doi: 10.1128/MCB.00839-13.
Epub 2013 Nov 4.
A reactive oxygen species-mediated, self-perpetuating loop persistently activates platelet-derived growth factor receptor α
Affiliations
- PMID: 24190966
- PMCID: PMC3911282
- DOI: 10.1128/MCB.00839-13
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A reactive oxygen species-mediated, self-perpetuating loop persistently activates platelet-derived growth factor receptor α
Mol Cell Biol.
2014 Jan.
Abstract
The platelet-derived growth factor (PDGF) receptors (PDGFRs) are central to a spectrum of human diseases. When PDGFRs are activated by PDGF, reactive oxygen species (ROS) and Src family kinases (SFKs) act downstream of PDGFRs to enhance PDGF-mediated tyrosine phosphorylation of various signaling intermediates. In contrast to these firmly established principles of signal transduction, much less is known regarding the recently appreciated ability of ROS and SFKs to indirectly and chronically activate monomeric PDGF receptor α (PDGFRα) in the setting of a blinding condition called proliferative vitreoretinopathy (PVR). In this context, we made a series of discoveries that substantially expands our appreciation of epigenetic-based mechanisms to chronically activate PDGFRα. Vitreous, which contains growth factors outside the PDGF family but little or no PDGFs, promoted formation of a unique SFK-PDGFRα complex that was dependent on SFK-mediated phosphorylation of PDGFRα and activated the receptor's kinase activity. While vitreous engaged a total of five receptor tyrosine kinases, PDGFRα was the only one that was activated persistently (at least 16 h). Prolonged activation of PDGFRα involved mTOR-mediated inhibition of autophagy and accumulation of mitochondrial ROS. These findings reveal that growth factor-containing biological fluids, such as vitreous, are able to tirelessly activate PDGFRα by engaging a ROS-mediated, self-perpetuating loop.
Figures
RV induced a unique SFK-PDGFRα relationship, which was required for activation of PDGFRα and downstream signaling events. (A) Near-confluent, serum-starved Fα, F2, and R627 (a kinase inactive point mutant [74]) cells were stimulated with PDGF-A (50 ng/ml) or normal rabbit vitreous (RV) for 10 min. Where indicated, the cells were pretreated with vehicle or SU6656 for 30 min. The cells were lysed, the lysates were immunoprecipitated with an anti-Src antibody, and the resulting samples were subjected to Western blotting using an anti-PDGFRα antibody (27P) and an anti-Src antibody. The fold values are the PDGFRα/Src ratio or the p-Akt/Akt ratio. The position of the 50-kDa heavy chain of the immunoprecipitating antibody (H-chain) is indicated. The data presented are representative of three independent experiments. (B) Serum-deprived F, Fα, and F2 cells were treated with PDGF-A (50 ng/ml) or RV for 10 min and lysed, and PDGFRα was then immunoprecipitated with an anti-PDGFRα antibody (27P). The immunoprecipitates were subjected to an in vitro kinase assay in which the substrate was a GST-PLCγ fusion protein. The extent of phosphorylation was monitored by Western blotting using an antiphosphotyrosine antibody (pY20). The membranes were stripped and reprobed with antibodies against PDGFRα or GST. The experimental results presented are representative of three independent experiments. p-PDGFRα, phosphorylated PDGFRα. (C) (Left) SFKs act upstream of PDGFR; ROS-activated SFKs phosphorylate monomeric PDGFRα and subsequently associate with it. The kinase activity of SFKs is required for the formation of the complex. (Right) SFKs act downstream of PDGFR; PDGF dimerizes PDGFRs and thereby triggers autophosphorylation that enables stable association of SFKs. The kinase activity of PDGFRα is required for formation of the complex. P, phosphate group. (D) Serum-starved F, F2, and Fα cells were stimulated with PDGF-A (50 ng/ml) or RV for 10 min. Their lysates were subjected to Western blot analysis using the following antibodies: anti-PDGFRα pY742 for p-PDGFRα and anti-Akt pS473 for p-Akt. The fold values are the p-PDGFRα/PDGFRα ratio or the p-Akt/Akt ratio. The data presented are representative of three independent experiments. F cells express no PDGFRs. (E) Serum-deprived Fα cells were treated with PDGF-A (50 ng/ml) or RV for 10 min (10 m) or 16 h and lysed, and the resulting lysates were immunoprecipitated with an anti-Src antibody (mouse origin) or a nonimmune IgG as a control (C). The resulting immunoprecipitates were subjected to Western blot analysis using antibodies against phospho-Src or PDGFRα. The membrane was reprobed with an anti-Src antibody. The data presented are representative of three experiments.
PDGFRα was the only RTK that was activated persistently in response to RV. (A) ARPE19α cells were cultured in medium containing 10% FBS until reaching 90% confluence and then starved in serum-free medium overnight. The cells were stimulated with vitreous from healthy rabbits (which contains low or undetectable levels of active PDGFs [37, 38]) for 10 min or 2 or 16 h. The lysates were subjected to a phospho-RTK array following the manufacturer's instructions. Short and long exposures are presented. C, control; p-HGFR, phosphorylated hepatocyte growth factor receptor; p-InsR, phosphorylated insulin receptor. (B) Summary of the results for all RTKs that were assayed. VEGFR1, vascular endothelial growth factor receptor 1; SCFR, stem cell growth factor receptor; M-CSFR, macrophage colony-stimulating factor receptor; FGFR1, fibroblast growth factor receptor 1; MSPR, macrophage-stimulating factor receptor; MuSK, muscle-specific kinase; DDR1, discoidin domain receptor kinase 1; ROR1, RTK-like orphan receptor 1; Insulin R, insulin receptor; HGFR, hepatocyte growth factor receptor; PYK, proline-rich tyrosine kinase. Coordinates in parentheses refer to the blot in panel A. (C and D) Lysates from cells treated as described above for panel A were immunoprecipitated with the indicated antibody and then subjected to Western blot analysis with an antibody recognizing the RTK (bottom blot) or antiphosphotyrosine (top blot). The fold values are the p-PDGFRα/PDGFRα ratio or the p-EGFR/EGFR ratio. The data presented are representative of three independent experiments. RV, rabbit vitreous. (E) Lysates from cells treated as described above for panel A were subjected to Western blotting using the indicated antibodies. RasGAP was included as a loading control.
Vitreous-driven activation of PDGFRα was selective, prolonged, and dependent on ROS. (A) Serum-starved ARPE19α cells were pretreated with NAC (10 mM) for 30 min and then stimulated with buffer (−), with PDGF-A, EGF, or IGF-1 (IGF) (each at 50 ng/ml), or with RV for 10 min. The lanes designated C1 and C2 were stimulated with buffer and RV, respectively. The cells were lysed, and the resulting lysates were immunoprecipitated using antibodies against PDGFRα, EGFR, IGF-1R, or nonimmune IgG (C1 and C2). The resulting immunoprecipitates were subjected to Western blot analysis using an antiphosphotyrosine antibody (pY20). The membranes were reprobed with antibodies against PDGFRα, EGFR, or IGF-1R. The fold values are the p-PDGFRα/PDGFRα ratio, the p-EGFR/EGFR ratio, or the p-IGF-1R/IGF-1R ratio. The data are representative of three experiments; similar results were obtained when the experiment was repeated with primary human corneal fibroblasts instead of ARPE19α cells (data not shown). (B) Serum-deprived ARPE19α cells were pretreated with NAC (10 mM) for 30 min before stimulation with PDGF-A (50 ng/ml) or RV for 10 min. For the 16-h stimulation with PDGF-A or RV, the 30-min NAC (10 mM) treatment was from 15.5 to 16 h. Following stimulation, the cells were lysed and subjected to Western blotting using the indicated antibodies. The fold values are the p-PDGFRα/PDGFRα ratio or the p-Akt/Akt ratio. The blots presented are representative of three experiments. (C) Serum-starved ARPE19α cells were treated with PDGF-A (50 ng/ml) or RV for 10 min, acid washed (to remove the growth factors), and harvested either at 10 min or at 15 h and 50 min later. The lysates were subjected to Western blot analysis using a phospho-PDGFRα Y754 antibody. The membrane was reprobed with a PDGFRα antibody (27P). The fold values are the p-PDGFRα/PDGFRα ratio. The data presented are representative of three experiments.
Non-PDGFs in vitreous persistently activated PDGFRα by triggering a ROS-mediated self-perpetuating loop. Non-PDGFs in vitreous engage their own receptors and thereby acutely activate NADPH oxidase at the plasma membrane (19, 56, 57). The resulting rise in ROS activates SFKs (59, 60), which derepress the kinase activity of monomeric PDGFRα (13, 17). This priming event involves autophosphorylation of PDGFRα and activation of the PI3K/Akt pathway that engages mTORC1 and suppresses autophagy (17, 49, 50, 52). These events lead to an accumulation of ROS from mitochondrial sources, which sustain a pool of activated SFKs and thereby close an intracellular, ROS-driven autocrine loop that results in enduring activation of PDGFRα.
RV stimulated mTORC1-dependent elevation of ROS, which was required for persistent activation of PDGFRα. (A and B) Near-confluent, serum-starved ARPE19α cells were pretreated with vehicle or rapamycin (100 nM) for 30 min and then stimulated with rabbit vitreous (RV) for 10 min or 16 h. The cells were harvested, and the resulting lysates were subjected to Western blot analysis with the following antibodies: pSer 2448 for p-mTOR and p-p70 S6 kinase for p-S6K. The fold values are the p-PDGFRα/PDGFRα ratio, the p-mTOR/mTOR ratio, or the p-S6K/S6K ratio. The data presented are representative of three independent experiments. (C) Cells were treated as described above for panel B and then washed, and the ROS level was determined as described in Materials and Methods. Values that are statistically significantly different (P < 0.05) are indicated by an asterisk and bar. Values that are not statistically significantly different are indicated by NS and a bar. (D) Near-confluent, serum-starved ARPE19α cells that stably expressed shRNA directed against gfp or raptor were treated with RV for 10 min or 16 h. The cells were lysed, and the resulting lysates were subjected to Western blot analyses using the indicated antibodies. The fold values are the p-PDGFRα/PDGFRα ratio or the Raptor/RasGAP ratio. The data presented are representative of three independent experiments. (E) Same as panel D, except that instead of lysing the cells for Western blot analysis, the level of ROS was determined as described in Materials and Methods. Values that are statistically significantly different (P < 0.05) are indicated by an asterisk and bar. Values that are not statistically significantly different are indicated by NS and a bar. (F) Serum-starved ARPE19α cells were cultured in medium containing RV, rapamycin (Rapa) (100 nM), or NAC (10 mM) for 3 days. The medium was refreshed daily, and the cells were counted at the end of day 3. These data are means ± standard deviations (SD) from three independent experiments. Values that are statistically significantly different (P < 0.05) are indicated by an asterisk and bar. Similar results were obtained when this experiment was repeated using primary human corneal fibroblasts instead of ARPE19α cells (data not shown).
Inhibition of autophagy was necessary for RV-mediated, persistent activation of PDGFRα. (A) ARPE19α cells were permitted to reach 70% confluence, serum starved, exposed to RV for 10 min or 16 h, and then lysed. The resulting lysates were subjected to Western blot analysis using the indicated antibodies. RasGAP served as a loading control. The fold values are the beclin-1/RasGAP ratio. The data presented are representative of three independent experiments. (B) Near-confluent, serum-starved ARPE19α cells that stably expressed shRNA directed against gfp or atg5 were treated with RV for 10 min or 16 h. The cells were lysed, and the resulting lysates were subjected to Western blot analyses using the indicated antibodies. (C) Near-confluent, serum-starved ARPE19α cells were pretreated with THC (5 μM) for 110 min, after which time RV was added and the cells were lysed 10 min later; the cells were exposed to drug and RV for 120 and 10 min, respectively. To monitor the effect of THC at the 16-h time point, the cells were first exposed to RV for 14 h and then THC was added and the cells were lysed 120 min later; the cells were exposed to THC and RV for 120 min and 16 h, respectively. The lysates were subjected to Western blot analyses using the indicated antibodies. In panels B and C, the fold values are the p-PDGFRα/PDGFRα ratio, the p-Akt/Akt ratio, the p-mTOR/mTOR ratio, the p-S6K/S6K ratio, or the Atg5/RasGAP ratio. The data presented are representative of three independent experiments. (D and E) Same as panels B and C, respectively, except that instead of lysing the cells for Western blot analysis, the level of ROS was determined as described in Materials and Methods. Values that are statistically significantly different (P < 0.05) are indicated by an asterisk and bar. Values that are not statistically significantly different are indicated by NS and a bar.
Mitochondrial ROS was required for persistent activation of PDGFRα in response to RV. (A and B) Near-confluent, serum-starved ARPE19α cells were pretreated with antimycin A (0.5 μM) (a mitochondrial electron transport inhibitor) or diphenyleneiodonium chloride (DPI) (5 μM) (an NADPH oxidase [NOX] inhibitor) for 20 min, after which time RV was added and the cells were lysed 10 min later; the cells were exposed to drug and RV for 30 and 10 min, respectively. To monitor the effects of the inhibitors at the 16-h time point, the cells were first exposed to RV for 15.5 h, the inhibitors were added, and the cells were lysed 30 min later; the cells were exposed to drug and RV for 30 min and 16 h, respectively. The lysates were subjected to Western blot analyses using the indicated antibodies. The fold values are the p-PDGFRα/PDGFRα ratio, the p-Akt/Akt ratio, the p-mTOR/mTOR ratio, or the p-S6K/S6K ratio. The data presented are representative of three independent experiments. Similar results were observed when carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (50 μM) (a mitochondrial electron transport inhibitor) and acetovanillone (apocynin) (10 μM) (a NOX inhibitor) were used instead of antimycin and DPI (data not shown). (C) Near-confluent, serum-starved ARPE19α cells stably expressing a mitochondrion-localized redox-sensitive GFP mutant (31) were treated with DTT (1 mM) for 1 h, RV for 10 min or 16 h, or H2O2 (1 mM) for 30 min. The panels are photos of representative cells that were illuminated with an excitation wavelength of 400 nm or 484 nm and an emission wavelength of 525 nm. Five exposures were measured of different areas (consisting of the three to five cells) for each experimental condition. Values in the bar graph are means ± SD from at least 3 independent experiments. Values that are statistically significantly different (P < 0.05) are indicated by an asterisk and bar. Values that are not statistically significantly different are indicated by NS and a bar.
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