NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

Abstract

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys¹⁸ in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.

Fig. 1.. Betweenness centrality analysis identifies NEDD9 as a critical node regulating the transition between adaptive and pathogenic fibrosis.

(A) Protein-protein interaction network analysis was used to clarify the molecular pathways that regulate two functionally distinct fibrosis subtypes: adaptive and pathogenic. Genes related to dermal and vascular fibrosis were collected from the curated literature, focusing on pulmonary arterial hypertension (PAH) as a human disease correlate to these findings in silico. Genes specifically associated with lung fibrosis (n = 362) were excluded from the analysis to limit the probability of analyzing pathways responsible for lung parenchymal fibrosis rather than pulmonary vascular fibrosis. (B) The gene products (proteins) associated with adaptive fibrosis (blue), pathogenic fibrosis (red), or both adaptive and pathogenic fibrosis (blue with red border) were mapped to the consolidated human protein-protein interactome (12), resulting in the fibrosome. (C) An aldosterone (ALDO)-fibrosome protein-protein interactome subnetwork resulting from associations involving fibrosis genes connected to ALDO-regulated genes. Arrow points to the SMAD3 target and Cas protein NEDD9. (D) Betweenness centrality (BC), a measure of importance in information transfer across the network of a node (protein) based on the shortest paths, was used to identify NEDD9 as a critical node in the phenotype transition from adaptive to pathogenic fibrosis. BC of an ALDO-regulated gene (a_i) in connecting adaptive and pathogenic fibrosis (F₁ and F₂, respectively) is the sum of the fraction of all fibrosis gene pairs’ shortest paths in the interactome that pass through this ALDO-regulated gene (t) in the interactome, and σ(s,t|a_i) is the number of those shortest paths in the interactome that pass through ALDO-regulated gene a_i. The BC score for NEDD9 was ranked 6^th of 86 proteins.

Fig. 2.. Oxidation of NEDD9-Cys18 impairs NEDD9-SMAD3 binding.

(A) Hill curves and EC₅₀ values of purified wild type (WT) human NEDD9 (0.06 nM–2 μM) or (B) mutant NEDD9 containing a substitution of cysteine for alanine at position 18 (C18A-NEDD9, 0.03 nM–1 μM), which is resistant to oxidant stress, incubated with fluorescently labeled SMAD3 (20 nM) in the presence of H₂O₂ (500 μM, 20 min). Data are expressed as EC₅₀ ± EC₅₀ confidence interval. n = 2. (C) Immunoprecipitation of COS-7 cells transfected with human DNA coding SMAD3 and WT-NEDD9 or C18A-NEDD9 after treatment with vehicle (V) control, which in this experiment was phosphate-buffered saline, or H₂O₂ (500 μM) for 5 min. NEDD9-SMAD3 binding was calculated by comparing the densitometry ratio of SMAD3 to NEDD9 assessed by immunoblot. Data are from representative immunoblots and expressed as the ratio of SMAD3/NEDD9 in densitometry arbitrary units (a.u.). Means ± SE, n = 3–4, Student’s unpaired two-tailed t-test. (D) In-gel trypsin digestion on NEDD9 immunoprecipitated from human pulmonary artery endothelial cells (HPAECs) treated with H₂O₂ (250 μM) for 20 min. Arrow, doubly charged y8 ion corresponding to NEDD9-Cys18 with the addition of three oxygens (-SO₃H) at retention time of 77.3 min and m/z value of 944.425 (n = 3). (E) Immunoprecipitation of NEDD9 from HPAECs treated with vehicle (V) control (DMSO 10⁻⁷ mol/L), H₂O₂ (250 μM) for 20 min, or aldosterone (ALDO) (10⁻⁷ mol/L) for 24 hr. Immunoblotting used an antibody against dimedone, detected upon reaction with cysteine sulfenic acid (R-SOH). Data from representative immunoblots expressed as the ratio of oxidized NEDD9 (NEDD9-SOH)/NEDD9 and the ratio of SMAD3/NEDD9-SOH in a.u.. Means ± SE, n = 3, Student’s unpaired two-tailed t-test. (C-E) Representative immunoblots and spectra are shown. MS, in-tandem liquid chromatography-mass spectrometry; m/z, mass/charge; IB, immunoblot; IP, immunoprecipitation.

Fig. 3.. NEDD9 modulates pulmonary endothelial cell collagen synthesis and fibrosis.

(A) Immunoblot and quantitation in densitometry arbitrary units (a.u.) of NEDD9 form human pulmonary artery endothelial cells (HPAECs) treated with vehicle (V) control, aldosterone (ALDO) (10⁻⁷ mol/L) for 24 hr, spironolactone (SP) (10 μM) for 24 hr to inhibit ALDO-induced oxidant stress, or hypoxia (FiO₂ 0.2%) for 24 hr. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons. The orientation of the hypoxia samples was adjusted for consistency with ALDO samples; however, all bands are from the same blot. (B) NEDD9 expression in HPAECs treated with the pan-TGF-β ligand neutralizing antibody TGF-βRII-FC (2.3 μg/mL) for 24 hr prior to treatment with ALDO (10⁻⁷ mol/L) for 24 hr. Means ± SE, n = 3, Student’s unpaired two-tailed t-test. (C) NEDD9 expression in HPAECs pre-treated with the proteasome inhibitor MG-132 (50 μM) for 0–4 hr prior to H₂O₂ (250 μM) for 20 min. Means ± SE, n = 3, Student’s unpaired two-tailed t-test. The orientation of the si-Scr and si-NEDD9 samples was adjusted for consistency; however, all bands are from the same blot. (D) Co-immunoprecipitation and quantitation for NEDD9 and the COL3A1-targeting transcription factor NKX2–5 from HPAECs incubated with vehicle or ALDO (10⁷ mol/L) for 24 hr. Means ± SE, n = 3, Student’s unpaired two-tailed t-test. In (E), HPAECs were transfected with si-RNA to SMAD3 (si-SMAD3) and the ratio of NKX2–5/NEDD9 is presented in a.u.. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons. (F) Chromatin immunoprecipitation of cell lysates using an anti-histone H3 antibody (positive control), immunobeads (negative control), and an anti-NKX2–5 antibody was followed by polymerase chain reaction amplification of the Col3A1 region containing the NKX2–5 binding site. Data were normalized to positive control and expressed as fold-change for NKX2–5 over negative control. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons. (G) MMP-2 proteolytic activity by SDS-PAGE zymography, collagen III (Col III) expression, and quantitation. (N9, NEDD9); (H) images and quantitation of 3-D collagen Matrigel contraction (scale bar = 17.4 mm); (I) images and quantitation of collagen fiber content area in um² (scale bar = 100 μm); and (J) stiffness assessed by atomic force microscopy from HPAECs transfected with si-NEDD9 or si-scrambled (negative) control (si-Scr) and treated with ALDO (10⁻⁷ mol/L) for 24 hr. (G-J) Means ± SE, n = 3–6, one-way ANOVA and Tukey’s multiple comparisons. *P < 0.05 versus V; **P < 0.05 versus ALDO. (A-I) Representative immunoblots, gels, and micrographs shown. MMP, matrix metalloproteinase; Un, untreated.

Fig. 4.. Pulmonary endothelial NEDD9-collagen III is increased in PAH in vivo.

(A) Immunofluorescence for NEDD9 and collagen III in tissue sections from animal models of PAH [IL-6 TG+ (n = 4), Schisto (n = 4), SU-5416/Hypoxia (n = 5), MCT (n = 5)] and PAH patients (n = 9). Samples from control mice (n = 4) and rats (n = 5), and donor tissue from non-diseased control patients (n = 5) were used for comparison. Insets are merged images for DAPI (blue) and NEDD9 (green) to show nuclear NEDD9 expression; representative images from samples within each condition are provided. Colocalization of collagen III and NEDD9 is expressed as percent double positive area/sum total area of stain for each protein in pulmonary arterioles. Means ± SE, Student’s unpaired two-tailed t-test. (B) Immunofluorescence for collagen III and NEDD9 in human pulmonary artery endothelial cells (HPAECs) isolated from PAH patients (PAH-HPAEC). Number of NEDD9+ cells per high powered field is quantitated. Co-immunoprecipitation of NEDD9 and NKX2–5 and immunoblot of NEDD9 and SMAD3, with quantitation in densitometry arbitrary units (a.u.). Means ± SE, n = 3–6/group. Student’s unpaired two-tailed t-test. (C) Collagen III-vWF co-localization in remodeled PAH pulmonary arterioles, expressed as percent double positive area/sum total area of stain for each protein in pulmonary arterioles. Means ± SE, n = 3–5/group, Student’s unpaired two-tailed t-test. IP, immunoprecipitation; IB, immunoblot; vWF, Von Willebrand Factor; PAH, pulmonary arterial hypertension; h.p.f., high powered field (200×). Scale bar = 50 μm, Inset scale bar = 4 μm. Representative micrographs and immunoblots shown.

Fig. 5.. Exosomes from ALDO-treated HPAECs increase NEDD9 and fibrillar collagen in HPASMCs.

(A) Schematic of culture conditions and representative immunoblots of NEDD9 from human pulmonary artery smooth muscle cells (HPASMCs) co-cultured with human pulmonary artery endothelial cell (HPAECs), treated with aldosterone (ALDO) (10⁻⁷ mol/L) for 24 hr. Data are expressed as densitometry arbitrary units (a.u.). Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons. (B) Quantification of NEDD9 positivity in HPASMCs, normal human lung fibroblasts (NHLFs), and dermal fibroblasts from patients with systemic sclerosis without PAH (SSc-DFBs) that were untreated (Un) or treated with exosomes isolated from vehicle (V) (DMSO 10⁻⁷ mol/L)-treated or aldosterone (ALDO) (10⁻⁷ mol/L)-treated HPAECs. Data are expressed as % of cells that were NEDD9+ per h.p.f. (200×) (white arrows). exo-HPAEC, exosomes isolated from HPAECs. Inset, representative NEDD9+ cell. Means ± SE, n = 3–4, one-way ANOVA and Tukey’s multiple comparisons. Scale bar = 100 μm for representative micrographs, scale bar for inset=10 μm. *P < 0.01 versus NHLF exo-HPAEC ALDO; **P < 0.001 versus HPASMC Un. (C) Colocalization of NEDD9 and collagen III in the perinuclear regions of cells undergoing mitotic division in HPASMCs and NHLFs treated with exosomes from ALDO-treated HPAECs (exo-ALDO-HPAEC). n = 3. Scale bar = 20 μm. (D) Immunoblot of NEDD9 and collagen I in HPASMCs treated with exo-ALDO-HPAEC. Data are expressed in densitometry a.u.. Means ± SE, n = 4 for NEDD9; n = 4 for collagen I, Student’s unpaired two-tailed t-test. (E) Representative immunoblots of exosomal TGF-β-LAP (TGF-β latent complex) secreted by HPAECs treated with V control and ALDO (10⁻⁷ mol/L) for 24 hr. Means ± SE, n = 3. Student’s unpaired two-tailed t-test.

Fig. 6.. Gene ablation or molecular inhibition of NEDD9 prevented vascular fibrosis and PAH in vivo.

(A) Right ventricular systolic pressure (RVSP) and RV mass by Fulton index (RV/LV + S) in male and female C57BL (WT), C57BL/NEDD9^+/−, and C57BL/NEDD9^−/− mice injected with Sugen-5416 (20 mg/kg) every 7 days during a 3-week period of hypoxia treatment (FiO₂ 10%). Means ± SE, n = 6–9 mice/condition for RVSP, n =5–10 mice/condition for heart weight, one-way ANOVA and Tukey’s multiple comparisons. (B) Representative images of Gomori trichrome staining to assess vascular fibrosis burden and vWF-collagen III colocalization, expressed as percent double positive area/sum total area of stain for each protein, of pulmonary arterioles (20–50 μm in diameter, adjacent to terminal bronchiole) from WT, NEDD9^+/−, and NEDD9^−/− mice treated with or without SU-5416/Hypoxia. Means ± SE, n = 6–10 mice/condition for trichrome; n = 4–7 mice/condition for vWF-collagen III, one-way ANOVA and Tukey’s multiple comparisons. Scale bar = 50 μm. Col III, collagen III. (C) NEDD9-collagen III and NEDD9-vWF colocalization in lung tissue from male Sprague Dawley rats administrated monocrotaline (MCT) (50 mg/kg) and treated with Staramine-mPEG (1.5 mg/kg) formulated with NEDD9 siRNA (si-NEDD9). Means ± SE, n = 4–5 rats/condition, one-way ANOVA and Tukey’s multiple comparisons Scale bar = 50 μm. (D) The number of hypertrophic vessels/high powered field (red arrow) and percent vascular fibrillar collagen in paraffin-embedded lung sections was analyzed using anti-α-smooth muscle actin (SMA) immunohistochemistry and Picrosirius Red staining, respectively, from vehicle-, MCT-, and MCT + si-NEDD9-treated rats. Means ± SE, n = 4–5 rats/condition, one-way ANOVA and Tukey’s multiple comparisons. Scale bar = 100 μm. Inset, representative hypertrophic vessel magnified, scale bar = 50 μm. (E) Table of phenotypic changes in V, MCT, and MCT + si-NEDD9-treated rats. Means ± SE, n = 5–7 rats/condition. P values in column represent one-way ANOVA. *P < 0.05 versus V, **P < 0.001 versus MCT; ***P = 0.02 versus MCT by Tukey’s multiple comparisons. (F) Representative RV pressure-volume loops are show to quantify RV-pulmonary artery coupling, assessed by the ratio of end-systolic elastance (Ees)/pulmonary vascular elastance (Ea) in MCT and MCT + si-NEDD9-treated rats. Means ± SE, n = 3 rats/condition, Student’s unpaired two-tailed t-test. h.p.f, high power fields (200×). N9, NEDD9; HR, heart rate; MAP, mean arterial pressure; RAP, right atrial pressure; CI, cardiac index; CO, cardiac output, indexed pulmonary vascular resistance (PVRi) in Wood units.