Figure 1.
HGF stimulates peripheral MT growth.
HPAEC grown on coverslips were stimulated with HGF (50 ng/ml, 10 min) with or without pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) followed by A: Immunofluorescence staining with an antibody against β-tubulin; B: Immunostaining with anti-EB1 antibody. Insets show high magnification images of cell periphery areas with microtubules or EB1-positive microtubule tips. Bar = 5 µm. Results are representative of five independent experiments. Bar graphs depict results of quantitative analysis of peripheral microtubules (A, right panel) and peripheral EB1 (B, right panel) in methanol-fixed HPAEC; *P<0.05; n = 4; 6 images from each experiment. C: Live cell imaging of HPAEC expressing GFP-EB1 stimulated with HGF with or without pretreatment with c-Met inhibitor. Projection analysis of 20 consecutive images before and after HGF treatment shows changes in GFP-EB1 track length. Bar = 2 µm. Quantification of GFP-EB1 track length is presented on right panels. Each pair of dots represents the median track length in a cell before and after thrombin treatment. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.
Figure 2.
Role of Rac1 in HGF-induced stimulation of peripheral MT network formation.
A: Cells grown on coverslips were transfected with non-specific RNA or Rac1-specific siRNA and stimulated with HGF (50 ng/ml, 10 min) followed by immunofluorescence staining with an antibody against β-tubulin. Bar = 5 µm. Magnified images (insets) show details of MT structure. Results are representative of four independent experiments. B: Fraction of peripheral MT was quantified as described in Methods; *P<0.05; n = 4; 6 images from each experiment. C: Projection analysis of 20 consecutive images in control (top panel) and Rac1 knockdown (bottom panel) live cells before and after HGF treatment shows changes in GFP-EB1 track length. Bar = 2 µm. Quantification of GFP-EB1 track length is presented on right panels. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.
Figure 3.
Involvement of Rac pathway in HGF-induced MT-associated signaling.
A–D: HPAEC were subjected to pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) or knockdown of Rac1 or PAK1 as described in Methods and stimulated with HGF (50 ng/ml) for the indicated periods of time. A: Rac activation was determined by Rac-GTP pulldown assay. The content of activated Rac was normalized to the total Rac content in EC lysates. B: Time-dependent stimulation of stathmin phosphorylation and increase in tubulin acetylation was detected by western blot. C: Effect of preincubation with c-Met inhibitor on HGF-induced stathmin phosphorylation was evaluated by western blot with phospho-S63-stathmin antibody. D and E: HGF-induced stathmin phosphorylation and tubulin acetylation in cells with Rac1 (D) and PAK1 (E) knockdown were evaluated by western blot. siRNA-induced target protein depletion was confirmed by membrane probing with Rac1 or PAK1 antibody. Equal protein loading in all assays was confirmed by membrane probing with β-actin antibody. F: Bar graphs depict the quantitative densitometry analysis of western blot data from four independent experiments; *P <0.05, RDU: relative density units.
Figure 4.
Expression of phosphorylation-deficient stathmin attenuates HGF-induced EC barrier enhancement.
A: Endothelial monolayers transfected with phosphorylation-deficient stathmin (STMN-S63A) or empty vector (Em. Vec.) were stimulated with HGF (50 ng/ml). A: TER measurements were performed over 1.5 hrs. Bar graphs depict results of quantitative analysis of permeability data; n = 5; *P<0.05. B: Cortactin phosphorylation at Y421 and tubulin acetylation at indicated time points of HGF treatment was monitored by Western blot. Probing for β-tubulin was used as a normalization control. Results are representative of three independent experiments. Bar graphs depict the quantitative densitometry analysis of western blot data; n = 4; *P <0.05, RDU: relative density units.
Figure 5.
Expression of phosphorylation-deficient stathmin attenuates HGF-induced stimulation of peripheral MT network formation.
Cells grown on coverslips were transfected with empty vector (Em. Vec.) or STMN-S63A and stimulated with HGF (50 ng/ml, 10 min). A: MT network was visualized by immunofluorescence staining of methanol-fixed cells with an antibody against β-tubulin. Transfected cells were detected by staining with His-tag antibody. Insets show magnified images with details of MT structure in non-transfected and STMN-S63A transfected cells. Bar = 10 µm. Results are representative of three independent experiments. B: Bar graphs depict results of quantitative analysis of peripheral microtubules; n = 3; 10 cells from each experiment; *P<0.05.
Figure 6.
Phosphorylation-deficient stathmin attenuates HGF protective effects against thrombin-induced MT disassembly and activation of Rho signaling.
Cells transfected with empty vector (Em. Vec.) or STMN-S63A were stimulated with thrombin (0.5 U/ml, 15 min) or HGF (50 ng/ml, 10 min) + thrombin. A: After separation of MT-enriched fraction and fraction with soluble β-tubulin, β-tubulin content in each fraction was determined by western blot. Determination of β-tubulin content in total cell lysates was used as a normalization control. B: Rho activity in total cell lysates was evaluated by RhoGTP pulldown assay (upper panel) and normalized to total Rho content in cell lysates (middle panel). C: Activation of the Rho pathway was evaluated by western blot analysis of phospho-MYPT and diphospho-MLC levels. Reprobing with β-actin antibody was used as the normalization control. D: GEF-H1 activation in HPAEC stimulated with thrombin or HGF + thrombin was evaluated by GEF pulldown assay. Western blot detection of GEF-H1 in corresponding total lysates was used as normalization control. E: Bar graphs depict the quantitative densitometry analysis of western blot data from three independent experiments; *P <0.05, RDU: relative density units.
Figure 7.
Phosphorylation-deficient stathmin attenuates HGF protective effects against thrombin-induced permeability and cytoskeletal remodeling.
Endothelial monolayers transfected with phosphorylation-deficient stathmin (STMN-S63A) or empty vector (Em. Vec.) were treated with thrombin or HGF (50 ng/ml) + thrombin (0.5 U/ml). A: TER measurements were performed over 1.5 hrs. Results are representative of four independent experiments. Bar graphs depict results of quantitative analysis of TER data; *P<0.05. B-C: F-actin remodeling (B, bar = 5 µm) and MT remodeling (C, bar = 10 µm) in EC expressing STMN-S63A was performed by double immunofluorescence staining with either Texas Red phalloidin, or β-tubulin antibody and His-tag antibody to detect STMN-S63A expressing cells. The magnified images (insets) show the details of actin and MT structure in non-transfected and STMN-S63A expressing cells. Results are representative of three independent experiments. Bar graphs (C, lower right panel) depict results of quantitative image analysis of peripheral microtubules from three independent experiments; 10 cells from each experiment. *P<0.05.
Figure 8.
Signaling mechanism of HGF-induced attenuation of GEF-H1 activity and barrier disruptive Rho signaling via Rac1-dependent stimulation of peripheral microtubule network.
HGF engages c-Met receptor and triggers Rac1 activity, leading to activation of PAK1. PAK1-mediated phosphorylation of stathmin inhibits its MT destabilizing activity and promotes the growth of the peripheral MT network leading to immobilization and inactivation of Rho-specific guanine nucleotide exchange factor GEF-H1. As a result, MT-dependent GEF-H1 inactivation attenuates agonist induced Rho signaling leading to recovery of peripheral actin cytoskeleton and restoration of endothelial barrier.