The free fatty acid receptor 1 promotes airway smooth muscle cell proliferation through MEK/ERK and PI3K/Akt signaling pathways

Abstract

Obesity is a risk factor for asthma and influences airway hyperresponsiveness, which is in part modulated by airway smooth muscle proliferative remodeling. Plasma free fatty acids (FFAs) levels are elevated in obese individuals, and long-chain FFAs act as endogenous ligands for the free fatty acid receptor 1 (FFAR1), which couples to both G_q and G_i proteins. We examined whether stimulation of FFAR1 induces airway smooth muscle cell proliferation through classical MEK/ERK and/or phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. The long-chain FFAs (oleic acid and linoleic acid) and a FFAR1 agonist (GW9508) induced human airway smooth muscle (HASM) cell proliferation, which was inhibited by the MEK inhibitor U0126 and the PI3K inhibitor LY294002 . The long-chain FFAs and GW9508 increased phosphorylation of ERK, Akt, and p70S6K in HASM cells and freshly isolated rat airway smooth muscle. Downregulation of FFAR1 in HASM cells by siRNA significantly attenuated oleic acid-induced phosphorylation of ERK and Akt. Oleic acid-induced ERK phosphorylation was blocked by either the Gα_i-protein inhibitor pertussis toxin or U0126 and was partially inhibited by either the Gα_q-specific inhibitor YM-254890 or the Gβγ signaling inhibitor gallein. Oleic acid significantly inhibited forskolin-stimulated cAMP activity, which was attenuated by pertussis toxin. Akt phosphorylation was inhibited by pertussis toxin, the ras inhibitor manumycin A, the Src inhibitor PP1, or LY294002 . Phosphorylation of p70S6K by oleic acid or GW9508 was significantly inhibited by LY294002 , U0126, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. In conclusion, the FFAR1 promoted airway smooth muscle cell proliferation and p70S6K phosphorylation through MEK/ERK and PI3K/Akt signaling pathways.

Keywords: FFAR1; G protein-coupled receptor; airway remodeling; cell proliferation; free fatty acid.

Fig. 1.

A: effects of 48-h treatment of the long-chain free fatty acids (oleic acid and linoleic acid) or a selective agonist of FFAR1 (GW9508) on primary cultured human airway smooth muscle (HASM) cells proliferation. Cell proliferation was assayed by the bromodeoxyuridine (BrdU) incorporation assay. Data are shown as percentages of cell proliferation compared with no treatment controls and represent means ± SE. Numbers of experiments are shown in parentheses. B and C: effect of pretreatment of HASM cells with the MEK inhibitor U0126 (5 µM for 2 h) (B) or the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (3 µM for 60 min) (C) on HASM cells proliferation stimulated by long-chain free fatty acids (oleic acid or linoleic acid; 10 µM each) or a selective agonist of FFAR1 (GW9508; 20 µM) for 48 h. Data are shown as percentages of cell proliferation compared with no treatment controls and represent means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, compared with no treatment control. #P < 0.05; ##P < 0.01; ###P < 0.001, compared with FFAR1 agonist alone. D: HASM cell viability analysis with MTT assay after 48-h treatment with U0126 (5 µM), LY294002 (3 µM), oleic acid (10 µM), linoleic acid (10 µM), or GW9508 (20 µM); n = 5. Data are shown as percentages of absorbance at 570 nm compared with no treatment control and represent means ± SE. *P < 0.05; ***P < 0.001, compared with no treatment control.

Fig. 2.

Effects of long-chain free fatty acids (oleic acid or linoleic acid) or a selective agonist of FFAR1 (GW9508) on the phosphorylation of ERK and Akt in cultured HASM cells. Cells were stimulated with oleic acid, linoleic acid, or GW9508, and subsequently cell lysates were processed to detect phosphorylated (A, top) and total (A, bottom) levels of ERK or Akt by immunoblot. A: concentration-dependent effect of oleic acid, linoleic acid, and GW9508 (0.5–20 µM; 10 min) on the phosphorylation of ERK (i) and Akt (ii) in cultured HASM cells. B: time-course effect of oleic acid (10 µM), linoleic acid (10 µM), or GW9508 (20 µM) on the phosphorylation of ERK (i) and Akt (ii) in cultured HASM cells. Data are shown as a ratio of phosphorylated to total ERK or Akt and expressed relative to basal (i.e., no treatment control) ratios and are expressed as means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, compared with basal (time 0). Numbers of experiments are shown in parentheses.

Fig. 3.

A: representative immunoblot analysis of oleic acid-stimulated phosphorylation of ERK and Akt in primary cultured HASM cells from 3 different donors (subject 1: 60-yr-old male Caucasian; subject 2: 56-yr-old male Caucasian; and subject 3: 27-yr-old male asthmatic Caucasian). Images are representative of at least 3 independent immunoblot analyses. B: effects of oleic acid or GW9508 on the phosphorylation of ERK (i) and (ii) in rat tracheal smooth muscle. Rat tracheal smooth muscle tissue was stimulated with oleic acid (20 µM) or GW9508 (20 µM) for 20 min, and subsequently the tissue homogenates were processed to detect phosphorylated (top) and total (bottom) levels of ERK or Akt by immunoblot. Phosphorylation of ERK or Akt is presented as a ratio of phosphorylated to total ERK or Akt and expressed relative to basal ratios. Data represent means ± SE. *P < 0.05, compared with basal.

Fig. 4.

A–D: involvement of FFAR1 in oleic acid-induced phosphorylation of ERK and Akt in HASM cells. HASM cells were transfected with either control nontargeting small-interfering RNA (siRNA), FFAR1-specific siRNA, or FFAR4-specific siRNA 3 days before analyses and then stimulated with oleic acid (10 µM, 10 min). The cell lysates were processed to detect phosphorylated (Ai–Hi, top) and total (Ai–Hi, bottom) levels of ERK or Akt by immunoblot. Aii–Hii: graphical analysis. A and B: the effect of downregulation of FFAR1 by siRNA on oleic acid (10 µM, 10 min)-induced phosphorylation of ERK (n = 4) (A) and Akt (n = 3) (B) in HASM cells. *P < 0.05; **P < 0.01, compared with the HASM cells transfected with nontargeting siRNA control without oleic acid stimulation. C and D: the effect of downregulation of FFAR4 by siRNA on oleic acid (10 µM, 10 min)-induced phosphorylation of ERK (n = 6) (C) and Akt (n = 8) (D) in HASM cells. ***P < 0.001, compared with the nontransfected HASM cells without oleic acid stimulation. E–H: effects of inhibitors of either Gα_q or Gα_i protein on oleic acid-induced phosphorylation of ERK and Akt in HASM cells. HASM cells were pretreated with or without inhibitors and then stimulated with oleic acid (10 µM, 10 min). The cell lysates were processed to detect phosphorylated (top) and total (bottom) levels of ERK or Akt by immunoblot. E and F: effect of Gα_q-specific inhibitor YM-254890 (1 µM for 30 min) on oleic acid-induced phosphorylation of ERK (n = 4) (E) or Akt (n = 6) (F) in HASM cells. G and H: effect of Gα_i-specific inhibitor pertussis toxin (PTX; 100 ng/ml for 4 h) on oleic acid-induced phosphorylation of ERK (n = 8) (G) or Akt (n = 5) (H) in HASM cells. Phosphorylation of ERK and Akt is presented a ratio of phosphorylated to total ERK or Akt and expressed relative to basal ratios. Data represents means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, compared with basal. White vertical spaces between the lanes in (C and D) indicate that these lanes were located on the same immunoblot but were not located in neighboring lanes on the original gel and immunoblot image.

Fig. 5.

Effect of inhibitors of Gβγ subunits, phospholipase C-β (PLC-β), protein kinase C (PKC), or Src on oleic acid-induced phosphorylation of ERK in HASM cells. Cells were pretreated with Gβγ signaling inhibitor gallein (10 µM for 60 min; n = 8) (A), phospholipase C-β inhibitor U73122 (5 µM for 30 min; n = 7) (B), PKC inhibitor GF109203X (100 nM for 60 min; n = 4) (C), or Src inhibitor PP1 (10 µM for 60 min; n = 5) (D) before treatment with oleic acid (10 µM) for 10 min. The cell lysates were processed to detect phosphorylated (Ai–Di, top) and total (Ai–Di, bottom) levels of ERK by immunoblot. Aii–Dii: graphical analysis. Phosphorylation of ERK is presented as a ratio of phosphorylated to total ERK and expressed relative to basal ratios. Data represent means ± SE. **P < 0.01; ***P < 0.001, compared with basal. White vertical spaces between the lanes in C indicate that these lanes were located on the same immunoblot but were not located in neighboring lanes on the original gel and immunoblot image.

Fig. 6.

Effect of oleic acid (10 µM) on forskolin (10 µM)-stimulated cAMP activity in HASM cells. Cells were pretreated with pertussis toxin (PTX; 100 ng/ml) for 4 h before simultaneous treatment with oleic acid and forskolin for 15 min. n = 6. Data represent means ± SE. **P < 0.01; ***P < 0.001, compared with forskolin alone. #P < 0.05, compared with forskolin + oleic acid.

Fig. 7.

Effects of oleic acid on phosphorylation of c-Raf in cultured HASM cells. Cells were stimulated with oleic acid, and subsequently cell lysates were processed to detect phosphorylated and total levels of c-Raf by immunoblot. For A–D: i: immunoblot analysis; ii: graphical analysis. A: effects of oleic acid (10 µM) on the phosphorylation of c-Raf at either Ser 338 (n = 6) or Ser 259 (n = 5) in HASM cells. B–D: effects of inhibitors of either Gα_i or Gα_q protein or Gβγ subunits on oleic acid-induced phosphorylation of c-Raf at Ser 338 in HASM cells. Cells were pretreated with the Gα_i-specific inhibitor pertussis toxin (PTX; 100 ng/ml for 4 h; n = 5; B), Gα_q-specific inhibitor YM-254890 (1 µM for 30 min; n = 8; C), or Gβγ signaling inhibitor gallein (10 µM for 30 min; n = 7; D) before treatment with oleic acid (10 µM) for 10 min. Phosphorylation of c-Raf is presented as a ratio of phosphorylated to total c-Raf and then normalized to basal levels. Data represent means ± SE. *P < 0.05; ***P < 0.001, compared with basal.

Fig. 8.

Effects of inhibitors of Gβγ subunits, ras, or Src on oleic acid-induced phosphorylation of Akt in HASM cells. Cells were pretreated with the Gβγ signaling inhibitor gallein (10 µM for 60 min; n = 7) (A), Src inhibitor PP1 (10 µM for 60 min; n = 8) (B), or ras inhibitor manumycin A (C) (3 µM for 3 h; n = 3) before treatment with oleic acid (10 µM) for 10 min. The cell lysates were processed to detect phosphorylated (Ai–Ci, top) and total (Ai–Ci, bottom) levels of Akt by immunoblot. Aii–Cii: graphical analysis. Phosphorylation of Akt is presented a ratio of phosphorylated to total Akt and expressed relative to basal ratios. Data represent means ± SE. *P < 0.05; **P < 0.01, compared with basal. White vertical spaces between the lanes in C indicate that these lanes were located on the same immunoblot but were not located in neighboring lanes on the original gel and immunoblot image.

Fig. 9.

Effects of inhibitors of MEK or phosphoinositide 3-kinase (PI3K) on FFAR1-mediated phosphorylation of ERK and Akt in HASM cells. Cells were pretreated with or without inhibitors, and then stimulated with oleic acid (10 µM, 10 min) or GW9508 (20 µM, 10 min). The cell lysates were processed to detect phosphorylated (Ai–Di, top) and total (Ai–Di, bottom) levels of ERK or Akt by immunoblot. Aii–Dii: graphical analysis. A and B: effect of MEK inhibitor U0126 (5 µM for 2 h) on oleic acid- or GW9508-induced phosphorylation of ERK (A) or Akt in HASM cells (B). C and D: effect of the PI3K inhibitor LY294002 (3 µM for 60 min) on oleic acid- or GW9508-induced phosphorylation of ERK (C) or Akt (D) in HASM cells. Phosphorylation of ERK and Akt are presented as a ratio of phosphorylated to total ERK or Akt and expressed relative to basal ratios. Data represent means ± SE. *P < 0.05; **P < 0.01, compared with basal. Numbers of experiments are shown in parentheses.

Fig. 10.

Effects of oleic acid, linoleic acid, or GW9508 on phosphorylation of p70S6K and S6 ribosomal protein in cultured HASM cells. Cells were stimulated with oleic acid, linoleic acid, or GW9508, and subsequently cell lysates were processed to detect phosphorylated and total levels of p70S6K or S6 ribosomal protein by immunoblot. For A–E: i: immunoblot analysis; ii: graphical analysis A: concentration-dependent effect of oleic acid, linoleic acid, and GW9508 (0.5–0 µM; 10 min) on the p70S6K phosphorylation in cultured HASM cells. B: time-course effect of oleic acid (10 µM), linoleic acid (10 µM), and GW9508 (20 µM) on the p70S6K phosphorylation in cultured HASM cells. C: effect of the PI3K inhibitor LY294002 (3 µM for 60 min) or the MEK inhibitor U0126 (5 µM for 2 h) on oleic acid (10 µM; 10 min)- or GW9508 (20 µM; 10 min)-induced p70S6K phosphorylation in HASM cells. D: effect of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (1 µM for 60 min) on oleic acid- or GW9508-induced phosphorylation of p70S6K in HASM cells. E: effect of rapamycin (1 µM for 60 min) on oleic acid- or GW9508-induced phosphorylation of S6 ribosomal protein in HASM cells. Phosphorylation of p70S6K and S6 ribosomal protein are presented as a ratio of phosphorylated to total p70S6K or S6 and expressed relative to basal ratios. Data are shown as percentages of basal cell phosphorylation and are expressed as means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, compared with basal (time 0). Numbers of experiments are shown in parentheses.

Fig. 11.

Schematic drawing of FFAR1-stimulated signaling cascade of ERK and Akt in HASM cells. G_i-coupled FFAR1 inhibits adenylyl cyclase activity, which inhibits cAMP production. Reduced cAMP production leads to decreased PKA activity. The decreased PKA activity in turn relieves the inhibitory effect of PKA on c-Raf, thus potentiating the c-Raf/MEK/ERK signaling. Both the Gα_q subunit and Gβγ subunit, which is dissociated from both G_i and G_q following FFAR1 activation, contribute to ERK phosphorylation, although the signaling pathway does not involve PLC-β, PKC, or Src. G_i-coupled FFAR1 also activates the PI3K/Akt pathway, which is mediated through Gα_i/ras/Src signaling. The MEK/ERK pathway directly induces cell proliferation, and the PI3K/Akt pathway induces p70S6K phosphorylation via mTORC1. The MEK/ERK pathway also cross activates mTORC1 and induces phosphorylation of p70S6K. p70S6K phosphorylates S6 ribosomal protein and leads to HASM cell proliferation.