No abstract

No abstract

The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H 2 O 2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains colocalized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell. Activation of the epidermal growth factor (EGF)2 receptor (EGFR) by EGF results in the initiation of signal transduction cascades involved in cellular survival and proliferation. Therefore, to control cellular growth and tumorigenesis, the activation of the EGFR has to be tightly regulated in a process that includes degradation of the receptor. Binding of EGF to EGFR is rapidly followed by internalization of the membrane-bound receptor mainly through clathrin-coated pits and into early endosomes, which develop into late endosomes. There, the EGFR is either targeted to lysosomes for degradation or recycled to the plasma membrane (1-3). The inability of the EGFR to be down-regulated via clathrinmediated endocytosis and degradation has been linked to its oncogenicity (4, 5).H 2 O 2 -induced oxidative stress has been shown to activate and aberrantly phosphorylate the EGFR, which impedes the clathrin-mediated endocytosis and subsequent lysosomal degradation of the receptor (6 -8). This results in prolonged downstream activation of proliferative molecules such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) (9), and the lack of receptor turnover has been shown to mediate tumor promotion in non-neoplastic rat liver epithelial cells (10). To gain more insight into H 2 O 2 -induced EGFR signaling and hyperplastic responses, we examined the trafficking of the receptor under oxidative stress.Huang and Sorkin (11) have recently reported that knock-down of Grb2 by RNA interference inhibits clat...

Exposure to hydrogen peroxide (H2O2), one of the reactive oxidants in the gas phase of cigarette smoke (CS), induces aberrant phosphorylation of the epidermal growth factor receptor (EGFR), resulting in the lack of ubiquitination by c-Cbl, and impaired degradation. EGFR activation without the feedback regulation of normal degradation leads to uncontrolled cell growth and tumor promotion. Using immunoprecipitation, immunoblotting, and confocal microscopy, we now demonstrate that the pattern of EGFR activation by CS is similar to H2O2. We found that exposure of human airway epithelial cells to CS, as with exposure to H2O2, not only results in an increase in EGFR activation over time, but the EGFR activated by H2O2 or CS is neither ubiquitinated nor subsequently degraded due to its inability to bind the E3 ubiquitin ligase, c-Cbl, either directly or indirectly via the Grb2 adapter protein. Moreover, the stabilized H2O2- and CS-activated EGFR remains plasma membrane-bound, while a population of the receptor is trafficked to a perinuclear region. Concomitantly, CS exposure results in the activation of downstream Akt and ERK1/2 survival and proliferation pathways. Therefore, exposure to CS, like exposure to H2O2, results in prolonged signaling by the EGFR and may contribute to uncontrolled lung cell growth.

Cigarette smoke (CS) induces a rapid, sustained upregulation of ceramide production in human bronchial epithelial cells, leading to increased apoptosis. Using loss-of-function and overexpression analyses, we show that neutral sphingomyelinase 2 (nSMase2) is required for CS-mediated ceramide generation and apoptosis. Glutathione (GSH), a crucial antioxidant in lung defense, blocks nSMase2 activity and thus inhibits apoptosis triggered by CS. We found that the exposure to CS, as with exposure to H(2)O(2), results in increased nSMase2 activation leading to ceramide generation and therefore increased apoptosis. Interestingly, exposure of cells to GSH abolishes nSMase2 activation caused by CS and leads to a decrease in CS-induced apoptosis. This suggests that the effects of CS oxidants on nSMase2 are counteracted by GSH. Our data support a model where CS induces nSMase2 activation thereby increasing membrane-sphingomyelin hydrolysis to ceramide. In turn, elevated ceramide enhances airway epithelial cell death, which causes bronchial and alveolar destruction and lung injury in pulmonary diseases.