Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor

Abstract

Growth factors modify the structure of the glycosaminoglycan (GAG) chains on biglycan leading to enhanced LDL binding. G-protein receptor-coupled agonists such as thrombin, signal changes the structure of proteoglycans produced by vascular smooth muscle cells (VSMCs). One component of classical G-protein-coupled receptor (GPCR) signaling invokes transactivation of protein tyrosine kinase receptors such as the epidermal growth factor receptor. Serine/threonine receptor growth factors such as transforming growth factor-(TGF)-beta are potent activators of proteoglycan synthesis. We have used the model of proteoglycan synthesis to demonstrate that the signaling paradigm of GPCR signaling can be extended to include the transactivation of serine/threonine receptor, specifically the TGF-beta type I receptor (TbetaRI) also known as activin-like kinase (ALK) V. Thrombin stimulated elongation of GAG chains and increased proteoglycan core protein expression and these responses were blocked by the TbetaRI antagonist, SB431542 and TbetaRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-beta is increased C-terminal phosphorylation of the transcription factor Smad2 generating phospho-Smad2C (phosphorylation of Smad2 C-terminal region). Thrombin stimulated increased phospho-Smad2C levels, and the response was blocked by SB431542 and JNJ5177094. The proteolytically inactive thrombin mimetic thrombin-receptor activating peptide also stimulated an increase in cytosolic phospho-Smad2C. Signaling pathways for growth factor regulated proteoglycan synthesis represent therapeutic targets for the prevention of atherosclerosis, but the novel finding of a GPCR-mediated transactivation of a serine/threonine growth factor receptor almost certainly has implications well beyond the synthesis of proteoglycans.

FIGURE 1.

Thrombin-mediated proteoglycan synthesis is inhibited by TGF-β receptor ALK V inhibition, which is not due to the release and autocrine/paracrine action of TGF-β. VSMCs were treated with SB431542 (SB; 0.1–3 μm) in the presence of thrombin (10 units/ml) and [³⁵S]SO₄ (50 μCi/ml) for 24 h. JNJ5177094 (JNJ; 30 μm) was used as a positive control. a, harvested medium containing secreted proteoglycans was spotted onto Whatman paper and run through cetyl pyridinium chloride precipitation, as outlined under “Experimental Procedures,” to assess radiolabel incorporation. b, secreted proteoglycans were isolated using DEAE-loaded ion exchange chromatography followed by concentration using ethanol/potassium acetate precipitation. Electrophoretic mobility relating to the overall size of complete proteoglycans was assessed by SDS-PAGE over a 4–13% acrylamide gradient gel. The gel reveals biglycan as the proteoglycan of interest and is representative of three identical experiments. c and d, VSMCs were transfected with ALK V siRNA for 48 h followed by treatment with TGF-β (2 ng/ml) or thrombin (10 units/ml) for 24 h. Radiolabel incorporation and electrophoretic mobility were assessed as described above. e, VSMCs were treated with SB431542 (3 μm) or JNJ5177094 (30 μm) in the presence of thrombin (10 units/ml) and [³⁵S]-Met/Cys (50 μCi/ml) for 24 h to asses proteoglycan core protein synthesis. TGF-β alone and with SB431542 (3 μm) was used as a positive control. Radiolabeled incorporation was assessed as described above. f, VSMCs were treated with a pan-TGF-β neutralizing antibody (Ab) with or without SB431542 (3 μm) in the presence of thrombin (10 units/ml) or TGF-β (2 ng/ml) and [³⁵S]SO₄ (50 μCi/ml) for 24 h. Radiolabeled incorporation was assessed as described above. Results are the mean ± S.E. of data normalized to control from three separate experiments in triplicate. **, p < 0.01 and *, p < 0.05 versus thrombin or TGF-β alone and ##, p < 0.01 versus control, using a one-way ANOVA.

FIGURE 2.

A thrombin-mimetic, TRAP-mediated proteoglycan synthesis is blocked by inhibition of ALK V. VSMCs were treated with SB431542 (3 μm) or JNJ5177094 (10 μm) in the presence of TRAP (500 μm) and [³⁵S]SO₄ (50 μCi/ml) for 24 h. a, radiolabeled incorporation was assessed as described in Fig. 1A. Results are the mean ± S.E. of data normalized to control from three separate experiments in triplicate. **, p < 0.01 versus TRAP alone and ##, p < 0.01 versus control using a one-way ANOVA. b, complete proteoglycans were isolated and separated over SDS-PAGE (4–13% acrylamide gradient) as described in Fig. 1b. The gel is a representative of three independent experiments.

FIGURE 3.

Thrombin stimulates Smad2 phosphorylation over 24 h. Thrombin causes nuclear translocation of phospho-Smad2. a, VSMCs were treated with thrombin (10 units/ml) for up to 24 h. VSMCs stimulated with TGF-β (2 ng/ml) for 1 h (TGF-β) were used as a positive control. Cell lysates were collected and proteins (50 μg) were resolved on SDS-PAGE 10% acrylamide gel and then transferred to a PVDF membrane. The membrane was then incubated with anti-Smad2(Ser-465/467) monoclonal antibody (1:1000) followed by peroxidise-labeled anti-rabbit IgG secondary antibody. The membrane was then reprobed with unphosphorylated-Smad2 and anti-GAPDH monoclonal antibody's (1:1000) followed by peroxidise labeled anti-rabbit IgG secondary antibody to determine equal loading. b, VSMCs were treated with thrombin (10 units/ml) for up to 24 h. TGF-β (2 ng/ml) stimulation at 1 h was used as a positive control. Nuclear fractions were collected using cellular disruption and centrifugation and separated (50 ng/ml) by SDS-PAGE on a 10% acrylamide gel. Proteins were transferred and probed as described in a. The gel is a representation of three separate experiments. Histograms represent band density expressed as fold over basal from at least three separate experiments. **, p < 0.01 versus untreated control using a one-way ANOVA. a and b do not show quantitation of TGF-β bands as they appear off of the scale.

FIGURE 4.

Blockade of PAR-1 and ALK V inhibits thrombin stimulated phosphorylation of Smad2. a, VSMCs were treated with SB431542 (SB; 3 μm) or JNJ5177094 (JNJ; 30 μm for thrombin and 10 μm for TRAP) in the presence of thrombin (10 units/ml) or TRAP (500 μm) and cellular lysates collected at 4 h. Lysate proteins (50 ng/ml) were resolved over 10% acrylamide SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with anti-Smad2 (Ser-465/467) monoclonal antibody (1:1000) followed by peroxidise-labeled anti-rabbit IgG secondary antibody (Ab). Reprobing with anti-smooth muscle α actin (1:1000) followed by peroxidase-labeled anti-mouse IgG secondary antibody indicated equal loading of proteins. The gel is a representation of three separate experiments. b, VSMCs were preincubated for 30 min with monoclonal anti-PAR-1 antibody (5–25 μg/ml) before addition of thrombin (10 units/ml). TGF-β stimulation for 4 h was used as a positive control. Cellular lysates were collected at 4 h and separated (50 ng/ml) by SDS-PAGE on a 10% acrylamide gel. Proteins were transferred and probed as described in Fig. 3a. The gel is a representation of three separate experiments. c, VSMCs were treated with SCH79797 (SCH; 1–10 μm) in the presence of thrombin (10 units/ml). SCH79797 (10 μm) in the presence of TGF-β (2 ng/ml) was used as a positive control. Cellular lysates were collected at 4 h and separated (50 ng/ml) by SDS-PAGE on a 10% acrylamide gel. Proteins were transferred and probed as described in Fig. 3a. The gel is a representation of three separate experiments. d, VSMCs were preincubated for 15 min with 5× molar excess hirudin before addition of thrombin (10 units/ml). TGF-β alone and in the presence of hirudin (5× molar excess) was used as a positive control. Cellular lysates were collected at 4 h and separated (50 ng/ml) by SDS-PAGE on a 10% acrylamide gel. Proteins were transferred and probed as described in Fig. 3a. The gel is a representation of three separate experiments. Histograms represent band density expressed as fold over basal from at least three separate experiments. ##, p < 0.01 versus untreated control, *, p < 0.05 versus thrombin or TRAP alone, and **, p < 0.01 versus thrombin or TRAP alone using a one-way ANOVA. b, c, and d do not show quantitation of TGF-β bands as they appear off the scale.

FIGURE 5.

The respective inhibitors of ALK V and PAR-1 do not cross react with each others receptors. TGF-β does not activate IP₃ production. a, VSMCs were incubated with myo-[3H]inositol (6.25 μCi/ml) for 24 h in IP-free medium. Cells were then washed and pretreated with SB431542 (SB; 0.3–3 μm) for 30 min before stimulation with thrombin (10 units/ml) for 5 min. JNJ5177094 (JNJ; 30 mm) in the presence of thrombin (10 units/ml) was used as a positive control. Whole cell lysates were collected, and IP₃ was isolated and measured using Dowex 1 loaded ion-exchange chromatography. Results are the mean ± S.E. of data normalized to control from three separate experiments in triplicate. *, p < 0.05 versus thrombin alone using a one-way ANOVA. b, VSMCs were treated with SB431542 (3 μm) or JNJ5177094 (30 μm) in the presence of TGF-β (2 ng/ml). Whole cell lysates were collected at 4 h, and proteins (50 ng/ml) were resolved by SDS-PAGE using a 10% acrylamide gel. Proteins were transferred and probed as described in Fig. 3a. c, VSMCs were incubated with myo-[3H]inositol (6.25 μCi/ml) for 24 h in IP-free medium. Cells were then washed and treated with TGF-β (2 ng/ml) for up to 30 min. A 30-min stimulation with PDGF (50 ng/ml) was used as a positive control. Whole cell lysates were collected, and IP₃ accumulation was assessed as described in the legend to Fig. 4. Results are the mean ± S.E. of data normalized to control from one experiment in duplicate.