Transgelin-2 as a therapeutic target for asthmatic pulmonary resistance

Abstract

There is a clinical need for new bronchodilator drugs in asthma, because more than half of asthmatic patients do not receive adequate control with current available treatments. We report that inhibition of metallothionein-2 protein expression in lung tissues causes the increase of pulmonary resistance. Conversely, metallothionein-2 protein is more effective than β₂-agonists in reducing pulmonary resistance in rodent asthma models, alleviating tension in tracheal spirals, and relaxing airway smooth muscle cells (ASMCs). Metallothionein-2 relaxes ASMCs via transgelin-2 (TG2) and induces dephosphorylation of myosin phosphatase target subunit 1 (MYPT1). We identify TSG12 as a nontoxic, specific TG2-agonist that relaxes ASMCs and reduces asthmatic pulmonary resistance. In vivo, TSG12 reduces pulmonary resistance in both ovalbumin- and house dust mite-induced asthma in mice. TSG12 induces RhoA phosphorylation, thereby inactivating the RhoA-ROCK-MYPT1-MLC pathway and causing ASMCs relaxation. TSG12 is more effective than β₂-agonists in relaxing human ASMCs and pulmonary resistance with potential clinical advantages. These results suggest that TSG12 could be a promising therapeutic approach for treating asthma.

Fig. 1.. MT-2 ameliorates asthmatic pulmonary resistance.

(A) Western blot analyses of metallothionein-2 (MT-2) protein in control and asthmatic lung tissues from ovalbumin (OVA)–sensitized and OVA-challenged rats. Lower panel: Mean density of MT-2 normalized to β-actin (*P < 0.05 versus control, t test; n = 5 per group). (B) Pulmonary resistance (R) in wild-type (WT) and metallothionein knockout (MT-KO) OVA-sensitized mice challenged with vehicle (control) or OVA. (*P < 0.05 versus control and #P < 0.05 versus WT mice challenged with OVA, n = 8 per group). (C) Gel of purified rat MT-2 recombinant protein and molecular weight markers (M). (D) Pulmonary resistance peak value sum of 2 to 4 min (sR_2–4) for OVA-challenged rats without treatment (Con) or treated with terbutaline (TB; 0.055 mg/kg), hydrocortisone (HC; 15 mg/kg), or MT-2 (1, 10, and 100 ng/kg). (*P < 0.05 versus Con, n = 8 per group). (E) Isometric tension of isolated tracheal spirals at baseline (control without challenge or treatment), challenged with acetylcholine without treatment (ACh) or treated with TB (1500 nM) or MT-2 (15, 30, and 60 nM) (*P < 0.05 versus ACh and ^#P < 0.05 versus TB, n = 5 per group). (F) Collagen gel contraction assays of rat airway smooth muscle cells (ASMCs) at baseline (control without treatment) or treated with TB (10,000 nM) or MT-2 (15, 30, and 60 nM) (*P < 0.05 versus baseline, n = 3 per group). (G) Real-time cell analysis of rat ASMCs at baseline (control without challenge or treatment), challenged with acetylcholine without treatment (ACh) or treated with TB (10,000 nM) or MT-2 (60 nM) (*P < 0.05 versus ACh and #P < 0.05 versus TB, n = 4 per group). All data are expressed as means ± SEM. Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Games-Howell/least significant difference (LSD) post hoc test.

Fig. 2.. MT-2 relaxes ASMCs specifically via transgelin-2.

(A) Confocal images of rat ASMCs showing the cellular membrane [DiIC₁₈(3), red], nucleus (Hoechst, blue), and MT-2 [FITC (fluorescein isothiocyanate), green] (scale bar, 10.0 μm). (B) Scatchard analysis of the binding of MT-2 to rat ASMCs (n = 3 per concentration). CPM, counts per minute. (C) Gel and Western blots using anti-His or anti–MT-2 antibody of immunoprecipitates of rat ASMCs lysates incubated with nickel–nitrilotriacetic acid beads linked to 6×His tag MT-2 recombinant protein used as a probe. (D) The competitive binding assays were performed with nonlabeled cold MT-2 (MT) or blocking antibodies against transgelin-2 (TG2), enolase-1 (ENO), D2 subclass of dopamine receptor (D2DR), or bovine serum albumin (BSA), whereas ¹²⁵I-MT-2 was used to quantify the specific binding of MT-2 to rat ASMCs (*P < 0.05 versus control without treatment, n = 3 per group). (E) Western blots of TG2 and β-actin of rat ASMCs treated with control or TG2 small interfering RNA (siRNA). Lower panel: Scatchard analysis of the binding of MT-2 to TG2-deficient rat ASMCs (n = 3 per concentration). (F) Confocal images of rat ASMCs showing TG2 (TG2, Cy3; red), nucleus (Hoechst, blue), and MT-2 (FITC, green) (scale bar, 10.0 μm). (G) Gel of purified rat recombinant TG2 protein and molecular weight markers (M). (H) Kinetic analysis of MT-2 by Biacore surface plasmon resonance with TG2 immobilized on the CM5 sensor chip. RU, response units. (I) Kinetic analysis of TG2 in Biacore surface plasmon resonance with MT-2 immobilized on the CM5 sensor chip. The response signals were recorded over the indicated time periods. All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.

Fig. 3.. Biological function of TG2 and the interaction with MT-2.

(A) Collagen gel contraction assays of rat ASMCs at baseline (control without treatment) or treated with TB (10,000 nM) or MT-2 (60 nM) (*P < 0.05 versus baseline and #P < 0.05 versus TB, n = 3 per group). (B) Comparison of control and TG2-deficient rat ASMCs challenged with acetylcholine without treatment (ACh) or treated with TB (10,000 nM) or MT-2 (60 nM) (*P < 0.05 versus ACh and #P < 0.05 versus TB, n = 4 per group). (C) Western blot analyses of TG2 and β-actin in lung tissue from WT or TG2 knockout (TG2-KO) mice. Lower panel: Mean density of TG2 protein normalized to β-actin protein (*P < 0.05 versus TG2-KO by ttest, n = 3 per group). (D) Pulmonary resistance (R) in WT and TG2-KO mice sensitized to OVA and challenged with vehicle (control) or OVA. (*P < 0.05 versus control and #P < 0.05 versus WT mice challenged with OVA, n = 8 per group). (E) Western blot analyses of MT-2 on the phosphorylation and total protein of ezrin, myosin phosphatase target subunit 1 (MYPT1) in rat ASMCs. Right panel: Corresponding densitometric analyses showing phosphorylated/total protein (%) treated with vehicle (control) or MT-2 (30 nM) (*P < 0.05 versus control, t test; n = 3 per group). (F) Western blots of TG2 and ezrin in total lysates or immunoprecipitates with TG2 antibody (IP: TG2) or control immunoglobulin G (IgG) of lung tissue from WT or TG2-KO mice. (G) Western blots of TG2 and ezrin in total lysates or immunoprecipitates with ezrin antibody (IP: Ezrin) or control IgG of lung tissue from WT or TG2-KO mice. (H) Kinetic analysis of ezrin by Biacore surface plasmon resonance with TG2 immobilized on the CM5 sensor chip. The response signals were recorded over the indicated time periods. All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.

Fig. 4.. TSG12 is a specific TG2 agonist in ASMCs.

(A) Kinetic analysis of TSG12 by Biacore surface plasmon resonance with TG2 immobilized on the CM5 sensor chip. The response signals were recorded for the indicated time. (B) Schematic interaction of the molecular docking of TSG12 and TG2 (Protein Data Bank code: 1WYM). (C) Comparison of control and TG2-deficient rat ASMCs challenged with acetylcholine without treatment (ACh) or treated with TB (10,000 nM) or TSG12 (50 nM) (*P < 0.05 versus ACh and #P < 0.05 versus TB, n = 4 per group). (D) Pulmonary resistance (R) in OVA-induced asthma WT and TG2-KO mice treated with vehicle (control) or TSG12 (100 ng/kg). (*P < 0.05 versus control and #P < 0.05 versus WT mice treated with TSG12, n = 8 per group). (E) TSG12 organ and serum distribution after inhalation. The distribution of TSG12 in different tissues after inhalation of TSG12 in mice (n = 3 per group) was determined by liquid chromatography-electrospray ionization tandem mass spectrometry and expressed as picograms per milligram of tissue or picograms per microliter of serum. N.D. means not detected or the concentration is lower than the limit of detection (0.1 pg/mg tissue). (F) The changed cross-sectional area (µm²) in precision-cut lung slices (PCLS) of arteries incubated with potassium chloride (KCL, 150 mM), sodium nitroprusside (SNP, 100 µM), or TSG12 (100 nM; n = 4 per group). (G) Decreased cross-sectional area (µm²) in PCLS of airways challenged with acetylcholine without treatment (ACh) or treated with isoproterenol (ISO; 100 µM); TSG12 (10 and 100 nM) or inactive control compound (CC; 100 nM). (*P < 0.05 versus ACh, n = 4 per group). (H) Western blot analyses of TSG12 on the phosphorylation and total protein of MYPT1 in rat tracheal spirals. Lower panel: Corresponding densitometric analyses showing phosphorylated/total protein (%) treated with vehicle (control) or TSG12 (25 µM). (*P < 0.05 versus control, t test; n = 3 per group). (I) Western blot analyses of TSG12 on the phosphorylation and total protein of RhoA, ROCK, MYPT1, and myosin light chain (MLC) in murine ASMCs. Right panels: Corresponding densitometric analyses showing phosphorylated/total protein (%) treated with vehicle (control) or TSG12 (25 µM). (*P < 0.05 versus control, t test; n = 3 per group). All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.

Fig. 5.. TG2 agonist TSG12 relaxes ASMCs and ameliorates pulmonary resistance.

(A) Pulmonary resistance (R) in OVA-challenged mice treated with vehicle (OVA), TB, or TSG12 (*P < 0.05 versus OVA and #P < 0.05 versus TB, n = 15 per group). (B) Pulmonary resistance (R) at baseline [control without house dust mite (HDM)] and HDM-challenged mice treated with vehicle (HDM), ISO (10 mg/kg), or TSG12 (100 ng/kg) or inactive CC (100 ng/kg). (*P < 0.05 versus HDM and #P < 0.05 versus baseline, n = 8 per group). Mch, methacholine. (C) Western blot analyses of TG2 (TG2) and β-actin in human ASMCs treated with control or specific siRNA inhibiting TG2 expression. Lower panel: Real-time cell analysis of control and TG2-deficient human ASMCs challenged with acetylcholine without treatment (ACh) or treated with TB (10,000 nM) or TSG12 (50 nM). (*P < 0.05 versus ACh and #P < 0.05 versus TB, n = 4 per group). (D) Optical magnetic twisting cytometry of human ASMCs challenged with acetylcholine without treatment (ACh) or with ISO (100 mM), TSG12 (10 and 100 nM) or, inactive CC (100 nM). (*P < 0.05 versus ACh and #P < 0.05 versus ISO, n = 4 per group). (E) Phase-contrast images and traction maps of human ASMCs (scale bars, 20.0 mm). (F) Traction force of human ASMCs treated with vehicle (control) or treated with TSG12 (100 nM), ISO (100 mM), or inactive CC (100 nM). Bars are the geometric means of the respective group. Cell traction data are converted to log scale before the statistical analyses. (*P < 0.05 versus control, n = 23 per group). (G) Real-time cell analysis of human ASMCs challenged with acetylcholine without treatment (ACh) or treated with salbutamol (SAL; 10 µM) or with a second treatment of either salbutamol (SAL/SAL; 10 μM) or TSG12 (SAL/TSG12; 10 nM) within 24 hours. (*P < 0.05 versus ACh and #P < 0.05 versus human ASMCs with β₂-adrenoceptor desensitization, n = 4per group). (H) Real-time cell analysis of human ASMCs challenged with acetylcholine without treatment (ACh) or treated with TSG12 (10 nM) or with a second treatment (TSG12/TSG12; 10 nM) within 24 hours (*P < 0.05 versus ACh, n = 4 per group). All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.