Arsenite-induced cytotoxicity is regulated by poly-ADP ribose polymerase 1 activation and parthanatos in p53-deficient H1299 cells: The roles of autophagy and p53.

Arsenic is a double-edged sword metalloid since it is both an environmental carcinogen and a chemopreventive agent. Arsenic cytotoxicity can be dependent or independent of the tumor suppressor p53. However, the effects and the underlying molecular mechanisms of arsenic cytotoxicity in p53-deficient cells are still unclear. Here, we report a distinctive cell death mode via PARP-1 activation by arsenic in p53-deficient H1299 cells. H1299 (p53-/-) cells showed higher sensitivity to sodium arsenite (NaAR) than H460 (p53+/+) cells. H460 cells induced canonical apoptosis through caspase-dependent poly-ADP ribose polymerase 1 (PARP-1) cleavage and induced the expression of phospho-p53 and p21. However, H1299 cells induced poly-ADP-ribose (PAR) polymer accumulation and caspase-independent parthanatos, which was inhibited by 3-aminobenzamide (AB) and nicotinamide (NAM). Fractionation studies revealed the mitochondrial translocation of PAR polymers and nuclear translocation of the apoptosis-inducing factor (AIF). Although the exposure of NaAR to p53-overexpressing H1299 cells increased the PAR polymer levels, it inhibited parthanatos by inducing p21 and phospho-p53 expression. LC3-II and p62 accumulated in a NaAR dose- and exposure time-dependent manner, and this accumulation was further enhanced by autophagy inhibition, indicating that arsenic inhibits autophagic flux. p53 overexpression led to a decrease in the p62 levels, an increase in the LC3-II levels, and reduced parthanatos, indicating that arsenic induces p53-dependent functional autophagy. These results show that the NaAR-induced cytotoxicity in p53-deficient H1299 cells is regulated by PARP-1 activation-mediated parthanatos, which is promoted by autophagy inhibition. This suggests that PARP-1 activation could be used as an effective therapeutic approach for arsenic toxicity in p53-deficient cells.

Arsenite-induced cytotoxicity is regulated by p38-SQSTM1/p62 and JNK-BNIP3L/Nix signaling in lung cancer cells.

Arsenic is a potent carcinogen in humans. However, the molecular mechanisms underlying its toxicity in lung cancer remain unclear. Here, we report that arsenite-induced cytotoxicity is regulated by SQSTM1/p62 and BNIP3L/Nix signaling in non-small-cell lung cancer H460 cells. Arsenite exposure resulted in dose-dependent growth inhibition, which was associated with apoptosis, as demonstrated by depolarized mitochondrial membrane potential and cleavage of caspase-8, caspase-3, PARP-1, and Bax. The autophagy adaptor p62 was detected in the monomeric and multiple high-molecular-weight (HMW) forms, and protein levels were upregulated depending on both arsenite concentrations (≤45 µM) and exposure times (<24 h). LC3-II, an autophagy marker, was upregulated as early as 1 h after arsenite treatment. Expression of Nix, a mitochondrial outer membrane protein, continued to increase with arsenite concentration and exposure time; it was detected in the monomeric and multiple HMW forms. Soon after arsenite exposure, p62 colocalized with Nix in the cytoplasm, and p62 knockdown reduced the Nix levels and increased the LC3-II levels. In contrast, Nix knockdown did not affect the p62 and LC3-II levels but reduced caspase-8, caspase-3, and Bax cleavage, indicating that Nix accumulation resulted from its reduced autophagic degradation and promoted apoptosis. p38 inhibition markedly increased arsenite-induced Nix protein and reduced p62 protein levels, resulting in increased autophagy and apoptosis. Furthermore, c-Jun NH2-terminal kinase inhibition reduced Nix and Bax cleavage, and both signaling pathways were suppressed by N-acetylcysteine treatment. Our results suggest that arsenite-induced cytotoxicity is modulated by the coordinated action of p62 and Nix through MAPK.

Autophagy-mediated cytoplasmic accumulation of p53 leads to apoptosis through DRAM-BAX in cadmium-exposed human proximal tubular cells.

The tumor suppressor p53 is involved in cadmium (Cd)-induced apoptosis and autophagy. However, the regulatory mechanisms of p53 in Cd-induced kidney injury are not well established. Here, we report the role of autophagy in Cd-induced p53 induction in human proximal tubular cells (HK-2). HK-2 cells treated with Cd induced the expression of p53, DNA damage autophagy modulator (DRAM), and Bcl-2-associated X protein (BAX), as well as caused poly [ADP-ribose] polymerase 1 (PARP-1) cleavage. Cd exposure also induced autophagy with the accumulation of monomeric p62 and multiple high molecular weight form (HMW)-p62. The expression levels of p53, p62, microtubule-associated protein 1A/1B-light chain 3 (LC3)-1, and LC3-II were similar in the sense that they increased up to 12 h and then gradually decreased. DRAM and BAX levels began to increase post autophagy induction and continued to increase, indicating that autophagy preceded apoptosis. While the genetic knockdown of p53 downregulated HWM-p62, DRAM, and BAX, the expression levels of these proteins were upregulated by p53 overexpression. The genetic knockdown of p62 downregulated p53, autophagy, DRAM, and BAX. The inhibition of autophagy through pharmacological and genetic knockdown reduced p53 and inhibited Cd-induced apoptosis. Collectively, Cd induces apoptosis through p53-mediated DRAM-BAX signaling, which can be regulated by autophagy.

Cytoplasmic sirtuin 6 translocation mediated by p62 polyubiquitination plays a critical role in cadmium-induced kidney toxicity.

Sirtuin 6 (Sirt6) is important for maintaining kidney homeostasis and function. Cd exposure increases the risk of developing kidney diseases. However, the role of Sirt6 in kidney disease mechanisms is unclear. Here, we evaluated the role of Sirt6 in Cd-induced kidney toxicity. After Cd exposure, p62/sequestosome-1 (SQSTM1), an autophagy substrate, accumulated in mouse kidney mesangial cells in monomeric and polyubiquitinated (polyUb) forms. Sirt6 accumulated in response to Cd treatment at concentrations below the half-maximal inhibitory concentration and decreased after 12 h of treatment. Sirt6 and p62 co-localized in the nucleus and redistributed to the cytosol after Cd treatment. Sirt6 was mainly present in nuclei-rich membrane fractions. Sirt6 interacted with p62. Ub, and microtubule-associated protein light chain 3 (LC3). Knockdown of p62 promoted Sirt6 nuclear accumulation and inhibited apoptosis. Sirt6 overexpression altered levels of polyUb-p62 and apoptosis. At earlier times during Cd treatment, polyubiquitination of p62 and apoptosis were reduced. Cytoplasmic translocation of Sirt6 occurred later, with increased polyubiquitination of p62 and apoptosis. Bafilomycin 1 (BaF1) treatment promoted cytosolic Sirt6 accumulation, increasing cell death. Silencing autophagy related 5 (Atg5) increased nuclear Sirt6 levels, reduced polyUb-p62, and inhibited cell death, indicating that autophagy was necessary for Sirt6 redistribution. Cd resistance was associated with reduced polyUb-p62 and persistent Sirt6 expression. Cd treatment in mice for 4 weeks promoted p62, Sirt6, and LC3-II accumulation, inducing apoptosis in kidney tissues. Overall, our findings show that polyUb-p62 targeted Sirt6 to autophagosomes, playing a crucial role in Cd-induced cell death and kidney damage.

Salinomycin suppresses TGF-ß1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer.

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-ß1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-ß1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-ß1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-ß1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-ß1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-ß1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-ß1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Correction to: Cytoplasmic sirtuin 6 translocation mediated by p62 polyubiquitination plays a critical role in cadmium-induced kidney toxicity.

In the original publication the grant number is incorrectly published.

Poly-ubiquitinated p62/SQSTM1-mediated hemeoxygenase-1 stabilization plays a critical role in cadmium-induced apoptosis of mouse monocyte Raw264.7 cells.

Cadmium (Cd) is a toxic heavy metal that can affect many organs, leading to serious pathological disorders through immune suppression. Here, we investigated the molecular mechanisms underlying the response of monocytes to Cd exposure. Cd treatment of Raw264.7 cells activated antioxidant enzymes, such as hemeoxygenase-1 (HO-1), superoxide dismutase, and catalase. Cd exposure upregulated p53, p53 phosphorylation, p21, and γH2AX phosphorylation. Cd exposure also induced poly ADP-ribose polymerase 1 (PARP-1) cleavage. These findings indicated that Cd induces apoptosis through oxidative stress-mediated DNA damage. Furthermore, upregulation of microtubule-associated protein 1 light chain 3B-II (LC3B-II), an indicator of autophagy, was found to depend on Cd concentration. Accumulation of an autophagy substrate p62/SQSTM1 in monomeric p62 and polyubiquitinated (polyUb)-p62 forms, was suppressed upon N-acetylcysteine treatment Cd-exposed Raw264.7 cells, indicating an impairment of autophagic degradation during oxidative stress. Knockdown of p62 in Raw264.7 cells using small interfering RNA (siRNA) downregulated HO-1 expression and reduced apoptosis. HO-1 knockdown suppressed apoptosis by decreasing the poly-ubiquitination of p62. Treatment with hemin and MG132 enhanced Cd-mediated increases in HO-1 and polyUb-p62 levels, resulting in increased apoptosis, which indicated that Cd-induced HO-1 accumulation is associated with polyUb-p62 formation. p62 and HO-1 interactions were demonstrated by immunofluorescence and immunoprecipitation assays. Additionally, p62 was downregulated in Raw264.7 cells in response to H2O2 and a low level of HO-1 was induced. Cells that were highly sensitive to Cd did not form polyUb-p62, resulting in insufficient HO-1 accumulation. These results suggest that maintenance of HO-1 stability via poly-ubiquitination of p62 in Cd-exposed monocytes promotes apoptosis, which could be involved in immune suppression.

Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.

Sirtuin 2 (Sirt2) is known to negatively regulate anoxia-reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia-reperfusion (I/R)-injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild-type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild-type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild-type and Sirt2 knockout mice were subjected to hypoxia-reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus-injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen-activated protein kinase phosphatase-1 and consequent activation of mitogen-activated protein kinases. CONCLUSION: Sirt2 is an aggravating factor during hepatic I/R injury. (Hepatology 2017;65:225-236).

Nefopam downregulates autophagy and c-Jun N-terminal kinase activity in the regulation of neuropathic pain development following spinal nerve ligation.

BACKGROUND: Neurodegeneration is associated with changes in basal cellular function due to the dysregulation of autophagy. A recent study introduced the involvement of autophagy during spinal nerve ligation (SNL). Nefopam has shown potential for reducing neuropathic pain, but the underlying mechanisms are unknown. Here, we investigated the effects of nefopam on neuropathic pain development following SNL, focusing on the involvement of autophagy. METHODS: The functional role of nefopam in capsaicin-induced autophagy was assessed by human glioblastoma M059 K cells. The neuropathic pain model was used to determine whether the effect of nefopam on pain control was mediated through autophagy control. Neuropathic pain was induced by L5 and L6 SNL in male rats randomized into three groups: Group S (sham-operated), Group C (received normal saline), and Group E (received nefopam). A behavioral test using a von Frey was examined. Expression changes of autophagy in response to nefopam was analyzed in spinal cord tissues (L4-L6) by immunoblotting and immunohistochemistry. RESULTS: The paw withdrawal threshold examined on days 3, 5, 7, and 14 post-SNL was significantly higher in Group E than in Group C. SNL increased the levels of microtubule-associated protein 1 light chain 3B (LC3B-1), with concomitant reduction of sequestosome 1 (SQTSM1/p62), compared with Group S, indicating that SNL induced autophagy. These effects were reversed by nefopam injection, and the results were confirmed by immunohistochemistry for LC3-I/II. Furthermore, SNL-mediated JNK activation was markedly decreased following nefopam injection. Hematoxylin and eosin staining on Day 14 post-SNL revealed that SNL caused lymphocyte infiltration and oligodendrocyte localization in the substantia gelatinosa of the dorsal gray horn, which were reduced by nefopam injection. CONCLUSION: Collectively, the mode of action of nefopam on neuropathic pain appears to be associated with downregulation of phospho-JNK and autophagy, as well as modulation of the immune response.

Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells.

BACKGROUND: Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action. METHODS: Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin. CONCLUSIONS: Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

MAPK/JNK1 activation protects cells against cadmium-induced autophagic cell death via differential regulation of catalase and heme oxygenase-1 in oral cancer cells.

Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress.

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice.

Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP-activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY-C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer-binding protein ß (C/EBPß) binding to the C/EBP-response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet-fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36-specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver.

Heme oxygenase-1-mediated apoptosis under cadmium-induced oxidative stress is regulated by autophagy, which is sensitized by tumor suppressor p53.

Heme oxygenase-1 (HO-1) is a stress-inducible cytoprotective enzyme. It is often overexpressed in different types of cancers and promotes cell survival. However, the role of HO-1 and the underlying molecular mechanism of cadmium (Cd)-induced oxidative stress in cancer cells remain undefined. Here we show that the role of HO-1 under Cd-induced oxidative stress is dependent upon autophagy, which is sensitized by the tumor suppressor p53. The sensitivity to Cd was 3.5- and 14-fold higher in p53-expressing YD8 and H460 cells than in p53-null YD10B and H1299 cells, respectively. The levels of p53 in YD8 and H460 cells decreased in a Cd concentration-dependent manner, which was inhibited by pretreatment with N-acetylcysteine. In both cell lines, Cd exposure resulted in caspase-3-mediated PARP-1 cleavage and the induction of CHOP, LC3-II, and HO-1, which were limited in YD10B and H1299 cells exposed to high concentrations of Cd. Cd exposure to p53-overexpressing YD10B cells enhanced Cd-induced HO-1 and LC3-II levels, whereas genetic knockdown of p53 in YD8 cells resulted in the suppression of Cd-induced levels of HO-1 and LC3-II, indicating that p53 is required in the sensing of HO-1 and induction of autophagy. The inhibition of autophagy using small interfering RNA (siRNA) for the autophagy-related gene atg5 enhanced HO-1, CHOP, and PARP-1 cleavage induced by Cd. However, transfection with HO-1 siRNA increased Cd-induced LC3-II, and suppressed the expression of CHOP and cleavage of PARP-1. Collectively, the role of HO-1 in apoptosis could be modulated by autophagy, which is sensitized by p53 expression in human cancer cell lines.

Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αß involved in protection of oral squamous cell carcinoma against cadmium toxicity.

Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose-response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC50 of 45 µM. Expression of Pin1 was decreased at or above the Cd IC50 value and was inversely correlated with the level of phospho(p)-Ser-GSK3αß. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αß; this was reversed by overexpression of Pin1. However, suppression of GSK3αß inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3ß. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αß and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αß can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αß at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis.

Transcriptional regulation, stabilization, and subcellular redistribution of multidrug resistance-associated protein 1 (MRP1) by glycogen synthase kinase 3αß: novel insights on modes of cadmium-induced cell death stimulated by MRP1.

Cadmium (Cd) resistance is associated with the suppression of autophagy in H460 lung cancer cells, which is regulated by phospho(p)serine-glycogen synthase kinase (GSK) 3αß. However, the involvement of multidrug resistance (MDR) in this signaling pathway and its underlying mechanisms remain to be elucidated. In this study, we used Cd-resistant cells (RH460), developed from H460 lung cancer cells, to demonstrate that the induction of MDR-associated protein (MRP1) in response to Cd is enhanced in H460 cells compared to RH460. Treating RH460 cells with Cd induced large cytoplasmic vacuoles, which was inhibited by the autophagy inhibitor 3-methyladenine. MRP1 was detected in the nuclear-rich membrane fractions and redistributed from the perinuclear to the cytoplasmic compartment following exposure to Cd. Cd-induced MRP1, p-Ser/p-Tyr GSK3αß, and LC3-II were all suppressed by the GSK3 inhibitor SB216763, but increased by lithium. Furthermore, MRP1 was upregulated by the Ser/Thr phosphatase inhibitor okadaic acid and downregulated by the tyrosine phosphatase inhibitor vanadate, suggesting that MRP1 protein was stabilized by p-Ser GSK3αß. In addition, co-immunoprecipitation and co-localization analyzes revealed a physical interaction between MRP1 and p-Ser GSK3αß. Genetic knockdown of GSK3ß decreased Cd-induced MRP1 mRNA and protein levels, whereas its overexpression upregulated MRP1 protein expression. MRP1 also co-localized with lysosomal membrane protein-2, which may cause lysosomal membrane permeabilization and the subsequent release of cathepsins into the cytosol. In mice chronically injected with Cd, MRP1 localized to the perinuclear region of bronchial and alveolar epithelial cells. Collectively, these data suggest that Cd toxicity is regulated by the transcriptional regulation, stabilization, and subcellular redistribution of MRP1 via the posttranslational modification of GSK3αß. Therefore, the serine phosphorylation of GSK3αß plays a critical role in MRP1-induced cell death.

Sodium arsenite-induced cytotoxicity is regulated by BNIP3L/Nix-mediated endoplasmic reticulum stress responses and CCPG1-mediated endoplasmic reticulum-phagy.

We elucidated the BNIP3L/Nix and SQSTM1/p62 molecular mechanisms in sodium arsenite (NaAR)-induced cytotoxicity. Considerable changes in the morphology and adhesion of H460 cells were observed in response to varying NaAR concentrations. NaAR exposure induced DNA damage-mediated apoptosis and Nix accumulation via proteasome inhibition. Nix targets the endoplasmic reticulum (ER), inducing ER stress responses. p62 and Nix were colocalized and their expressions were inversely correlated. Autophagy inhibition upregulated Nix, p62, cell cycle progression gene 1 (CCPG1), heme oxygenase (HO)- 1, and calnexin expression. Nix knockdown decreased the NaAR-induced ER stress and microtubule-associated protein 1 A/1B light-chain 3 (LC3) B-II levels and increased the CCPG1 and calnexin levels. p62 knockdown upregulated Nix, LC3-II, and CCPG1 expressions and the ER stress responses, indicating that p62 regulates Nix levels. Nix downstream pathways were mitigated by Ca2+ chelators. We demonstrate the critical roles of Nix and p62 in ER stress and ER-phagy in response to NaAR.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3ß.

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3ß (GSK3ß) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3ß at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3ß(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3ß(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3ß(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.

The role of autophagy in cadmium-induced acute toxicity in glomerular mesangial cells and tracking polyubiquitination of cytoplasmic p53 as a biomarker.

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.

Role of the AMPK/SREBP-1 pathway in the development of orotic acid-induced fatty liver.

Orotic acid (OA), an intermediate in pyrimidine metabolism, has been used for a variety of purposes, such as dietary supplements. Although it is well documented that OA induces fatty liver in a species-specific manner, the precise molecular mechanisms remain unclear. The present study investigated the role of the adenosine monophosphate-activated protein kinase (AMPK)-sterol regulatory element-binding protein-1 (SREBP-1) pathway in the OA-induced fatty liver. Treatment with OA suppressed the phosphorylation of AMPK via proteasomal degradation of upstream kinase LKB1 and induced activation of SREBP-1 in both human hepatoma cell lines and primary rat hepatocytes. OA-induced SREBP-1 transcriptional activity was suppressed by cotreatment with aminoimidazole carboxamide ribonucleotide (AICAR) or metformin, or by overexpression of constitutively active AMPK (CA-AMPK) in the human hepatoma cell line. Importantly, in vivo data corroborated these results. Feeding 1% OA with diet decreased the phosphorylation of AMPK and increased the maturation of SREBP-1 and the expression of SREBP-responsive genes in the rat liver. OA-induced lipid accumulation was also completely inhibited by rapamycin. Mouse hepatocytes and mice were resistant to OA-induced lipogenesis because of little if any response in AMPK and downstream effectors. In conclusion, OA induces hepatic lipogenesis, mediated predominantly by the AMPK/SREBP-1 pathway in rat hepatocytes and human hepatoma cell lines.