Lysophosphatidic acid modulates c-Met redistribution and hepatocyte growth factor/c-Met signaling in human bronchial epithelial cells through PKC delta and E-cadherin

Abstract

Previously we demonstrated that ligation of lysophosphatidic acid (LPA) to G protein-coupled LPA receptors induces transactivation of receptor tyrosine kinases (RTKs), such as platelet-derived growth factor receptor beta (PDGF-Rbeta) and epidermal growth factor receptor (EGF-R), in primary cultures of human bronchial epithelial cells (HBEpCs). Here we examined the role of LPA on c-Met redistribution and modulation of hepatocyte growth factor (HGF)/c-Met pathways in HBEpCs. Treatment of HBEpCs with LPA-induced c-Met serine phosphorylation and redistribution to plasma membrane, while treatment with HGF-induced c-Met internalization. Pretreatment with LPA reversed HGF-induced c-Met internalization. Overexpression of dominant negative (Dn)-PKC delta or pretreatment with Rottlerin or Pertussis toxin (PTx) attenuated LPA-induced c-Met serine phosphorylation and redistribution. Co-immnuoprecipitation and immunocytochemistry showed that E-cadherin interacted with c-Met in HBEpCs. LPA treatment induced E-cadherin and c-Met complex redistribution to plasma membranes. Overexpression of Dn-PKC delta attenuated LPA-induced E-cadherin redistribution and E-cadherin siRNA attenuated LPA-induced c-Met redistribution to plasma membrane. Furthermore, pretreatment of LPA attenuated HGF-induced c-Met tyrosine phosphorylation and downstream signaling, such as Akt kinase phosphorylation and cell motility. These results demonstrate that LPA regulates c-Met function through PKC delta and E-cadherin in HBEpCs, suggesting an alternate function of the cross-talk between G-protein-coupled receptors (GPCRs) and RTKs in HBEpCs.

Figure 1. LPA induces serine phosphorylation of c-Met

A. HBEpCs (70-80 % confluence in 100 mm dishes) were treated with LPA (1 μM) for 2, 10, or 30 min or HGF (25 ng/ml) for 10 min. Cell lysates were immunoprecipitated with anti-c-Met antibody and blotted with anti-phosphoserine and anti-c-Met antibodies. B. HBEpCs (70-80 % confluence in 35 mm-dishes) were treated with LPA (1 μM) for 2, 10, or 30 min or HGF (25 ng/ml) for 10 min. Cell lysates were analyzed by Western blotting with anti-phosphotyrosine (1230/1245/1246)-c-Met and anti-c-Met antibodies. C. HBEpCs (70-80 % confluence in 100 mm dishes) were pretreated with or without PTx (100 ng/ml) for 4 h, then further challenged with or without LPA (1 μM) for 10 min. Lysates were immunoprecipitated with anti-c-Met antibody and blotted with anti-phosphoserine and anti-c-Met antibodies. Shown are representative blots of three independent experiments.

Figure 2. Overexpression of Dn-PKC δ or rottlerin attenuates LPA-induced serine phosphorylation of c-Met

HBEpCs (50 % confluence in 100 mm dishes) were infected with adenoviral empty vector (20 MOI) or adenoviral vector containing Dn-PKC δ (1, 10, and 20 MOI) for 48 h or pretreated with DMSO (0.1 %) or Rottlerin (5 μM) for 1 h, then challenged with LPA (1 μM) for 15 min. Cell lysates were either processed for blotting with anti-PKC δ antibody (A), or immunoprecipitated with anti-c-Met antibody and blotted with anti-phosphoserine and anti-c-Met antibodies (B and C). Shown are representative blots of three independent experiments.

Figure 3. LPA induces c-Met intracellular traffic in HBEpCs

A. HBEpCs (70-80 % confluence in cover slips) were treated with LPA (1 μM) for 15, 30, or 60 min, and immunostained with anti-c-Met antibody. B. HBEpCs (70-80 % confluence in cover slips) were pretreated with BIM (1 μM) or 0.1 % DMSO for 1 h, then challenged with LPA (0.1, 1.0, and 5.0 μM) for an additional 15 min. Cells were immunostained with anti-c-Met antibody. C. HBEpCs (50 % confluence in cover slips) were infected with adenoviral empty vector (20 MOI) or adenoviral vector containing Dn-PKC δ (1, 10, and 20 MOI) for 48 h. Cells were challenged with LPA (1 μM) for 15 min. Cells were immunostained with anti-c-Met antibody. Shown are representative images of three independent experiments.

Figure 4. c-Met associates with E-cadherin in HBEpCS

A. HBEpCs (70-80 % confluence in cover slips) were treated with LPA (1 μM) for 5, 15, or 30 min, and double immunostained with anti-c-Met (green) and anti-E-cadherin (red) antibodies. Combined images shown in lower pattern. Shown are representative images of three independent experiments. B. HBEpCs (70-80 % conference in 100 mm dishes) were treated with LPA (1 μM) for 10 or 30 min. Lysates were immunoprecipitated with anti-E-cadherin antibody and blotted with anti-E-cadherin or anti-c-Met antibody. Shown are representative blots of three independent experiments.

Figure 5. Overexpression of Dn-PKC δ attenuates LPA-mediated E-cadherin redistribution in HBEpCS

HBEpCs (50 % confluence in cover slips) were infected with adenoviral empty vector (20 MOI) or adenoviral vector containing Dn-PKC δ (20 MOI) for 48 h. Cells were challenged with or without LPA (1 μM) for 15 min. Cells were immunostained with anti-E-cadherin antibody. Shown are representative images of three independent experiments.

Figure 6. E-cadherin siRNA attenuates LPA-induced c-Met redistribution in HBEpCs

A. HBEpCs (50 % confluence in cover slips) were transfected with scrambled and E-cadherin siRNA (100 nM) for 72 h, then challenged with or without LPA (1 μM) for 15 min. Cells were immunostained with anti-c-Met antibody. Shown are representative images of three independent experiments. B. HBEpCs (50 % confluence in 6-well plates) were transfected with scrambled and E-cadherin siRNA (100 nM) for 72 h. Lysates were blotted with anti-E-cadherin or anti-c-Met antibody. Shown are representative blots of three independent experiments.

Figure 7. LPA attenuates HGF-induced tyrosine phosphorylation of c-Met

A. HBEpCs (70-80 % confluence in cover slips) were treated with or without LPA (1 μM) for 15 min, then challenged with or without HGF (25 ng/ml) for an additional 30 min. Cells were immunostained with anti-c-Met antibody. Shown are representative images of three independent experiments. B. HBEpCs (70-80 % confluence in 6-well plates) were pretreated with or without LPA (1 μM) for 15 min, then challenged with HGF (25 ng/ml) for 15, 30, 60, or 120 min. Cell lysates were blotted with anti-phospho-c-Met or anti-c-Met antibody. Shown are representative blots of three independent experiments. C. HBEpCs (70-80 % confluence in 6-well plates) were pretreated with or without LPA (1 μM) for 15 min, then challenged with HGF (10 and 25 ng/ml) for 30 min. Cell lysates were blotted with anti-phospho-c-Met and anti-c-Met antibodies. Shown are representative blots of three independent experiments.

Figure 8. LPA attenuates HGF / c-Met-mediated signaling and cell motility

A. HBEpCs (70-80 % confluence in 6-well plates) were pretreated with or without LPA (1 μM) for 15 min, then challenged with HGF (25 ng/ml) for an additional 30, 60, or 120 min. Cell lysates were blotted with anti-phospho-Akt and anti-Akt antibodies. Shown are representative blots of three independent experiments. B. HBEpCs colonies (grown on 100 mm dishes) were pretreated with or without LPA (1 μM) for 15 min, then challenged with or without HGF (25 ng/ml) for 3 h. Shown are representative images of five independent experiments. C. Cells area was measured with NIH imageJ software.

Figure 9. Mechanisms of regulation of HGF / c-Met signaling by LPA in HBEpCs

Ligation of LPA to G-coupled LPA receptors induces c-Met serine phosphorylation and redistribution through activation of PKC δ and interaction with E-cadherin, resulting in attenuation of HGF / c-Met-mediated phosphorylation of c-Met and Akt and cell motility.