QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 5 June 2007
doi: 10.1242/jcs.003038

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dada, L. A. Articles by Sznajder, J. I. Search for Related Content PubMed PubMed Citation Articles by Dada, L. A. Articles by Sznajder, J. I.

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA

View larger version (18K): [in this window] [in a new window]   Fig. 1. Effects of hypoxia on Na,K-ATPase endocytosis in A549 cells. (A) Serum-starved A549 cells were exposed to 21 or 1.5% O2 for 60 minutes and the protein levels of the Na,K-ATPase-1 subunit or the transferrin receptor in the plasma membrane were studied by cell surface biotinylation followed by streptavidin pull-down and western blot analysis using specific antibodies. A representative western blot for each protein is shown, mean ± s.e.m. (n=4). **P<0.01. (B) COS-7 cells were transiently transfected with vector or GFF-Dyn K44A, serum-starved and exposed to 21 or 1.5% O2 for 60 minutes. Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific anti 1-subunit antibody. Top: bars represent mean ± s.e.m. (n=3), **P<0.01. Bottom: a representative western blot for 1-Na,K-ATPase endocytosis and GFP-dynamin expression levels.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effect of cytoskeleton-disrupting agents on hypoxia-induced Na,K-ATPase endocytosis. Serum-starved A549 cells were pretreated in the presence or absence of 0.1 µM LB (15 minutes; A), 2 µM Cyt D (15 minutes; B), 1 µM phalloidin (overnight, C), 1 µM Colchicine (4 hours, D) and then exposed to 21 or 1.5% O2 for 60 minutes. Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific anti 1-subunit antibody. A representative western blot for each treatment is shown, mean ± s.e.m. (n=3). **P<0.01.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Hypoxia-induced activation of RhoA in A549 cells. (A) Serum-starved A459 cells were exposed to hypoxia for 15, 30 or 60 minutes, 1% Triton X-100-soluble membrane fractions were obtained and RhoA translocation was evaluated by western blot. Top: a representative western blot for RhoA translocation and total 1-Na,K-ATPase as loading control; Bottom: points on the curve represent mean ± s.e.m. (n=3). *P<0.05; **P<0.01. (B) A549 cells were exposed to hypoxia for 5, 15, 30 or 60 minutes and cell lysates were subjected to a pull-down assay with a GST-tagged fusion protein rhotekin Rho binding (RBD) domain. A representative western blot of RhoA bound to GTP (top) and total RhoA (bottom) is shown (n=3). (C) A549 cells were plated onto glass coverslips and exposed to 21 or 1.5% for 15 or 60 minutes. After incubation cells were washed, fixed, stained with rhodamine-phalloidin and evaluated using fluorescence microscopy. Representative images are shown.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Effect of mitochondria-generated ROS on RhoA activity and protein levels in A549 cells during hypoxia. (A) A549 cells were infected with null adenovirus (Sham) or adenovirus expressing Myc-tagged GPX1 (AdGPx) and subsequently exposed to 21% O2 or 1.5% O2 for 15 minutes. 1% Triton X-100-soluble membrane fractions were obtained and RhoA translocation was evaluated by western blot. Mean ± s.e.m. (n=3), **P<0.01. A representative western blot is shown for RhoA and Myc. (B) WT and (0)-A549 cells were exposed to 21% O2 or 1.5% O2 for 15 minutes and then processed as described above. Mean ± s.e.m. (n=3), **P<0.01. (C) WT and (0)-A549 cells were exposed to 21% O2 or 1.5% O2 for 15 minutes and cell lysates were subjected to a pull-down assay with a GST-tagged fusion protein RBD and processed as described. Mean ± s.e.m. (n=3), **P<0.01. (D) A549 cells stably transfected with RNAi against Drosophila HIF (dHIF) or the Rieske iron sulphur protein (FeS) were exposed to 21% O2 or 1.5% O2 for 15 minutes and then processed as described above. Mean ± s.e.m. (n=3), **P<0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 5. H2O2 induced RhoA activation in A549 cells. (A) Serum-starved A459 cells were treated with 100 µM t-H2O2 for 5, 15 and 30 minutes. 1% Triton X-100-soluble membrane fractions were obtained and RhoA translocation was evaluated by western blot. Top: points on the curve represent mean ± s.e.m. (n=3). **P<0.01. Bottom: a representative western blot for RhoA translocation and 1-Na,K-ATPase as loading control. (B) A549 cells were exposed to 100 µM t-H2O2 for 15 minutes; stress fiber formation was assessed in cells fixed and stained with rhodamin-palloidin and evaluated by fluorescence microscopy. Representative photomicrographs of control cells (V), and t-H2O2 treated cells are shown.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Dominant negative RhoA prevents hypoxia-induced Na,K-ATPase endocytosis. Permanently transfected A549 cells expressing either vector or dominant negative RhoA (RhoAN19) were grown in the presence of G418. (A) Actin stress fiber formation was assessed in DN-RhoA cells fixed and stained with rhodamin-palloidin and evaluated by using fluorescence microscopy. Representative photomicrographs of control cells (21% O2), and hypoxia (1.5% O2)-treated cells are shown. (B) Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific antibody after exposure to 1.5% O2 for 60 minutes. Top: bars represent mean ± s.e.m. (n=3). **P<0.01. Bottom: a representative western blot. (C) Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific antibody after treatment with 100 µM H2O2 for 30 minutes. Top: bars represent mean ± s.e.m. (n=3). **P<0.01. Bottom: a representative western blot.

View larger version (21K): [in this window] [in a new window]   Fig. 7. ROCK activation is necessary in hypoxia-induced Na,K-ATPase endocytosis. (A) A549 cells were exposed to hypoxia in the presence or absence of 10 µM Y-27632 and cell lysates were obtained. Equal amount of proteins were separated by SDS-PAGE and immunoblotted with either phospho-MYPT (Thr696) antibody or pan-MYPT antibody. A representative western blot is shown (n=3). (B) A549 cells were pre-incubated in the presence of vehicle (V) or 10 µM Y-27632 and then exposed to hypoxia for 60 minutes. Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins followed by streptavidin pull-down and western blot analysis using a specific antibody. Top: bars represent mean ± s.e.m. (n=3), **P<0.01. Bottom: a representative western blot. (C) COS-7 cells were transiently transfected with vector, DN-ROCK, and Na,K-ATPase endocytosis was studied by biotin labeling of surface proteins. Top: bars represent mean ± s.e.m. (n=3), **P<0.01. Bottom: a representative western blot for 1-Na,K-ATPase endocytosis and Myc-ROCK expression levels.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Hypoxia-mediated translocation of PKC is independent of RhoA. Serum-starved WT and DN-RhoA-A549 cells were exposed to 21% O2 or 1.5% O2 for 20 minutes. Triton X-100-soluble membrane fractions were obtained and PKC translocation was evaluated by western blot. Top: bars represent mean ± s.e.m. (n=3). **P<0.01. Bottom: a representative western blot for PKC translocation and total 1-Na,K-ATPase as loading control.

View larger version (9K): [in this window] [in a new window]   Fig. 9. A signaling model for hypoxic activation of RhoA and Na,K-ATPase regulation in alveolar epithelial cells. Based on our current findings we propose that hypoxia stimulates oxidant production within mitochondria. These oxidants activate the RhoA/ROCK pathway, resulting in stress fiber formation and a decrease in Na,K-ATPase protein levels at the plasma membrane.