Epithelial cells utilize cortical actin/myosin to activate latent TGF-β through integrin α(v)β(6)-dependent physical force

Abstract

Transforming Growth Factor Beta (TGF-β) is involved in regulating many biological processes and disease states. Cells secrete cytokine as a latent complex that must be activated for it to exert its biological functions. We previously discovered that the epithelial-restricted integrin α(v)β(6) activates TGF-β and that this process is important in a number of in vivo models of disease. Here, we show that agonists of G-protein coupled receptors (Sphingosine-1-Phosphate and Lysophosphatidic Acid) which are ligated under conditions of epithelial injury directly stimulate primary airway epithelial cells to activate latent TGF-β through a pathway that involves Rho Kinase, non-muscle myosin, the α(v)β(6) integrin, and the generation of mechanical tension. Interestingly, lung epithelial cells appear to exert force on latent TGF-β using sub-cortical actin/myosin rather than the stress fibers utilized by fibroblasts and other traditionally "contractile" cells. These findings extend recent evidence suggesting TGF-β can be activated by integrin-mediated mechanical force and suggest that this mechanism is important for an integrin (α(v)β(6)) and a cell type (epithelial cells) that have important roles in biologically relevant TGF-β activation in vivo.

fig. 1. S1P induces α_vβ₆-mediated TGF-β activation via Rho Kinase

(A) Western blot for pSmad2 or Total Smad2 from lysates of NHBE cells treated with methanol (control) or S1P (1–30 μM) in the presence or absence of an anti-β₆ antibody (40 μg/mL). (B) Western blots, as above, of lysates from NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Rho Kinase inhibitor (Y-27632, 100 μM), and anti-TGF-β antibody (40 μg/mL). (C) Western blots of lysates of air-liquid interface cultures of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Y-27632 (100 μM), and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding dose groups. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the standard error of the mean (s.e.m.).

fig. 1. S1P induces α_vβ₆-mediated TGF-β activation via Rho Kinase

(A) Western blot for pSmad2 or Total Smad2 from lysates of NHBE cells treated with methanol (control) or S1P (1–30 μM) in the presence or absence of an anti-β₆ antibody (40 μg/mL). (B) Western blots, as above, of lysates from NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Rho Kinase inhibitor (Y-27632, 100 μM), and anti-TGF-β antibody (40 μg/mL). (C) Western blots of lysates of air-liquid interface cultures of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Y-27632 (100 μM), and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding dose groups. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the standard error of the mean (s.e.m.).

fig. 1. S1P induces α_vβ₆-mediated TGF-β activation via Rho Kinase

(A) Western blot for pSmad2 or Total Smad2 from lysates of NHBE cells treated with methanol (control) or S1P (1–30 μM) in the presence or absence of an anti-β₆ antibody (40 μg/mL). (B) Western blots, as above, of lysates from NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Rho Kinase inhibitor (Y-27632, 100 μM), and anti-TGF-β antibody (40 μg/mL). (C) Western blots of lysates of air-liquid interface cultures of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL), Y-27632 (100 μM), and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding dose groups. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the standard error of the mean (s.e.m.).

fig. 2. S1P and LPA induced α_vβ₆-mediated TGF-β activation requires cellular tension

(A) Western blots for pSmad2 and Total Smad2 of lysates of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (B) Western blots, as above, of lysates of NHBE cells treated with water (control), LPA (50 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (C) Western blots of lysates from NHBE cells cultured on various rigidities (0.025%, 0.05%, 0.1%, 0.2%) of polyacrylamide flexible substrates and treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL) and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding vehicle treated group. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the s.e.m.

fig. 2. S1P and LPA induced α_vβ₆-mediated TGF-β activation requires cellular tension

(A) Western blots for pSmad2 and Total Smad2 of lysates of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (B) Western blots, as above, of lysates of NHBE cells treated with water (control), LPA (50 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (C) Western blots of lysates from NHBE cells cultured on various rigidities (0.025%, 0.05%, 0.1%, 0.2%) of polyacrylamide flexible substrates and treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL) and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding vehicle treated group. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the s.e.m.

fig. 2. S1P and LPA induced α_vβ₆-mediated TGF-β activation requires cellular tension

(A) Western blots for pSmad2 and Total Smad2 of lysates of NHBE cells treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (B) Western blots, as above, of lysates of NHBE cells treated with water (control), LPA (50 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of Blebbistatin (20 μM). (C) Western blots of lysates from NHBE cells cultured on various rigidities (0.025%, 0.05%, 0.1%, 0.2%) of polyacrylamide flexible substrates and treated with methanol (control), S1P (30 μM), and active TGF-β1 (15 pg/mL) in the presence or absence of an anti-β₆ antibody (40 μg/mL) and anti-TGF-β antibody (40 μg/mL). *P < 0.05 for indicated comparison or versus the corresponding vehicle treated group. The ratio of pSmad2 density to that of Total Smad2 was analyzed. All results are representative of at least three independent experiments. Error bars represent the s.e.m.

fig. 3. S1P and LPA induced β₆-mediated TGF-β activation results in cortical actin formation

(A) Phalloidin staining of the actin cytoskeleton in NHBE cells treated with methanol (vehicle) or S1P (30 μM) in the presence or absence of Y-27632 (100 μM) and Blebbistatin (20 μM). (B) Phalloidin staining, as above, in NHBE cells treated with water (vehicle) or LPA (50 μM) in the presence or absence of Y-27632 (100 μM) and Blebbistatin (20 μM). Arrows indicate cortical actin structures. Scale bars: 20 μm