Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells.

We have reported that an environmental pollutant, cadmium, promotes cell death in the human renal tubular cells (RTCs) through hyperactivation of a serine/threonine kinase Akt. However, the molecular mechanisms downstream of Akt in this process have not been elucidated. Cadmium has a potential to accumulate misfolded proteins, and proteotoxicity is involved in cadmium toxicity. To clear the roles of Akt in cadmium exposure-induced RTCs death, we investigated the possibility that Akt could regulate proteotoxicity through autophagy in cadmium chloride (CdCl2)-exposed HK-2 human renal proximal tubular cells. CdCl2 exposure promoted the accumulation of misfolded or damaged proteins, the formation of aggresomes (pericentriolar cytoplasmic inclusions), and aggrephagy (selective autophagy to degrade aggresome). Pharmacological inhibition of Akt using MK2206 or Akti-1/2 enhanced aggrephagy by promoting dephosphorylation and nuclear translocation of transcription factor EB (TFEB)/transcription factor E3 (TFE3), lysosomal transcription factors. TFEB or TFE3 knockdown by siRNAs attenuated the protective effects of MK2206 against cadmium toxicity. These results suggested that aberrant activation of Akt attenuates aggrephagy via TFEB or TFE3 to facilitate CdCl2-induced cell death. Furthermore, these roles of Akt/TFEB/TFE3 were conserved in CdCl2-exposed primary human RTCs. The present study shows the molecular mechanisms underlying Akt activation that promotes cadmium-induced RTCs death.

Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells.

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC.

[Involvement of Notch1 and ALK4/5 Signaling Pathways in Renal Tubular Cell Death: Their Application to Clarification of Cadmium Toxicity].

Renal tubular cell death is caused by various extracellular stresses including toxic amounts of cadmium, an occupational and environmental pollutant metal, and is responsible for renal dysfunction. While cadmium exposure disrupts many intracellular signaling pathways, the molecular mechanism underlying cadmium-induced renal tubular cell death has not yet been fully elucidated. We have recently identified two important intracellular signaling pathways that promote cadmium-induced renal tubular cell death: the Notch1 signaling and activin receptor-like kinase (ALK) 4/5 signaling (also known as the activin-transforming growth factor ß receptor pathways). In this review paper, we introduce our previous experimental findings, focusing on Notch1 and ALK4/5 signaling pathways, which may uncover the molecular mechanisms involved in cadmium-induced renal tubular cell death.

Blockade of ALK4/5 signaling suppresses cadmium- and erastin-induced cell death in renal proximal tubular epithelial cells via distinct signaling mechanisms.

Various types of cell death, including apoptosis, necrosis, necroptosis, and ferroptosis, are induced in renal tubular epithelial cells following exposure to environmental stresses and toxicants such as osmotic stress, ischemia/reperfusion injury, cisplatin, and cadmium. This is known to cause renal dysfunction, but the cellular events preceding stress-induced cell death in renal tubules are not fully elucidated. The activin receptor-like kinase (ALK) 4/5, also known as activin-transforming growth factor (TGF) ß receptor, is involved in stress-induced renal injury. We, therefore, studied the role of ALK4/5 signaling in HK-2 human proximal tubular epithelial cell death induced by cisplatin, cadmium, hyperosmotic stress inducer, sorbitol, and the ferroptosis activator, erastin. We found that ALK4/5 signaling is involved in cadmium- and erastin-induced cell death, but not sorbitol- or cisplatin-induced apoptotic cell death. Cadmium exposure elevated the level of phosphorylated Smad3, and treatment with the ALK4/5 kinase inhibitors, SB431542 or SB505124, suppressed cadmium-induced HK-2 cell death. Cadmium-induced cell death was attenuated by siRNA-mediated ALK4 or Smad3 silencing, or by treatment with SIS3, a selective inhibitor of TGFß1-dependent Smad3 phosphorylation. Furthermore, ALK4/5 signaling activated Akt signaling to promote cadmium-induced HK-2 cell death. In contrast, siRNA-mediated Inhibin-bA silencing or treatment with TGFß1 or activin A had little effect on cadmium-induced HK-2 cell death. On the other hand, treatment with SB431542 or SB505124 attenuated erastin-induced ferroptosis by hyperactivating Nrf2 signaling in HK-2 cells. These results suggest that blockade of ALK4/5 signaling protects against cadmium- and erastin-induced HK-2 cell death via Akt and Nrf2 signaling pathways, respectively.

Silver nanoparticles induce lysosomal-autophagic defects and decreased expression of transcription factor EB in A549 human lung adenocarcinoma cells.

Although silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary damage is unclear. AgNPs are incorporated into cells and processed via the autophagy pathway. We examined the effects of AgNP exposure on autophagic flux and expression of transcription factor EB (TFEB) in A549 lung adenocarcinoma cells. In cells exposed to citrate-coated 60-nm AgNPs, confocal laser microscopic examination showed a decrease in the LysoTracker fluorescence signal and an increase in that of Cyto-ID, indicating lysosomal pH alkalization and autophagosome formation, respectively. The proteins p62 and microtubule-associated protein light chain 3B-II (LC3B-II) are both degraded by autophagy, and their levels increased depending on AgNP dose. Furthermore, AgNP-induced increase in LC3B-II was not enhanced by treatment with the autophagic inhibitor bafilomycin A1. TFEB mRNA levels, and protein levels in cytosolic and nuclear fractions, were suppressed by exposure to AgNPs, suggesting transcriptional inhibition of TFEB expression. Overexpression of TFEB did not suppress AgNP-induced LC3B-II accumulation and cellular damage, indicating that impairment of autophagic flux and cellular damage by AgNPs might not be primarily caused by reduced TFEB expression. The present study suggests that AgNP-induced lysosomal dysfunction plays a principal role in the autophagic flux defect.

Involvement of Notch1 signaling in malignant progression of A549 cells subjected to prolonged cadmium exposure.

Cadmium exposure is known to increase lung cancer risk, but the underlying molecular mechanisms in cadmium-stimulated progression of malignancy are unclear. Here, we examined the effects of prolonged cadmium exposure on the malignant progression of A549 human lung adenocarcinoma cells and the roles of Notch1, hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor 1 receptor (IGF-1R)/Akt/extracellular signal-regulated kinase (ERK)/p70 S6 kinase 1 (S6K1) signaling pathways. Exposing A549 cells to 10 or 20 µm cadmium chloride (CdCl2) for 9-15 weeks induced a high proliferative potential, the epithelial-mesenchymal transition (EMT), stress fiber formation, high cell motility, and resistance to antitumor drugs. Of note, the CdCl2 exposure increased the levels of the Notch1 intracellular domain and of the downstream Notch1 target genes Snail and Slug. Strikingly, siRNA-mediated Notch1 silencing partially suppressed the CdCl2-induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance. In addition, we found that prolonged CdCl2 exposure induced reduction of E-cadherin in BEAS-2B human bronchial epithelial cells and antitumor drug resistance in H1975 human tumor-derived non-small-cell lung cancer cells depending on Notch1 signaling. Moreover, Notch1, HIF-1α, and IGF-1R/Akt/ERK/S6K1 activated each other to induce EMT in the CdCl2-exposed A549 cells. These results suggest that Notch1, along with HIF-1α and IGF-1R/Akt/ERK/S6K1 signaling pathways, promotes malignant progression stimulated by prolonged cadmium exposure in this lung adenocarcinoma model.

The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

UNLABELLED: The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. SIGNIFICANCE STATEMENT: The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway.

PI3K signaling mediates diverse regulation of ATF4 expression for the survival of HK-2 cells exposed to cadmium.

Cadmium exposure causes endoplasmic reticulum (ER) stress and accumulation of activating transcription factor 4 (ATF4), an ER stress marker. To elucidate the role of phosphatidylinositol-3-kinase (PI3K) signaling in this process, we examined the effects of PI3K signaling on cadmium chloride (CdCl2) exposure-induced ATF4 expression in HK-2 human renal proximal tubular cells. ATF4 knockdown by siRNA enhanced CdCl2-induced cellular damage, indicating a cytoprotective function of ATF4. Treatment with LY294002, a PI3K inhibitor, suppressed CdCl2-induced ATF4 expression and Akt phosphorylation at Thr308 with little effect on phosphorylation of eukaryotic translation initiation factor 2 subunit α at Ser51. Activation of PI3K signaling with epidermal growth factor treatment enhanced CdCl2-induced Akt phosphorylation and ATF4 expression. Suppression of CdCl2-induced ATF4 expression by LY294002 treatment was markedly blocked by cycloheximide, a translation inhibitor, but not by MG-132, a proteasome inhibitor, or actinomycin D, a transcription inhibitor. CdCl2 exposure also induced phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448, glycogen synthase kinase-3α (GSK-3α) at Ser21, GSK-3ß at Ser9, and 90 kDa ribosomal S6 kinase 2 (RSK2) at Ser227 in HK-2 cells. Treatment with rapamycin, an mTOR inhibitor, MK2206, an Akt inhibitor, and BI-D1870, a RSK inhibitor, partially suppressed CdCl2-induced ATF4 expression. Conversely, SB216763, a GSK-3 inhibitor, markedly inhibited the potency of LY294002 to suppress CdCl2-induced ATF4 expression. These results suggest that PI3K signaling diversely regulates the expression of ATF4 in a translation-dependent manner via downstream molecules, including mTOR, GSK-3α/ß, and RSK2, and plays a role in protecting HK-2 cells from cadmium-induced damage.

Phosphorylation of FOXO3a by PI3K/Akt pathway in HK-2 renal proximal tubular epithelial cells exposed to cadmium.

We examined the effects of cadmium chloride (CdCl2) exposure on the phosphorylation and function of the forkhead box class O (FOXO) transcription factor FOXO3a in HK-2 human renal proximal tubular cells. Phosphorylation of FOXO3a (at Thr32 and Ser253) and its upstream kinase, Akt (at Thr308 and Ser473) were markedly increased following exposure to 10 or 20 µM CdCl2. Treatment with wortmannin (500 nM), an inhibitor of phosphoinositide-3-kinase (PI3K), suppressed CdCl2-induced phosphorylation of Akt and FOXO3a at their Akt phosphorylation sites. CdCl2-induced phosphorylation of FOXO3a was markedly suppressed by the epidermal growth factor receptor inhibitor, AG1478 (1 µM), the Ca(2+)/calmodulin-dependent kinase II inhibitor, KN-93 (10 µM), and the Src inhibitor, PP2 (10 µM), but only partially suppressed by the insulin-like growth factor-1 receptor inhibitor, PPP (2.5 µM). Furthermore, the p38 inhibitor, SB203580 (20 µM), suppressed CdCl2-induced phosphorylation of Akt and FOXO3a, suggesting possible cross-talk between p38 mitogen-activated protein kinase and Akt. Although phosphorylation of FOXO3a was associated with reduced levels of nuclear FOXO3a, this change in cellular localization was transient. Silencing of FOXO3a expression using short interfering RNA suppressed CdCl2-induced cellular damage and accumulation of cytoplasmic nucleosomes in HK-2 cells. These results show that cadmium exposure induces phosphorylation of FOXO3a through the PI3K/Akt signaling pathway and suggest that FOXO3a phosphorylation (inactivation) transiently promotes survival of HK-2 cells. Phosphorylation of FOXO3a by the PI3K/Akt pathway may regulate cell fate in proximal tubules exposed to cadmium.

Endocannabinoid-Goα signalling inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signalling.

The ability of neurons to regenerate their axons after injury is determined by a balance between cellular pathways that promote and those that inhibit regeneration. In Caenorhabditis elegans, axon regeneration is positively regulated by the c-Jun N-terminal kinase mitogen activated protein kinase pathway, which is activated by growth factor-receptor tyrosine kinase signalling. Here we show that fatty acid amide hydrolase-1, an enzyme involved in the degradation of the endocannabinoid anandamide (arachidonoyl ethanolamide), regulates the axon regeneration response of γ-aminobutyric acid neurons after laser axotomy. Exogenous arachidonoyl ethanolamide inhibits axon regeneration via the Goα subunit GOA-1, which antagonizes the Gqα subunit EGL-30. We further demonstrate that protein kinase C functions downstream of Gqα and activates the MLK-1-MEK-1-KGB-1 c-Jun N-terminal kinase pathway by phosphorylating MLK-1. Our results show that arachidonoyl ethanolamide induction of a G protein signal transduction pathway has a role in the inhibition of post-development axon regeneration.

The Caenorhabditis elegans Ste20-related kinase and Rac-type small GTPase regulate the c-Jun N-terminal kinase signaling pathway mediating the stress response.

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like MAPK signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPKK, and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.

Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway.

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.