of the fibrotic area, expressed as the fold change relative to the saline control group (n=5–7). c) Lung hydroxyproline content, an index of collagen deposition, was measured in saline-treated control mice and BLM-challenged mice administered either vehicle or AM-001 (n=5–7). d) mRNA expression levels of profibrotic markers transforming growth factor β1 ( TGF-β1 ), collagen type 1 ɑ1 ( COL1A1 ), collagen type 3 ɑ1 ( COL3A1 ) and connective tissue growth factor ( CTGF ) in lung tissues were measured by quantitative reverse-transcriptase PCR (qRT-PCR) in lung tissue from the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001 (n=3–7). e) The transcript levels of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-activating enzyme E1 ( NAE1 ), ubiquitin conjugating enzyme E2 M ( UBE2M ) and NEDD8 in lung tissues from both the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001 were analysed by qRT-PCR (n=3–7). f) Co-immunostaining of NEDD8 (red), forkhead box protein O3 (FoxO3a) (red) and α-smooth muscle actin (α-SMA) (green) in lung sections from the control saline-treated group and BLM-challenged mice treated with either vehicle or AM-001. Nuclei were stained with DAPI (blue). Scale bars: 100 μm. g) Representative immunoblot analysis and h) quantification of Epac1, phosphorylated FoxO3a (p-FoxO3a), total FoxO3a, NEDD8, phosphorylated SMAD2/3 (p-SMAD2/3) and total SMAD2/3 in lung homogenates from the indicated group of mice (n=3). The data are presented as mean± sem . ns : nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Article Snippet: Pevonedistat (MLN4924) is a potent and selective inhibitor of the NEDD8-activating enzyme (NAE) with a half-maximal inhibitory concentration of 4.7 nM, obtained from MedChemExpress (Monmouth Junction, NJ, USA; catalogue number: HY-70062).

Techniques: Inhibition, Injection, Staining, Control, Saline, Expressing, Reverse Transcription, Quantitative RT-PCR, Ubiquitin Proteomics, Immunostaining, Western Blot