Matrix Metalloproteinase (MMP)-1 Induces Lung Alveolar Epithelial Cell Migration and Proliferation,Protects from Apoptosis,and Represses Mitochondrial Oxygen Consumption

Idiopathic pulmonary fibrosis is a devastating lung disorder of unknown etiology. Although its pathogenesis is unclear, considerable evidence supports an important role of aberrantly activated alveolar epithelial cells (AECs), which produce a large variety of mediators, including several matrix metalloproteases (MMPs), which participate in fibroblast activation and lung remodeling. MMP-1 has been shown to be highly expressed in AECs from idiopathic pulmonary fibrosis lungs although its role is unknown. In this study, we explored the role of MMP-1 in several AECs functions. Mouse lung epithelial cells (MLE12) transfected with human Mmp-1 showed significantly increased cell growth and proliferation at 36 and 48 h of culture (p < 0.01). Also, MMP-1 promoted MLE12 cell migration through collagen I, accelerated wound closing, and protected cells from staurosporine- and bleomycin-induced apoptosis compared with mock cells (p < 0.01). MLE12 cells expressing human MMP-1 showed a significant repression of oxygen consumption ratio compared with the cells with the empty vector. As under hypoxic conditions hypoxia-inducible factor-1α (HIF-1α) mediates a transition from oxidative to glycolytic metabolism, we analyzed activation of HIF-1α. Ηigher activation of this factor was detected in MMP-1-transfected cells under normoxia and hypoxia. Likewise, a significant decrease of both total and mitochondrial reactive oxygen species was observed in MMP-1-transfected cells. Paralleling these findings, attenuation of MMP-1 expression by shRNA in A549 (human) AECs markedly reduced proliferation and migration (p < 0.01) and increased the oxygen consumption ratio. These findings indicate that epithelial expression of MMP-1 inhibits mitochondrial function, increases HIF-1α expression, decreases reactive oxygen species production, and contributes to a proliferative, migratory, and anti-apoptotic AEC phenotype.     

Diindolylmethane-mediated Gli1 Protein Suppression Induces Anoikis in Ovarian Cancer Cells in Vitro and Blocks Tumor Formation Ability in Vivo

Anoikis is a cell death that occurs due to detachment of a cell from the extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell that undergoes metastasis. In this study for the first time, we demonstrated the potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer cells by different concentrations of diindolylmethane (DIM), an active ingredient of cruciferous vegetables, reduced the anoikis resistance in a concentration-dependent manner. Reduction in anoikis resistance was associated with a decrease in the expression of Gli1 and an increase in the cleavage of poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only increased the expression of Gli1, but also blocked anoikis induced by DIM and abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells. Cyclopamine-mediated reduction in anoikis resistance was associated with reduced expression of Gli1 and induction of cleaved PARP. Shh treatment blocked cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies established that DIM- or cyclopamine-treated ovarian cancer cells under suspension culture conditions drastically lost their ability of tumor formation in vivo in mice. Taken together, our results establish that Gli1 is a critical player in anoikis resistance in ovarian cancer.     

Nilotinib Induces Autophagy in Hepatocellular Carcinoma through AMPK Activation

Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

Intermediate domain of receptor-interacting protein kinase 1 (RIPK1) determines switch between necroptosis and RIPK1 kinase-dependent apoptosis

Receptor-interacting protein kinase 1 (RIPK1) is an important component of the tumor necrosis factor receptor 1 (TNFR1) signaling pathway. Depending on the cell type and conditions, RIPK1 mediates MAPK and NF-κB activation as well as cell death. Using a mutant form of RIPK1 (RIPK1ΔID) lacking the intermediate domain (ID), we confirm the requirement of this domain for activation of these signaling events. Moreover, expression of RIPK1ΔID resulted in enhanced recruitment of caspase-8 to the TNFR1 complex II component Fas-associated death domain (FADD), which allowed a shift from TNF-induced necroptosis to apoptosis in L929 cells. Addition of the RIPK1 kinase inhibitor necrostatin-1 strongly reduced recruitment of RIPK1 and caspase-8 to FADD and subsequent apoptosis, indicating a role for RIPK1 kinase activity in apoptotic complex formation. Our study shows that RIPK1 has an anti-apoptotic function residing in its ID and demonstrates a cellular system as an elegant genetic model for RIPK1 kinase-dependent apoptosis that, in contrast to the Smac mimetic model, does not rely on depletion of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2).     

Nix Is Critical to Two Distinct Phases of Mitophagy,Reactive Oxygen Species-mediated Autophagy Induction and Parkin-Ubiquitin-p62-mediated Mitochondrial Priming

Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.     

Polyubiquitination of insulin-like growth factor I receptor (IGF-IR) activation loop promotes antibody-induced receptor internalization and down-regulation

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.     

NADPH Oxidase 4 Induces Cardiac Arrhythmic Phenotype in Zebrafish

Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked up-regulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation, which is accompanied by overproduction of reactive oxygen species (ROS). In this study, we investigated the impact on the cardiac phenotype of NOX4 overexpression in zebrafish. One-cell stage embryos were injected with NOX4 RNA prior to video recording of a GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24–31 h post-fertilization. Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean S.D. of beat-to-beat intervals was 0.027 s/beat in control embryos versus 0.038 s/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino co-injection, treatments of the embryos with polyethylene glycol-conjugated superoxide dismutase, or NOX4 inhibitors fulvene-5, 6-dimethylamino-fulvene, and proton sponge blue. Injection of NOX4-P437H mutant RNA had no effect on the cardiac phenotype or ROS production. In addition, phosphorylation of calcium/calmodulin-dependent protein kinase II was increased in NOX4 embryos but diminished by polyethylene glycol-conjugated superoxide dismutase, whereas its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies the development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia.     

Epidermal Growth Factor (EGF)-enhanced Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression Promotes Macrophage and Glioblastoma Cell Interaction and Tumor Cell Invasion

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCϵ- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.