Mary E. Choi

Autophagy Promotes Intracellular Degradation of Type I Collagen Induced by Transforming Growth Factor (TGF)-β1

Background: Autophagy is a process that cells use to degrade and recycle cellular proteins, however, the role of autophagy in kidney fibrosis remains largely unknown. Results: Autophagy is responsible for the intracellular degradation of type I collagen. Conclusion: Autophagy negatively regulates and prevents excess collagen accumulation in the kidney. Significance: Our findings implicate a novel role of autophagy as a cytoprotective mechanism against renal fibrosis. Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A1 also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-β1. We also show that TGF-β1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-β1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-β1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.

Transforming growth factor β1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages

Cell death and cell survival are central components of normal development and pathologic states. Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that regulates both cell growth and cell death. To better understand the molecular mechanisms that control cell death or survival, we investigated the role of TGF-β1 in the apoptotic process by dominant-negative inhibition of both TGF-β1 and mitogen-activated protein kinase (MAPK) signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis following serum deprivation, as determined by DNA laddering assay. However, apoptosis is prevented in serum-deprived macrophages by the presence of exogenous TGF-β1. Using stably transfected RAW 264.7 cells with the kinase-deleted dominant-negative mutant of TβR-II (TβR-IIM) cDNA, we demonstrate that this protective effect by TGF-β1 is completely abrogated. To determine the downstream signaling pathways, we examined TGF-β1 effects on the MAPK pathway. We show that TGF-β1 induces the extracellular signal-regulated kinase (ERK) activity in a time-dependent manner up to 4 h after stimulation. Furthermore, TGF-β1 does not rescue serum deprivation-induced apoptosis in RAW 264.7 cells transfected with a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW 264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken together, our data demonstrate for the first time that TGF-β1 is an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via TβR-II-mediated signaling and downstream intracellular MAPK signaling pathway.

Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells

We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH(2)-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH2-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.

TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3−/−) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.

Induction of apoptosis by particulate matter: role of TNF-α and MAPK

Particulate matter (PM) is a major by-product from the combustion of fossil fuels. The biological target of inhaled PM is the pulmonary epithelium and resident macrophages. In this study, we demonstrate that cultured macrophages (RAW 264.7 cells) exposed continously to a well-defined model of PM [benzo[ a]pyrene adsorbed on carbon black (CB+BaP)] exhibit a time-dependent expression and release of the cytokine tumor necrosis factor-α (TNF-α). CB+BaP also evoked programmed cell death or apoptosis in cultured macrophages as assessed by genomic DNA-laddering assays. The CB+BaP-induced apoptosis was inhibited when macrophages were treated with CB+BaP in the presence of a neutralizing antibody to TNF-α, suggesting that TNF-α plays an important role in mediating CB+BaP-induced apoptosis in macrophages. Interestingly, neither untreated carbon black nor benzo[ a]pyrene alone induced apoptosis or caused the release of TNF-α in RAW 264.7 cells. Moreover, we observed that TNF-α activates mitogen-activated protein kinase (MAPK) activity, the extracellular signal-regulated kinases p42/p44, in a time-dependent manner. RAW 264.7 cells treated with PD-098059, a selective inhibitor of MAPK kinase activity, did not exhibit CB+BaP-induced apoptosis and TNF-α secretion. Furthermore, cells treated with the MAPK kinase inhibitor did not undergo TNF-α-induced apoptosis. Taken together, our data suggest that TNF-α mediates PM-induced apoptosis and that the MAPK pathway may play an important role in regulating this pathway.

Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.

Oxidative stress and autophagy: Crucial modulators of kidney injury

Both acute kidney injury (AKI) and chronic kidney disease (CKD) that lead to diminished kidney function are interdependent risk factors for increased mortality. If untreated over time, end stage renal disease (ESRD) is an inevitable outcome. Acute and chronic kidney diseases occur partly due to imbalance between the molecular mechanisms that govern oxidative stress, inflammation, autophagy and cell death. Oxidative stress refers to the cumulative effects of highly reactive oxidizing molecules that cause cellular damage. Autophagy removes damaged organelles, protein aggregates and pathogens by recruiting these substrates into double membrane vesicles called autophagosomes which subsequently fuse with lysosomes. Mounting evidence suggests that both oxidative stress and autophagy are significantly involved in kidney health and disease. However, very little is known about the signaling processes that link them. This review is focused on understanding the role of oxidative stress and autophagy in kidney diseases. In this review, we also discuss the potential relationships between oxidative stress and autophagy that may enable the development of better therapeutic intervention to halt the progression of kidney disease and promote its repair and resolution.

Autophagy: Friend or Foe in Lung Disease?

Autophagy is a highly conserved process by which cells can recycle organelles and proteins by degrading them in the lysosomes. Although autophagy is considered a dynamic system responsible for cellular renovation and homeostasis under physiological conditions, it is increasingly clear that autophagy is directly relevant to clinical disease. During disease progression, autophagy not only serves as a cellular protective mechanism but also can represent a harmful event under certain conditions. In addition, although autophagy can act as a nonselective bulk degradation process, recent research shows that autophagy can selectively degrade specific proteins, organelles, and invading bacteria, in processes termed selective autophagy. Selective autophagy has drawn the attention of researchers because of its potential importance in clinical diseases. In this article, we outline the most recent studies implicating autophagy and selective autophagy in human lung diseases, including chronic obstructive pulmonary disease, pulmonary hypertension, idiopathic pulmonary fibrosis, and sepsis. We also discuss the relationship between autophagy and other molecular mechanisms related to disease progression, including programmed necrosis (necroptosis) and the inflammasome, an inflammatory signaling platform that regulates the secretion of IL-1β and IL-18. Finally, we examine the dual nature of autophagy and selective autophagy in the lung, which have both protective and injurious effects for human lung disease.

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells

Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.