BackgroundInflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology.ObjectiveWe sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells.MethodsMice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ.ResultsMice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release.ConclusionsMitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

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Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identifiedCbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl ؊/؊ mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization. Growth factor receptor tyrosine kinases (RTKs)1 play crucial roles in cellular proliferation, survival, migration, and differentiation. Epidermal growth factor receptor (EGFR/ErbB1) is a member of the ErbB family (ErbB1-4) of RTKs, which play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of RTKs leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways. The ligand-activated RTKs also undergo rapid endocytosis (1). The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. The receptors can be targeted to the lysosome for degradation, which terminates receptor signals. Alternatively, the internalized receptors can be recycled back to the cell surface for continued ligand binding and signaling (2-5). The relative efficiency of lysosomal sorting versus recycling is a key determinant of the signaling potency of RTKs (6). For example, EGFR is predominantly delivered to lysosomes when activated by EGF. In contrast, heregulin-activated ErbB2 is primarily recycled. The greater efficiency of the recycling process is thought to be a major determinant of the signaling superiority of ErbB2 over EGFR (7-9).Despite a critical role of endocytic sorting as a determinant of ErbB receptor down-regulation, the biochemical mechanisms that regulate this process have only recently begun to be elucidated. We, and others, have identified Cbl as one such regulator (10 -12). Cbl is recruited to the activated EGFR through both direct and indirect binding. Direct Cbl-EGFR interaction is mediated through the N-terminal tyrosine kinase-binding domain of Cbl, which binds to phosphorylated Tyr-1045 on EGFR (13). Indirect Cbl-E...

Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING ®nger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.

Studies over the last 4 years have now firmly established that Cbl, the product of the c-cbl proto-oncogene, is a component of early tyrosine kinase signaling cascades in a variety of cell types (reviewed in Refs. 1 and 2). For example, Cbl is an early and prominent target of tyrosine phosphorylation in response to activation of a number of cell surface receptors coupled to tyrosine kinase activation, including the lymphocyte antigen receptors, cytokine receptors, and growth factor receptors (1, 2). Cbl's extensive proline-rich region (amino acids 481-690) mediates interactions with the SH3 1 domains of Src family kinases and adapter proteins Grb2 and Nck to promote the formation of signaling protein complexes that are present in cells prior to receptor activation. Receptor activation-dependent phosphorylation of Cbl on tyrosine residues creates docking sites for several SH2 domain-containing proteins (3-5), leading to activation-induced association of Cbl with the p85 subunit of phosphatidylinositol 3-kinase, the Crk adapter proteins, and VAV (1, 2). Finally, we showed that the transforming N-terminal region of Cbl (Cbl-N, equivalent to Cbl sequences present in the v-cbl oncogene) harbors a phosphotyrosine-binding domain that selectively and directly interacts with the T cell tyrosine kinase ZAP-70 (6, 7). Subsequently, this domain was also shown to mediate binding to receptor tyrosine kinases EGFR and PDGFR␣ (1, 2, 8 -10). While these associations have promoted the notion that Cbl functions as a complex adapter protein that couples tyrosine kinases to downstream signaling pathways, a number of recent biochemical and genetic studies have identified Cbl as a potential negative regulator of Syk/ ZAP-70, as well as other PTKs (1, 2).The C. elegans homologue of Cbl, SLI-1, was identified through a genetic screen as a negative regulator of the LET-23 receptor tyrosine kinase, a homologue of the mammalian EGFR, which functions in the vulval development (11). Notably, the loss of function mutations in SLI-1 were mapped to the evolutionarily conserved N-terminal region, corresponding to the PTB domain. One of these was a point mutation (G315E); the analogous mutation in mammalian Cbl-N abrogated its PTB domain activity (6). Recently, a Drosophila Cbl homologue (D-Cbl) was identified and also shown to function as a negative regulator of the Drosophila EGFR-mediated R7 photoreceptor development (12, 13). Dominant oncogenic forms of mammalian Cbl introduced into NIH-3T3 cells induced an up-regulation of signaling downstream of the endogenous PDGFR␣ tyrosine * This work was supported in part by National Institutes of Health Grant CA76118 and American Cancer Society Grants . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The abbreviations used are: SH2 and -3, Src homology 2 and 3, respectively; Tyr(P) or pY, phosphotyrosine; CD, cluster of dif...

Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation–mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9–MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.