Role of Iron in the Mechanism of Asbestos-Induced Apoptosis in Human Lung and Pleural Target Cells

Occupational exposure to asbestos has been associated with increased incidence of pulmonary interstitial fibrosis, mesothelioma of the pleura, and bronchogenic carcinoma. Although the mechanism by which asbestos causes cancer remains unknown , iron associated with asbestos is thought to play a role in the pathogenic effects of fibers. The aim of this research was to examine and compare the asbestos-induced signaling phenomena in relevant human lung and pleural target cells, and to deter mine the role of iron from asbestos fibers in these events. Exposure of human airway epithelial (A549) cells, human pleural mesothelial (METSA) cells, and normal human small airway epithelial (SAEC) cells to asbestos resulted in a significant dephosphorylation and inactivation of epidermal growth factor receptor (EGFR). The effects of three types of asbestos, i.e. crocidolite, amosite and chrysotile, on the EGFR phosphorylation state in A549 cells appeared to be directly related to the amount of iron mobilized from these fibers. These results strongly suggest that iron plays an important role in asbestos induced inactivation of EGFR. We observed that exposure of A549 and SAEC cells to crocidolite , but not inert titanium dioxide , led to a significant time- and dose-dependent inactivation of the main EGFR signaling pathways, including Akt and extracellular signal-regulated kinase 1/2 (ERKI/2) pathways. Crocidolite also initiated apoptosis via pathways involving activation of p38 mitogen-activated protein kinase (MAPK), caspase -3 and -9, and cleavage of poly(ADP-ribose) polymerase (PARP). Prevention of these effects with an iron chelator or endocytosis inhibitors strongly suggests that iron mobilized from fibers inside the cells initiates the observed events. Inhibition of p38 MAPK with SB203580 prevented inactivation of EGFR , inactivation of EGFR-associated survival pathways, and initiation of apoptosis. Our results also suggest that p38 MAPK-dependent protein serine/threonine phosphatase activation plays an important role in the observed phenomena. Taken together , it appears that iron-dependent p38 MAPK activation, through a serine/threonine phosphatase-mediated mechanism , regulates asbestos-induced apoptosis in human lung epithelial cells. We speculate that apoptosis of human lung target cells induced by asbestos fibers is a pathologic feature in lung injury and may account for some of the pulmonary toxicity of the fibers

Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR

Scheme illustrating sustained activation of PKC induces PLD- and endocytosis- dependent phosphorylation of Thr-654 on EGFR and sequestration of EGFR to a cPKC- dependent subset of recycling compartment (pericentrion).

<p>Prolonged treatment with ATII or PMA could induce translocation of EGFR to pericentrion and phosphorylation of EGFR on Thr-654 on a cPKC- and PLD- dependent manner. Sequestration of EGFR to pericentrion protects EGFR accessed by EGF. </p

Effects of PMA on phosphorylation of EGFR and the role of the pericentrion.

<p>A, HEK293 cells were serum starved for 5 hours followed by 100nM PMA for 2 min, 5 min, 10 min, 30 min or 60 min. Phospho-Thr654 and total EGFR were determined as described above. B. HEK293 cells were starved for 5 hours and then pretreated with vehicle, Gö6976, 1-butanol, depleted of potassium (K-), or preincubated 400mM sucrose followed by 1-hour 100nM PMA treatment. The procedure for potassium-depletion is as described previously (30). Levels of P-Thr654 and total EGFR were determined as described. C, HEK293 cells were transfected with dominate negative constructs of PLD1 or PLD2. After 24 hours post-transfection, cells were starved for 5 hours and then treated with 100nM PMA for 1 hour. Levels of P-Thr654 and total EGFR were determined as shown before. D, HEK293 cells were starved for 5 hours and then pretreated with 100nM PMA for the indicated time followed by 5min 5ng/ml EGF treatment. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) and total EGFR were determined as described. E, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol, FIPI, or vehicle followed with 100nM PMA or vehicle for 1 hour and then treated with 5ng/ml EGF or vehicle for 5 min. P-Tyr1045 and EGFR were determined by western blotting. F, HEK293 cells were starved for 5 hours and then pretreated with vehicle or 100nM for 1 hour followed by treatment with 10ng/ml EGF for 2 min. Phospho-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.</p

ATII-induced sequestration and protection of EGFR loss require the pericentrion.

<p>A, HEK293 cells were transfected with WT-EGFR-GFP. After 24 hours, cells were starved for 5 hours and then treated with 100nM PMA for 5 or 60 min. After fixation and permeabilization, location of EGFR (green) and endogenous EEA1 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). B, HEK293 cells were starved for 5 hours and then pretreated with Gö6976, 1-butanol or vehicle followed by 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (green) and Lamp1 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). D, HEK293 cells were transfected with Rab11-GFP. After 24 hours, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation and permeabilization, location of Rab11 (green) and endogenous EGFR (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (Leica TSC SP8). E, HEK293 cells starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour following with 5ng/ml EGF or vehicle for 3 hours. Protein levels of EGFR and actin were determined by western blotting. F, HEK293 cells starved for 5 hours and then were pretreated with Gö6976, Bis, 1-butanol or vehicle followed with 100nM PMA for 1 hour and then treated with 5ng/ml EGF or vehicle for 3 hours. Protein levels of EGFR and actin were determined by western blotting. G, HEK293 cells or HEK293 cells stably overexpressing AT_1AR were starved for 5 hours and then treated with vehicle, 100nM PMA or 100nM ATII for 1 hour. After treatments, cells were collected immediately and the mRNA level of EGFR were determined by real-time PCR. Pictures are representative of at least three experiments. E: **** <i>P</i><0.0001 compared to control, two-way ANOVA.</p

Effects of ATII on localization and fate of EGFR.

<p>A, HEK293 cells stably transfected with AT_1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, location of AT_1AR (green) and endogenous Rab11 (red) were determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells stably transfected with AT_1AR (green) were serum starved for 5 hours followed by 100nM ATII or vehicle for 1 hour. After fixation and permeabilization, endogenous EGFR (red) was determined by immunofluorescence, and cells were analyzed by confocal microscopy (ZEISS 510). C, HEK293 cells stably transfected with AT_1AR were serum starved for 5 hours and treated with 2ng/ml of EGF for 3 hour with or without 1 hour pretreated with 100nM ATII. Protein level of EGFR was determined by Western Blotting. Blots were stripped and reprobed for Na⁺K⁺ATPase to normalize for loading. These results are representative of three independent experiments. Pictures are representative of at least three experiments.</p

Effects of ATII on phosphorylation of EGFR.

<p>A, HEK293 cells stably transfected with AT_1AR were serum starved for 5 hours followed by 100nM ATII for 10 min. Phosphorylation of EGFR on Thr-654 (P-Thr654) was determined by western blotting. Blots were stripped and reprobed for total EGFR to normalize for loading. B, HEK293 cells stably transfected with AT_1AR were serum starved for 5 hours and treated with 5ng/ml of EGF for 5 min with or without 1 hour pretreated with 100nM ATII. Phosphorylation of EGFR on Tyrosine 1045 (P-Tyr1045) was determined by western blotting. The blots were stripped and reprobed for total EGFR to normalize for loading. C, HEK293 cells stably transfected with AT_1AR were serum starved for 5hours and then pretreated with Gö6976, FIPI or vehicle followed with 100nM ATII or vehicle for 1 hour and then treated with 5ng/ml EGF for 5 min. P-Tyr1045 and EGFR were determined by western blotting. D, HEK293 cells stably transfected with AT_1AR were serum starved for 5hours and then pretreated with Gö6976, 1-butanol or vehicle followed with 100nM ATII or vehicle for 5min, P-Tyr1068 and EGFR were determined by western blotting. For all figures, * p <0.05, ***p <0.001 from at least three independent experiments.</p

Effects of PMA on EGFR T654A mutant.

<p>A, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then treated with 100nM PMA or vehicle for 1 hour. After fixation, cells were analyzed by confocal microscopy (ZEISS 510). B, HEK293 cells were transfected with WT-EGFR-GFP or TA-EGFR-GFP. 24 hours after transfection, cells were starved for 5 hours and then pretreated with 100nM PMA or vehicle for 1 hour followed by 5ng/ml EGF for 3 hours. The levels of EGFR, EGFR-GFP and actin were determined by western blotting. For all figures, * p <0.05, from at least three independent experiments.</p