Consensus subtypes of hepatocellular carcinoma associated with clinical outcomes and genomic phenotypes.

BACKGROUND AND AIMS: Although many studies revealed transcriptomic subtypes of HCC, concordance of the subtypes are not fully examined. We aim to examine a consensus of transcriptomic subtypes and correlate them with clinical outcomes. APPROACH AND RESULTS: By integrating 16 previously established genomic signatures for HCC subtypes, we identified five clinically and molecularly distinct consensus subtypes. STM (STeM) is characterized by high stem cell features, vascular invasion, and poor prognosis. CIN (Chromosomal INstability) has moderate stem cell features, but high genomic instability and low immune activity. IMH (IMmune High) is characterized by high immune activity. BCM (Beta-Catenin with high Male predominance) is characterized by prominent ß-catenin activation, low miRNA expression, hypomethylation, and high sensitivity to sorafenib. DLP (Differentiated and Low Proliferation) is differentiated with high hepatocyte nuclear factor 4A activity. We also developed and validated a robust predictor of consensus subtype with 100 genes and demonstrated that five subtypes were well conserved in patient-derived xenograft models and cell lines. By analyzing serum proteomic data from the same patients, we further identified potential serum biomarkers that can stratify patients into subtypes. CONCLUSIONS: Five HCC subtypes are correlated with genomic phenotypes and clinical outcomes and highly conserved in preclinical models, providing a framework for selecting the most appropriate models for preclinical studies.

Feasibility of Raman spectroscopic identification of gall bladder cancer using extracellular vesicles extracted from bile.

Extracellular vesicles (EVs), which are heterogeneous membrane-based vesicles with bilayer cell membrane structures, could be versatile biomarkers for the identification of diverse diseases including cancers. With this potential, this study has attempted the Raman spectroscopic identification of gall bladder (GB) cancer by directly measuring the EV solution extracted from human bile without further sample drying. For this purpose, bile samples were obtained from four normal individuals and 21 GB polyp, eight hepatocellular carcinoma (HCC), and five GB cancer patients, and EVs were extracted from each of the bile samples. The Raman peak shapes of the EVs extracted from the GB cancer samples, especially the relative intensities of peaks in the 1560-1340 cm-1 range, were dissimilar to those of the samples from the normal, GB polyp, and HCC groups. The intensity ratios of peaks at 1537 and 1453 cm-1 and at 1395 and 1359 cm-1 of the GB cancer samples were lower and higher, respectively, than those of the samples of the remaining three groups. The differences of peak intensity ratios were statistically significant based on the Mann-Whitney U test. DNA/RNA bases, amino acids, and bile salts contributed to the spectra of EVs, and their relative abundances seemed to vary according to the occurrence of GB cancer. The varied metabolite compositions and/or structures of EVs were successfully demonstrated by the dissimilar peak intensity ratios in the Raman spectra, thereby enabling the discrimination of GB cancer.

Hematopoietic progenitor kinase 1 down-regulates the oncogenic receptor tyrosine kinase AXL in pancreatic cancer.

The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL over-expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, bafilomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest-specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to the Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression (p < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC.

Transcriptional coactivator with PDZ-binding motif suppresses the expression of steroidogenic enzymes by nuclear receptor 4 A1 in Leydig cells.

Transcriptional coactivator with PDZ-binding motif (TAZ) plays crucial role in maintaining testicular structure and function via regulation of senescence of spermatogenic cells. However, it remains unclear whether TAZ is involved in testosterone biosynthesis in testicular Leydig cells. We found that TAZ deficiency caused aberrant Leydig cell expansion and increased lipid droplet formation, which was significantly associated with increased lipogenic enzyme expression. Additionally, the expression of key steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 (CYP) 11A1, CYP17A1, and 3ß-hydroxysteroid dehydrogenase, was greatly increased in TAZ-deficient testes and primary Leydig cells. Interestingly, the transcriptional activity of nuclear receptor 4 A1 (NR4A1) was dramatically suppressed by TAZ; however, the protein expression and the subcellular localization of NR4A1 were not affected by TAZ. TAZ directly associated with the N-terminal region of NR4A1 and substantially suppressed its DNA-binding and transcriptional activities. Stable expression of TAZ in the mouse Leydig TM3 cell line decreased the expression of key steroidogenic enzymes, whereas knockdown of endogenous TAZ in TM3 cells increased transcripts of steroidogenic genes induced by NR4A1. Consistently, testosterone production was enhanced within TAZ-deficient Leydig cells. However, TAZ deficiency resulted in decreased testosterone secretion caused by dysfunctional mitochondria and lysosomes. Therefore, TAZ plays essential role in NR4A1-induced steroidogenic enzyme expression and testosterone production in Leydig cells.

Pathological predictive factors for late recurrence of hepatocellular carcinoma in chronic liver disease.

BACKGROUND & AIMS: Late recurrence of hepatocellular carcinoma (HCC) is regarded as de novo HCC from chronic hepatitis. This study investigated clinicopathological and molecular factors to develop a nomogram for predicting late HCC recurrence (>2 years after curative resection). METHODS: The training and validation cohorts included HCC patients with a major aetiology of hepatitis B who underwent curative resection. Clinicopathological features including lobular and porto-periportal inflammatory activity, fibrosis and liver cell change were evaluated. Proteins encoded by genes related to late recurrence were identified using a reverse phase protein array of 95 non-tumourous liver tissues. Immunoexpression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), plasminogen activator inhibitor-1, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and spleen tyrosine kinase (SYK) was measured. RESULTS: Late recurrence occurred in 74/402 (18%) and 47/243 (19%) in the training and validation cohorts respectively. Cirrhosis, moderate/severe lobular inflammatory activity, and expression of pSTAT3, pERK1/2, and SYK proteins correlated to the gene signature of hepatocyte injury and regeneration were independently associated with late recurrence, with odds ratios (95% confidence intervals) of 2.0 (1.2-3.3), 21.1 (4.3-102.7) and 6.0 (2.1-17.7) respectively (P < .05 for all). A nomogram based on these variables (histological parameters and immunohistochemical marker combinations) showed high reliability in both the training and validation cohorts (Harrell's C index: 0.701 and 0.716; 95% confidence intervals: 0.64-0.76 and 0.64-0.79 respectively). CONCLUSIONS: The combination of pSTAT3, pERK1/2 and SYK immunoexpression with high lobular inflammatory activity and cirrhosis (fibrosis) predicts late HCC recurrence.

Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis.

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

TMP21 regulates autophagy by modulating ROS production and mTOR activation.

Autophagy is a catabolic cellular response to stress that has been liked to various human diseases. However, the precise involvement of autophagy in health and disease remains unclear. To explore the molecular mechanisms of autophagy, we investigated the effect of TMP21. We found that the down-regulation of TMP21 induced autophagy in SH-SY5Y cells. In addition, the enhanced autophagy observed upon TMP21 depletion was almost completely blocked in ATG5 knockout (KO) or ATG7-KO HeLa cells. Silencing of TMP21 in SH-SY5Y cells also increased the production of cellular reactive oxygen species (ROS). Accordingly, treatment with the ROS scavenger NAC suppressed autophagy activation as well as ROS production in TMP21-depleted cells. In addition, the inhibition of mTOR by treatment with Torin1 was mitigated in TMP21 overexpressing cells compared with that in control cells. Taken together, these results indicated that TMP21 could regulate autophagy by modulating ROS production and mTOR activation.

Prognostic significance of high metabolic activity in breast cancer: PET signature in breast cancer.

High metabolic activity, reflected in increased glucose uptake, is one of the hallmarks of many cancers including breast cancer. However, not all cancers avidly take up glucose, suggesting heterogeneity in their metabolic demand. Thus, we aim to generate a genomic signature of glucose hypermetabolism in breast cancer and examine its clinical relevance. To identify genes significantly associated with glucose uptake, gene expression data were analyzed together with the standardized uptake values (SUVmax) of 18F-fluorodeoxy-glucose on positron emission tomography (PET) for 11 breast cancers. The resulting PET signature was evaluated for prognostic significance in four large independent patient cohorts (n = 5417). Potential upstream regulators accountable for the high glucose uptake were identified by gene network analysis. A PET signature of 242 genes was significantly correlated with SUVmax in breast cancer. In all four cohorts, high PET signature was significantly associated with poorer prognosis. The prognostic value of this PET signature was further supported by Cox regression analyses (hazard ratio 1.7, confidential interval 1.48-2.02; P < 0.001). The PET signature was also strongly correlated with previously established prognostic genomic signatures such as PAM50, Oncotype DX, and NKI. Gene network analyses suggested that MYC and TBX2 were the most significant upstream transcription factors in the breast cancers with high glucose uptake. A PET signature reflecting high glucose uptake is a novel independent prognostic factor in breast cancer. MYC and TBX2 are potential regulators of glucose uptake.

Prevalence and molecular epidemiology of ESBLs, plasmid-determined AmpC-type ß-lactamases and carbapenemases among diarrhoeagenic Escherichia coli isolates from children in Gwangju, Korea: 2007-16.

OBJECTIVES: Young children could act as important carriers of cefotaxime-resistant Enterobacteriaceae. However, most studies on these bacteria have focused on hospitalized adults. Therefore, we determined the prevalence and characteristics of ESBL-, plasmid-determined AmpC-type ß-lactamase (PABL)- and carbapenemase-producing diarrhoeagenic Escherichia coli isolates mainly from infants and children in the south-west region of Korea over a 10 year period. METHODS: Non-duplicate E. coli clinical isolates were recovered from diarrhoeagenic patient specimens at 12 hospitals in Gwangju, Korea, between January 2007 and December 2016. Antimicrobial susceptibilities and molecular features of ESBL- and carbapenemase-producing isolates were determined. RESULTS: A total of 1047 pathogenic E. coli isolates were collected and 58 cefotaxime-resistant E. coli isolates (5.5%) were identified. The prevalence and types of ß-lactamase genes increased steadily from 5.7% in 2007 to 11.6% in 2016 with some fluctuations. CTX-M-14 (53.4%) was the predominant CTX-M genotype. PFGE revealed high genetic heterogeneities among diarrhoeagenic E. coli isolates, suggesting horizontal transfer of antibiotic resistance genes, which was also proved by conjugation assay. CONCLUSIONS: Progressive increases in carriage rates and the number of ß-lactamase types, and the possibility of community outbreaks of these food-borne bacteria in young children, may pose tangible public health threats.

Different epithelial cell response to membrane vesicles produced by Listeria monocytogenes cultured with or without salt stress.

We have previously shown that Listeria monocytogenes, a causative agent of listeriosis, can produce membrane vesicles (MVs) during in vitro culture. The aim of this study was to investigate the ability of MVs from L. monocytogenes cultured with or without salt stress to induce cytotoxicity and pro-inflammatory responses in colon epithelial Caco-2 cells. MVs were purified from wild-type L. monocytogenes 10403S strain and an isogenic ΔsigB mutant strain. MVs from both wild-type and ΔsigB mutant strains increased viability of Caco-2 cells regardless of salt stress. Both MVs from wild-type and ΔsigB mutant strains stimulated expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells. Expression levels of pro-inflammatory cytokine genes in cells treated with MVs from bacteria cultured without salt stress were significantly higher than those in cells treated with MVs from bacteria cultured with salt stress. However, expression levels of chemokine genes in cells treated with MVs from bacteria cultured with salt stress were significantly higher than those in cells treated with MVs from bacteria cultured without salt stress. In addition, expression levels of interleukin (IL)-1ß and IL-8 genes were partially inhibited by either lysozyme-treated MVs or ethylenediaminetetraacetic acid-treated MVs compared to those after treatment with intact MVs. Our results suggest that salt stress can affect the production of L. monocytogenes MVs, thus causing different pro-inflammatory responses in host cells.

Identification of prognostic biomarker in predicting hepatocarcinogenesis from cirrhotic liver using protein and gene signatures.

INTRODUCTION: Cirrhosis primes the liver for hepatocellular carcinoma (HCC) development. However, biomarkers that predict HCC in cirrhosis patients are lacking. Thus, we aimed to identify a biomarker directly from protein analysis and relate it with transcriptomic data to validate in larger cohorts. MATERIAL AND METHOD: Forty-six patients who underwent hepatectomy for HCC that arose from cirrhotic liver were enrolled. Reverse-phase protein array and microarray data of these patients were analyzed. Clinical validation was performed in two independent cohorts and functional validation using cell and tissue microarray (TMA). RESULTS: Systematic analysis performed after selecting 20 proteins from 201 proteins with AUROC >70 effectively categorized patients into high (n = 20) or low (n = 26) risk HCC groups. Proteome-derived late recurrence (PDLR)-gene signature comprising 298 genes that significantly differed between high and low risk groups predicted HCC well in a cohort of 216 cirrhosis patients and also de novo HCC recurrence in a cohort of 259 patients who underwent hepatectomy. Among 20 proteins that were selected for analysis, caveolin-1 (CAV1) was the most dominant protein that categorized the patients into high and low risk groups (P < .001). In a multivariate analysis, compared with other clinical variables, the PDLR-gene signature remained as a significant predictor of HCC (HR 1.904, P = .01). In vitro experiments revealed that compared with mock-transduced immortalized liver cells, CAV1-transduced cells showed significantly increased proliferation (P < .001) and colony formation in soft agar (P < .033). TMA with immunohistochemistry showed that tissues with CAV1 expression were more likely to develop HCC than tissues without CAV1 expression (P = .047). CONCLUSION: CAV1 expression predicts HCC development, making it a potential biomarker and target for preventive therapy.

RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif.

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy.

Inhibition of Drp1 Ameliorates Synaptic Depression, Aß Deposition, and Cognitive Impairment in an Alzheimer's Disease Model.

Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-ß (Aß) in neurons and neuropathology and cognitive functions in Aß precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aß-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aß deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aß-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD.SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimer's disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-ß (Aß)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aß deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.

Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy.

Salt stress affects global protein expression profiles of extracellular membrane-derived vesicles of Listeria monocytogenes.

Our previous study has suggested that Listeria monocytogenes produces extracellular membrane vesicles (MVs) and its general stress transcription factor sigma B (σB) affects the production of MVs under energy stress. The objective of this study was to evaluate the production of MVs and perform global protein profiling for MVs with or without salt stress to understand the function of MVs in the pathogenesis of L. monocytogenes. When cells of L. monocytogenes were grown under 0.5 M salt stress, protein concentrations of MVs derived from wild-type strain and its isogenic ΔsigB mutant were approximately doubled compared to those of MVs derived from cells without salt stress. Proteomic analyses showed that the number of MV proteins expressed in wild-type strain was similar to that in ΔsigB mutant under salt stress. However, global protein expression profiles were dramatically changed under salt stress compared to those without salt stress. Fifteen σB dependent proteins were expressed in MVs of wild-type strain under salt stress, including osmolyte transporter OpuCABCD. In addition, MVs produced by salt stressed wild-type and ΔsigB mutant inhibited biofilm formation abilities of both strains. Taken together, our results suggest that salt stress can promote the production of MVs involved in carnitine transporter proteins, with σB playing a pivotal role in biological event.

Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.

RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing ß,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the ß,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications.

Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress toward understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation.

Heterogeneous nuclear ribonucleoprotein A1 post-transcriptionally regulates Drp1 expression in neuroblastoma cells.

Excessive mitochondrial fission is associated with the pathogenesis of neurodegenerative diseases. Dynamin-related protein 1 (Drp1) possesses specific fission activity in the mitochondria and peroxisomes. Various post-translational modifications of Drp1 are known to modulate complex mitochondrial dynamics. However, the post-transcriptional regulation of Drp1 remains poorly understood. Here, we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates Drp1 expression at the post-transcriptional level. hnRNP A1 directly interacts with Drp1 mRNA at its 3'UTR region, and enhances translation potential without affecting mRNA stability. Down-regulation of hnRNP A1 induces mitochondrial elongation by reducing Drp1 expression. Moreover, depletion of hnRNP A1 suppresses 3-NP-mediated mitochondrial fission and dysfunction. In contrast, over-expression of hnRNP A1 promotes mitochondrial fragmentation by increasing Drp1 expression. Additionally, hnRNP A1 significantly exacerbates 3-NP-induced mitochondrial dysfunction and cell death in neuroblastoma cells. Interestingly, treatment with 3-NP induces subcellular translocation of hnRNP A1 from the nucleus to the cytoplasm, which accelerates the increase in Drp1 expression in hnRNP A1 over-expressing cells. Collectively, our findings suggest that hnRNP A1 controls mitochondrial dynamics by post-transcriptional regulation of Drp1.

Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation.

Chloroquine (CQ) and amodiaquine (AQ) have been used for treating or preventing malaria for decades, and their application has expanded into treating inflammatory disease in humans. CQ and AQ are applicable for controlling rheumatoid arthritis, but their molecular mechanisms of anti-inflammatory activity remain to be elucidated. In this study, we examined the effects of CQ and AQ on T cell activation and T cell-mediated immune response. CQ had no significant effect on T cell numbers, but decreased the population of T cells with a high division rate. However, AQ treatment significantly increased the number of cells with low division rates and eliminated cells with high division rates, resulting in the inhibition of T cell proliferation triggered by T cell receptor stimulation, of which inhibition occurred in developing effector T helper and regulatory T cells, regardless of the different exogenous cytokines. Interestingly, the cyclin-dependent kinase inhibitor p21 was significantly and dose-dependently increased by CQ, and more potently by AQ, while other cell cycle regulators were unchanged. Both CQ and AQ elevated the transcription level of p21 though the activation of p53, but also blocked p21 protein degradation in the presence of cycloheximide, causing p21 protein accumulation mainly in the nucleus. Sustained treatment of developing T cells with either CQ or AQ suppressed IFN-γ production in a dose dependent manner and potently inhibited the differentiation of IFN-γ-producing Th1 cells. These results demonstrate that CQ and AQ increase the expression level of p21 and inhibit T cell proliferation and the development of IFN-γ-producing Th1 cells, thereby revealing beneficial roles in treating a wide range of chronic inflammatory diseases mediated by inflammatory T cells.

Down-regulation of mortalin exacerbates Aß-mediated mitochondrial fragmentation and dysfunction.

Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aß-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aß-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aß-mediated mitochondrial fragmentation and dysfunction in AD.