Role of protein transamidation in serotonin-induced proliferation and migration of pulmonary artery smooth muscle cells

Abstract

Pulmonary hypertension is characterized by elevated pulmonary artery pressure and pulmonary artery smooth muscle cell (SMC) proliferation and migration. Clinical and experimental evidence suggests that serotonin (5-HT) is important in these responses. We previously demonstrated the participation of the 5-HT transporter and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and time-dependent increase in serotonylation of multiple proteins in both bovine and rat pulmonary artery SMCs. Inhibition of TGase with dansylcadaverin blocked this activity, as well as SMC-proliferative and migratory responses to 5-HT. Serotonylation of proteins also was blocked by 5-HT transporter inhibitors, and was enhanced by inhibition of monoamine oxidase, an enzyme known to degrade 5-HTi, indicating that 5-HTi levels regulate serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by TGase was fibronectin (FN). 5-HT-stimulated SMC serotonylation and proliferation were blocked by FN small interfering (si) RNA. These findings, together with previous observations that FN expression in the lung strongly correlates with the progression of pulmonary hypertension in both experimental animals and humans, suggest an important role of FN serotonylation in the pathogenesis of this disease.

Figure 1.

Schematic illustration of serotonylated modification of proteins by transglutaminase (TGases).

Figure 2.

Serotonin (5-hydroxytryptamine [5-HT])-induced protein serotonylation in bovine pulmonary artery smooth muscle cell (SMC) protein extract. SMCs were washed with Hanks' balanced salt solution, harvested, sonicated at 4°C for 10 seconds, and then centrifuged for 5 minutes at 10,000 × g. Supernatants containing cell extracts then were analyzed for protein serotonylation. (A) Proteins in Tris-buffered saline buffer were incubated with 0–1,000 μmol/L 5-HT at 37°C for 60 minutes in the presence or absence of the TGase inhibitor, dansylcadaverin (DSC; 200 μmol/L). Reactions were terminated by the addition of 5 mmol/L EGTA on ice. Proteins were concentrated and desalted with micro-filter columns (Pierce, Rockford, IL) by centrifugation at 4°C, and then analyzed by Western blotting using anti–5-HT–BSA–conjugated anti-serum (Sigma) for protein serotonylation. (B) Protein serotonylation was induced by 1 mmol/L 5-HT in SMC protein extract for 60 minutes and assessed by Western blotting. (C) Anti–5-HT–BSA anti-serum was preincubated with 10 mmol/L 5-HT for 2 hours to neutralize 5-HT–binding activity. Proteins from control (−) and 5-HT–treated cell lysates (+) were immunoblotted with 0.5 μg/ml 5-HT anti-serum control or neutralized 5-HT anti-serum. Protein molecular weight marker is shown to the left in all figures. Bar graphs represent means (±SD) (n = 3). *Significant difference from the untreated controls at (P < 0.05); ^#significant difference from 5-HT–treated cells (P < 0.05).

Figure 3.

5-HT–mediated protein serotonylation in SMCs. Bovine SMCs were washed with serum-free Dulbecco's modified Eagle's medium (DMEM) and then incubated with 5-HT. (A) Cells were incubated for 5–60 minutes with 5-HT (1 μmol/L). (B) Cells were incubated with 5-HT (–(1,000 μmol/L) for 60 minutes. (C) Rat cells were incubated with 5-HT (0–10 μmol/L) for 60 minutes. After incubation, cell extracts were obtained, electrophoresed, and subjected to immunoblotting, as noted in Materials and Methods. Bar graphs represent means (±SD) (n = 3). *Significant difference from the untreated controls (P < 0.05).

Figure 4.

Modulation of protein serotonylation in SMCs in culture by 5-HT transporter (5-HTT), monoamine oxidase (MAO), and TGase. Bovine SMCs were washed with serum-free DMEM and then pretreated with (A) 5-HTT inhibitor, imipramine or fluoxetine (1,10 μmol/L) (B) MAO inhibitor, phenelzine (50 μmol/L), or (C) TGase inhibitor, DSC (200 μmol/L) for 30 minutes. Protein serotonylation was then induced by 5-HT (1 μmol/L) for 30 minutes. Cells lysates underwent electrophoresis, and serotonylation was analyzed by Western blotting with anti–5-HT–BSA conjugate antibody. Bar graphs represent means (±SD) for n = 3. *Significant difference from the untreated controls (P < 0.05); ^#significant difference from 5-HT–treated cells (P < 0.05).

Figure 5.

5-HT–induced fibronectin (FN) serotonylation in SMCs. Bovine SMCs were washed with serum-free DMEM and incubated with 5-HT (1 μmol/L) for 1 hour. (A) 5-HT–induced FN serotonylation was assessed by immunoprecipitation (IP) of FN in whole-cell lysates, followed by electrophoresis and immunoblotting (IB) with anti–5-HT–BSA conjugate antibody. Total FN was measured with anti-FN antibody on the stripped membrane (lower panel). (B) Release of FN into the medium. Medium (4 ml) from control (−) and 5-HT–incubated (+) SMCs was concentrated and desalted. FN in the concentrated medium (50 μl) was assessed by Western blotting with anti-FN antibody. Lower panel shows FN in the cell lysate.

Figure 6.

Role of TGase in 5-HT–induced SMC proliferation and migration responses. Growth-arrested bovine SMCs were pretreated with TGase inhibitor, DSC (200 μmol/L) for 30 minutes. (A) To test the proliferation response, cells were stimulated with 5-HT (1 μmol/L) for 24 hours. Cell proliferation was analyzed by measuring [³H]-thymidine incorporation (n = 6). (B) Migration was assessed by a wound healing test. Wounding was performed by scraping through the cell monolayer with a sterile 1-ml pipette tip immediately after the addition of 5-HT (10 μmol/L). At 20 hours after 5-HT treatment, cell migration images were taken at four wound sites along the wounding scratch by Nikon phase-contrast microscopy at 100-fold. Triplicate results were obtained in three separate experiments, and a representative experiment is shown. The scale bar shown on the photomicrographs = 0.2 mm. (C) Cell migration was further accessed by Boyden chamber assay. Migrated cells on the bottom of the Transwell filter were stained with crystal violet 20 hours after 5-HT treatment. *Significant difference from control (P < 0.05); ^#significant difference from 5-HT alone (P < 0.05).

Figure 7.

Silencing of cellular FN inhibits SMC-proliferative response to 5-HT. (A) Effects of FN siRNA on its protein expression and serotonylation. Bovine SMCs in 60-mm plates were transfected with FN siRNA and control siRNA using lipofectamine 2,000 for 48 hours. Cells were then treated with 5-HT (1 μmol/L) for 40 minutes. FN serotonylation was evaluated by its immunoprecipitation. FN expression was analyzed in total cell lysates by Western blot and normalized with expression of tubulin on the stripped membrane. (B) Inhibition of 5-HT–induced SMC proliferation by FN siRNA. SMCs transfected with FN siRNA and control siRNA were growth arrested in serum-free medium for 72 hours before treatment with 5-HT (1 μmol/L). Cell proliferation was monitored by [³H]-thymidine incorporation for 24 hours. Data presented are means (±SD) (n = 3). *Significant difference from control (P < 0.05); ^#significant difference from control siRNA 50 nM + 5-HT (P < 0.05).