Nupr1-mediated vascular smooth muscle cell phenotype transformation involved in methamphetamine induces pulmonary hypertension.

AIMS: Nuclear protein 1 (Nupr1) is a multifunctional stress-induced protein involved in the regulation of tumorigenesis, apoptosis, and autophagy. However, its role in pulmonary hypertension (PH) after METH exposure remains unexplored. In this study, we aimed to investigate whether METH can induce PH and describe the role and mechanism of Nupr1 in the development of PH. METHODS AND RESULTS: Mice were made to induce pulmonary hypertension (PH) upon chronic intermittent treatment with METH. Their right ventricular systolic pressure (RVSP) was measured to assess pulmonary artery pressure. Pulmonary artery morphometry was determined by H&E staining and Masson staining. Nupr1 expression and function were detected in human lungs, mice lungs exposed to METH, and cultured pulmonary arterial smooth muscle cells (PASMCs) with METH treatment. Our results showed that chronic intermittent METH treatment successfully induced PH in mice. Nupr1 expression was increased in the cultured PASMCs, pulmonary arterial media from METH-exposed mice, and METH-ingested human specimens compared with control. Elevated Nupr1 expression promoted PASMC phenotype change from contractile to synthetic, which triggered pulmonary artery remodeling and resulted in PH formation. Mechanistically, Nupr1 mediated the opening of store-operated calcium entry (SOCE) by activating the expression of STIM1, thereby promoting Ca2+ influx and inducing phenotypic conversion of PASMCs. CONCLUSIONS: Nupr1 activation could promote Ca2+ influx through STIM1-mediated SOCE opening, which promoted METH-induced pulmonary artery remodeling and led to PH formation. These results suggested that Nupr1 played an important role in METH-induced PH and might be a potential target for METH-related PH therapy.

Nupr1 mediates renal fibrosis via activating fibroblast and promoting epithelial-mesenchymal transition.

Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.

Methamphetamine produces cardiac damage and apoptosis by decreasing melusin.

Methamphetamine (METH) is an amphetamine-type drug that is highly addictive and widely abused. Many studies have shown that METH exposure causes severe damage not only to the nervous system but also to the cardiovascular system. Melusin protein is a mechanotransducer that plays an important role in maintaining normal heart function. However, the role of melusin in METH-induced cardiotoxicity has not yet been reported. We hypothesized that methamphetamine can produce cardiac damage and apoptosis by decreasing the quantity of melusin. To test this hypothesis, we determined the protein expression of melusin and apoptosis markers in METH-treated rats and primary rat cardiomyocytes. We also established a melusin-overexpressing cell model to assess the importance of melusin in maintaining antiapoptotic pathways. To confirm our findings from the in vitro and animal models, we also evaluated the apoptotic index of cardiomyocytes and the protein expression of apoptotic markers in postmortem heart tissues from deceased METH abusers and age-matched control subjects. The results showed that the apoptosis of cardiomyocytes was increased significantly and that the protein expression of melusin was decreased after exposure to METH in primary rat cardiomyocytes, in rats and in humans. METH treatment also decreased the expression of the downstream proteins FAK, IQGAP1, p-AKT, p-GSK3ß, and p-ERK in primary rat cardiomyocytes and in vivo. After overexpression of melusin, the above effects were partially reversed in primary rat cardiomyocytes. We conclude that METH can produce cardiac damage and apoptosis by decreasing melusin, while melusin-activated signaling by phosphorylated AKT, phosphorylated GSK3ß, and ERK may be resistant to methamphetamine-induced myocardial apoptosis.

Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPß-related signaling pathway.

Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPß) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPß is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPß after Meth exposure and evaluated the effects of silencing C/EBPß, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPß protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPß expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPß in vitro. Further studies are needed to elucidate the role of C/EBPß in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q., Liu, C., Lin, Z., Xie, W.-B., Wang, H. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPß-related signaling pathway.

NDRG1 attenuates epithelial-mesenchymal transition of nasopharyngeal cancer cells via blocking Smad2 signaling.

N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial-mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.

Smad2 and myocardin-related transcription factor B cooperatively regulate vascular smooth muscle differentiation from neural crest cells.

RATIONALE: Vascular smooth muscle cell (VSMC) differentiation from neural crest cells (NCCs) is critical for cardiovascular development, but the mechanisms remain largely unknown. OBJECTIVE: Transforming growth factor-ß (TGF-ß) function in VSMC differentiation from NCCs is controversial. Therefore, we determined the role and mechanism of a TGF-ß downstream signaling intermediate Smad2 in NCC differentiation to VSMCs. METHODS AND RESULTS: By using Cre/loxP system, we generated a NCC tissue-specific Smad2 knockout mouse model and found that Smad2 deletion resulted in defective NCC differentiation to VSMCs in aortic arch arteries during embryonic development and caused vessel wall abnormality in adult carotid arteries where the VSMCs are derived from NCCs. The abnormalities included 1 layer of VSMCs missing in the media of the arteries with distorted and thinner elastic lamina, leading to a thinner vessel wall compared with wild-type vessel. Mechanistically, Smad2 interacted with myocardin-related transcription factor B (MRTFB) to regulate VSMC marker gene expression. Smad2 was required for TGF-ß-induced MRTFB nuclear translocation, whereas MRTFB enhanced Smad2 binding to VSMC marker promoter. Furthermore, we found that Smad2, but not Smad3, was a progenitor-specific transcription factor mediating TGF-ß-induced VSMC differentiation from NCCs. Smad2 also seemed to be involved in determining the physiological differences between NCC-derived and mesoderm-derived VSMCs. CONCLUSIONS: Smad2 is an important factor in regulating progenitor-specific VSMC development and physiological differences between NCC-derived and mesoderm-derived VSMCs.

STIM1 Mediates Methamphetamine-Induced Neuronal Autophagy and Apoptosis.

Methamphetamine (METH) is a widely abused amphetamine-type psychoactive drug that causes serious health problems. Previous studies have demonstrated that METH can induce neuron autophagy and apoptosis in vivo and in vitro. However, the molecular mechanisms underlying METH-induced neuron autophagy and apoptosis remain poorly understood. Stromal interacting molecule 1 (STIM1) was hypothesized to be involved in METH-induced neuron autophagy and apoptosis. Therefore, the expression of STIM1 protein was measured and the effect of blocking STIM1 expression with siRNA was investigated in cultured neuronal cells, and the hippocampus and striatum of mice exposed to METH. Furthermore, intracellular calcium concentration and endoplasmic reticulum (ER) stress-related proteins were determined in vitro and in vivo in cells treated with METH. The results suggested that STIM1 mediates METH-induced neuron autophagy by activating the p-Akt/p-mTOR pathway. METH exposure also resulted in increased expression of Orai1, which was reversed after STIM1 silencing. Moreover, the disruption of intracellular calcium homeostasis induced ER stress and up-regulated the expression of pro-apoptotic protein CCAAT/enhancer-binding protein homologous protein (CHOP), resulting in classic mitochondria apoptosis. METH exposure can cause neuronal autophagy and apoptosis by increasing the expression of STIM1 protein; thus, STIM1 may be a potential gene target for therapeutics in METH-caused neurotoxicity.

Blocking Sigmar1 exacerbates methamphetamine-induced hypertension.

AIM: Methamphetamine (METH) chronic exposure is an important risk factor for hypertension development. However, the mechanisms behind METH-induced hypertension remain unclear. Therefore, we aimed to reveal the potential mechanisms underlying METH-induced hypertension. METHODS AND RESULTS: We structured the mouse hypertension model by METH, and observed that METH-treated mice have presented vascular remodeling (large-and small-size arteries) with collagen deposit around the vessel and increasing blood pressure (BP) and Sigma1 receptor (Sigmar1) in vascular tissue. We hypothesized that Sigmar1 is crucial in METH-induced hypertension and vascular remodeling. Sigmar1 knockout (KO) mice and antagonist (BD1047) pretreated mice exposed to METH for six-week showed higher BP and more collagen deposited around vessels than wild-type (WT) mice exposed to METH for six-week, in contrast, mice pretreated with Sigmar1 agonist (PRE-084) had unchanged BP and perivascular collagen despite the six-week METH exposure. Furthermore, we found that METH exposure induced vascular smooth muscle cells (VSMCs) and mesenchymal stem cells to differentiate into the myofibroblast-like cell and secrete collagen into surrounding vessels. Mechanically, Sigmar1 can suppress the COL1A1 expression by blocking the classical fibrotic TGF-ß/Smad2/3 signaling pathway in METH-exposed VSMCs and mesenchymal stem cells. CONCLUSION: Our results suggest that Sigmar1 is involved in METH-induced hypertension and vascular fibrosis by blocking the activation of the TGF-ß/Smad2/3 signaling pathway. Accordingly, Sigmar1 may be a novel therapeutic target for METH-induced hypertension and vascular fibrosis.

Cell division cycle 7 is a novel regulator of transforming growth factor-ß-induced smooth muscle cell differentiation.

Smooth muscle cell (SMC) differentiation and proliferation occur simultaneously during embryonic development. The underlying mechanisms especially common factors regulating both processes, however, remain largely unknown. The present study has identified cell division cycle 7 (Cdc7) as one of the factors mediating both the proliferation and SMC differentiation. TGF-ß induces Cdc7 expression and phosphorylation in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. Cdc7 specific inhibitor or shRNA knockdown suppresses TGF-ß-induced expression of SMC early markers including α-SMA, SM22α, and calponin. Cdc7 overexpression, on the other hand, enhances SMC marker expression. Cdc7 function in inducing SMC differentiation is independent of Dumbbell former 4 or Dbf4, the catalytic subunit of Cdc7 critical for cell proliferation, suggesting that Cdc7 mediates SMC differentiation through a mechanism distinct from cell proliferation. Cdc7 regulates SMC differentiation via activating SMC marker gene transcription. Knockdown of Cdc7 by shRNA inhibits SMC marker gene promoter activities. Mechanistically, Cdc7 interacts with Smad3 to induce SMC differentiation. Smad3 is required for Cdc7 function in inducing SMC promoter activities and marker gene expression. Likewise, Cdc7 enhances Smad3 binding to SMC marker promoter via supporting Smad3 nuclear retention and physically interacting with Smad3. Taken together, our studies have demonstrated a novel role of Cdc7 in SMC differentiation.

Smad3-mediated myocardin silencing: a novel mechanism governing the initiation of smooth muscle differentiation.

Both TGF-ß and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-ß-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-ß did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-ß treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-ß-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-ß stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-ß to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-ß induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.

Response gene to complement 32 is essential for fibroblast activation in renal fibrosis.

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-ß (TGF-ß). TGF-ß is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-ß-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.

Response gene to complement 32 promotes vascular lesion formation through stimulation of smooth muscle cell proliferation and migration.

OBJECTIVE: The objectives of this study were to determine the role of response gene to complement 32 (RGC-32) in vascular lesion formation after experimental angioplasty and to explore the underlying mechanisms. METHODS AND RESULTS: Using a rat carotid artery balloon-injury model, we documented for the first time that neointima formation was closely associated with a significantly increased expression of RGC-32 protein. Short hairpin RNA knockdown of RGC-32 via adenovirus-mediated gene delivery dramatically inhibited the lesion formation by 62% as compared with control groups 14 days after injury. Conversely, RGC-32 overexpression significantly promoted the neointima formation by 33%. Gain- and loss-of-function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that RGC-32 is essential for both the proliferation and migration of RASMCs. RGC-32 induced RASMC proliferation by enhancing p34(CDC2) activity. RGC-32 stimulated the migration of RASMC by inducing focal adhesion contact and stress fiber formation. These effects were caused by the enhanced rho kinase II-α activity due to RGC-32-induced downregulation of Rad GTPase. CONCLUSIONS: RGC-32 plays an important role in vascular lesion formation following vascular injury. Increased RGC-32 expression in vascular injury appears to be a novel mechanism underlying the migration and proliferation of vascular smooth muscle cells. Therefore, targeting RGC-32 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Methamphetamine induces thoracic aortic aneurysm/dissection through C/EBPß.

AIMS: Thoracic aortic aneurysm/dissection (TAAD) is a life-threatening disease with diverse clinical manifestations. Although the association between methamphetamine (METH) and TAAD is frequently observed, the causal relationship between METH abuse and aortic aneurysm/dissection has not been established. This study was designed to determine if METH causes aortic aneurysm/dissection and delineate the underlying mechanism. METHODS AND RESULTS: A new TAAD model was developed by exposing METH to SD rats pre-treated with lysyl oxidase inhibitor ß-aminopropionitrile (BAPN). Combination of METH and BAPN caused thoracic aortic aneurysm/dissection in 60% of rats. BAPN+METH significantly increased the expression and activities of both matrix metalloproteinase MMP2 and MMP9, consistent with the severe elastin breakage and dissection. Mechanistically, METH increased CCAAT-enhancer binding protein ß (C/EBPß) expression by enhancing mothers against decapentaplegic homolog 3 (Smad3) and extracellular regulated protein kinase (ERK1/2) signaling. METH also promoted C/EBPß binding to MMP2 and MMP9 promoters. Blocking C/EBPß significantly attenuated METH+BAPN-induced TAAD and MMP2/MMP9 expression. Moreover, BAPN+METH promoted aortic medial smooth muscle cell (SMC) apoptosis through C/EBPß-mediated IGFBP5/p53/PUMA signaling pathways. More importantly, the expression of C/EBPß, MMP2/MMP9, and apoptosis-promoting proteins was increased in the aorta of human patients with thoracic aortic dissection, suggesting that the mechanisms identified in animal study could be relevant to human disease. CONCLUSIONS: Our study demonstrated that METH exposure has a casual effect on TAAD. C/EBPß mediates METH-introduced TAAD formation by causing elastin breakage, medial cell loss and degeneration. Therefore, C/EBPß may be a potential factor for TAAD clinical diagnosis or treatment.

Implications of alpha-synuclein nitration at tyrosine 39 in methamphetamine-induced neurotoxicity in vitro and in vivo.

Methamphetamine is an amphetamine-type psychostimulant that can damage dopaminergic neurons and cause characteristic pathological changes similar to neurodegenerative diseases such as Parkinson's disease. However, its specific mechanism of action is still unclear. In the present study, we established a Parkinson's disease pathology model by exposing SH-SY5Y cells and C57BL/6J mice to methamphetamine. In vitro experiments were performed with 0, 0.5, 1.0, 1.5, 2.0 or 2.5 mM methamphetamine for 24 hours or 2.0 mM methamphetamine for 0-, 2-, 4-, 8-, 16-, and 24-hour culture of SH-SY5Y cells. Additional experimental groups of SH-SY5Y cells were administered a nitric oxide inhibitor, 0.1 mM N-nitro-L-arginine, 1 hour before exposure to 2.0 mM methamphetamine for 24 hours. In vivo experiments: C57BL/6J mice were intraperitoneally injected with N-nitro-L-arginine (8 mg/kg), eight times, at intervals of 12 hours. Methamphetamine 15 mg/kg was intraperitoneally injected eight times, at intervals of 12 hours, but 0.5-hour after each N-nitro-L-arginine injection in the combined group. Western blot assay was used to determine the expression of nitric oxide synthase, α-synuclein (α-Syn), 5G4, nitrated α-synuclein at the residue Tyr39 (nT39 α-Syn), cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) in cells and mouse brain tissue. Immunofluorescence staining was conducted to measure the positive reaction of NeuN, nT39 α-Syn and 5G4. Enzyme linked immunosorbent assay was performed to determine the dopamine levels in the mouse brain. After methamphetamine exposure, α-Syn expression increased; the aggregation of α-Syn 5G4 increased; nT39 α-Syn, nitric oxide synthase, cleaved caspase-3, and cleaved PARP expression increased in the cultures of SH-SY5Y cells and in the brains of C57BL/6J mice; and dopamine levels were reduced in the mouse brain. These changes were markedly reduced when N-nitro-L-arginine was administered with methamphetamine in both SH-SY5Y cells and C57BL/6J mice. These results suggest that nT39 α-Syn aggregation is involved in methamphetamine neurotoxicity.

Lentivirus-mediated RNAi silencing targeting ABCC2 increasing the sensitivity of a human nasopharyngeal carcinoma cell line against cisplatin.

BACKGROUND: High resistance to drug is taken as a characteristic of human tumors, which is usually mediated by multidrug resistance-associated genes. ABCC2, an ATP-binding cassette multidrug resistance transporter, is found to be expressed in a variety of human cancers. In this study the effect of a RNAi construct targeting ABCC2 on the chemosensitivity of NPC cell line CNE2 against cisplatin was investigated. METHODS: Lentiviral vectors were constructed to allow an efficient expression of anti-ABCC2 siRNA. The effective target sequence comprised nucleotides 1707-1727 of the human ABCC2 mRNA. The cell clones expressing the construct were picked and expanded, followed by identification using qRT-PCR and western blot method. As control, lentiviral vector containing invalid RNAi sequence was transfected to CNE2 cells. In vitro, cellular accumulation of cisplatin was detected by HPLC. The capacity of cellular growth and sensitivity of cells against cisplatin were detected by MTT assay. In vivo, the sensitivity of the tumor tissues against cisplatin were evaluated by transplanted CNE2 nude mice model. RESULTS: Two CNE2 cell clones with reduced expression of targeted ABCC2 mRNA and protein for more than 70% by qRT-PCR and western blot were established, and no differences were shown in proliferation rates compared to control CNE2 cells by growth curves analysis. In vitro the accumulation of intracellular cisplatin in these CNE2 cell clones with reduced expression of ABCC2 increased markedly, accompanied by increased sensitivity against cisplatin. In vivo, the growth of CNE2 solid tumors with a stably transfected anti-ABCC2 siRNA construct was significantly inhibited by cisplatin in transplanted nude mice model. CONCLUSION: Our investigation demonstrated that lentivirus-mediated RNAi silencing targeting ABCC2 might reverse the ABCC2-related drug resistance of NPC cell line CNE2 against cisplatin.

CDK5-mediated tau accumulation triggers methamphetamine-induced neuronal apoptosis via endoplasmic reticulum-associated degradation pathway.

Overexposure to methamphetamine (METH) causes apoptosis in a number of cell types, particularly neuronal cells. However, the underlying mechanisms of METH-induced neuronal apoptosis remain to be elucidated. Accumulation of microtubule-associated protein Tau can lead to activation of multiple neurotoxic pathways, which is closely correlated with neuronal apoptosis. The aim of this study was to determine the role of Tau in METH-induced neuronal apoptosis. We determined the expression of two phosphorylated Tau proteins (serine 396 and threonine 231) in the human neuroblastoma SH-SY5Y cells and in the hippocampus of Sprague-Dawley rats treated with vehicle or METH using western blotting, immunohistochemical staining and immunofluorescence staining. We also measured the expression levels of the phosphorylated Tau protein, ubiquitination proteins, the intermediate products of proteasome degradation pathway, CD3-δ (a substrate of proteasome degradation pathway), endoplasmic reticulum stress signal molecule phosphorylated PERK (pPERK), and endoplasmic reticulum stress-specific apoptotic signal molecule caspase-12 in SH-SY5Y cells and in rats after inhibiting the expression of an upstream regulatory factor of phosphorylated Tau protein (CDK5) using siRNA or virus transfection. The results showed that exposure to METH significantly up-regulated the expression of phosphorylated Tau protein in vivo and in vitro and silencing the expression of CDK5 inhibited the up-regulation of phosphorylated Tau induced by METH exposure. METH exposure also significantly increased the expression of ubiquitination protein and CD3-δ and these effects were blocked by CDK5 silencing. In addition, METH exposure significantly elevated the levels of phosphorylated PERK and caspase-12 and these effects were suppressed after CDK5 silencing, which indicates that blockade of CDK5 expression can mitigate METH-induced neuronal apoptosis. These results suggest that METH can impair the endoplasmic reticulum-associated degradation (ERAD) pathway and induce neuronal apoptosis through endoplasmic reticulum stress, which is mainly mediated by abnormal CDK5-regulated Tau phosphorylation.

SUMOylation of Alpha-Synuclein Influences on Alpha-Synuclein Aggregation Induced by Methamphetamine.

Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (α-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of α-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of α-syn is involved in high-dose METH-induced α-syn aggregation. We measured the levels of α-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of α-syn SUMOylation on α-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of α-syn by transfecting a lentivirus containing the sequence of WT α-syn or K96/102R α-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT α-syn or K96/102R α-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of α-syn, although the expression of α-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in α-syn SUMOylation by UBC9 overexpression relieves METH-induced α-syn overexpression and aggregation, whereas the decrease in α-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of α-syn also aggravate α-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of α-syn plays a fundamental part in α-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes.

Methamphetamine (METH) is an amphetamine-typed stimulant drug that is increasingly being abused worldwide. Previous studies have shown that METH toxicity is systemic, especially targeting dopaminergic neurons in the central nervous system (CNS). However, the role of neuroinflammation in METH neurotoxicity remains unclear. We hypothesized that Toll-like receptor 4 (TLR4) and Caspase-11 are involved in METH-induced astrocyte-related neuroinflammation. We tested our hypothesis by examining the changes of TLR4 and Caspase-11 protein expression in primary cultured C57BL/6 mouse astrocytes and in the midbrain and striatum of mice exposed to METH with western blot and double immunofluorescence labeling. We also determined the effects of blocking Caspase-11 expression with wedelolactone (a specific inhibitor of Caspase-11) or siRNA on METH-induced neuroinflammation in astrocytes. Furthermore, we determined the effects of blocking TLR4 expression with TAK-242 (a specific inhibitor of TLR4) or siRNA on METH-induced neuroinflammation in astrocytes. METH exposure increased Caspase-11 and TLR4 expression both in vitro and in vivo, with the effects in vitro being dose-dependent. Inhibition of Caspase-11 expression with either wedelolactone or siRNAs reduced the expression of inflammasome NLRP3 and pro-inflammatory cytokines. In addition, blocking TLR4 expression inhibited METH-induced activation of NF-κB and Caspase-11 in vitro and in vivo, suggesting that TLR4-Caspase-11 pathway is involved in METH-induced neuroinflammation. These results indicate that Caspase-11 and TLR4 play an important role in METH-induced neuroinflammation and may be potential gene targets for therapeutics in METH-caused neurotoxicity.

Nupr1 Modulates Methamphetamine-Induced Dopaminergic Neuronal Apoptosis and Autophagy through CHOP-Trib3-Mediated Endoplasmic Reticulum Stress Signaling Pathway.

Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH exposure causes detrimental effects on multiple organ systems, primarily the nervous system, especially dopaminergic pathways, in both laboratory animals and humans. In this study, we hypothesized that Nuclear protein 1 (Nupr1/com1/p8) is involved in METH-induced neuronal apoptosis and autophagy through endoplasmic reticulum (ER) stress signaling pathway. To test this hypothesis, we measured the expression levels of Nupr1, ER stress protein markers CHOP and Trib3, apoptosis-related protein markers cleaved-caspase3 and PARP, as well as autophagy-related protein markers LC3 and Beclin-1 in brain tissues of adult male Sprague-Dawley (SD) rats, rat primary cultured neurons and the rat adrenal pheochromocytoma cells (PC12 cells) after METH exposure. We also determined the effects of METH exposure on the expression of these proteins after silencing Nupr1, CHOP, or Trib3 expression with synthetic small hairpin RNA (shRNA) or siRNA in vitro, and after silencing Nupr1 in the striatum of rats by injecting lentivirus containing shRNA sequence targeting Nupr1 gene to rat striatum. The results showed that METH exposure increased Nupr1 expression that was accompanied with increased expression of ER stress protein markers CHOP and Trib3, and also led to apoptosis and autophagy in rat primary neurons and in PC12 cells after 24 h exposure (3.0 mM), and in the prefrontal cortex and striatum of rats after repeated intraperitoneal injections (15 mg/kg × 8 injections at 12 h intervals). Silencing of Nupr1 expression partly reduced METH-induced apoptosis and autophagy in vitro and in vivo. These results suggest that Nupr1 plays an essential role in METH-caused neuronal apoptosis and autophagy at relatively higher doses and may be a potential therapeutic target in high-dose METH-induced neurotoxicity.