Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.     

Cell adaptive response to extracellular matrix density is controlled by ICAP-1-dependent beta1-integrin affinity

Cell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially understood. We provide evidence for a novel mechanistic insight into focal adhesion (FA) dynamics by demonstrating that integrin cytoplasmic domain-associated protein 1 (ICAP-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1-deficient mouse embryonic fibroblasts and cells expressing active beta(1) integrin, shows that the integrin high affinity state favored by talin is antagonistically controlled by ICAP-1. This affinity switch results in modulation in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the ICAP-1-dependent decrease in integrin affinity allows cell sensing of matrix surface density, suggesting that integrin conformational changes are important in mechanotransduction. Our results clarify the function of ICAP-1 in cell adhesion and highlight the central role it plays in the cell's integrated response to the extracellular microenvironment.     

Preferential binding of HIF-1 to transcriptionally active loci determines cell-type specific response to hypoxia

Background  

Hypoxia-inducible factor 1 (HIF-1) plays a key role in cellular adaptation to hypoxia. To better understand the determinants of HIF-1 binding and transactivation, we used ChIP-chip and gene expression profiling to define the relationship between the epigenetic landscape, sites of HIF-1 binding, and genes transactivated by hypoxia in two cell lines.     

HIF-1alpha response to hypoxia is functionally separated from the glucocorticoid stress response in the in vitro regenerating human skeletal muscle

Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1α (HIF-1α); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11β-hydroxysteroid dehydrogenases (11β-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1α small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1α-based response to hypoxia, and the GC response system composed of GRα and types 1 and 2 11β-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1α response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts.     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.     

HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.     

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1''s role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.     

WNK1 is an unexpected autophagy inhibitor

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.     

Thyrsiferol Inhibits Mitochondrial Respiration and HIF-1 Activation

The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at Complex I (IC(50) 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.