Activating Transcription Factor 3 Stimulates Follicle-Stimulating Hormone-ß Expression In Vitro But Is Dispensable for Follicle-Stimulating Hormone Production in Murine Gonadotropes In Vivo.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHß subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone ß (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LßT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

Transcription factor GATA2 may potentiate follicle-stimulating hormone production in mice via induction of the BMP antagonist gremlin in gonadotrope cells.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHß subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Skeletal muscle transcriptome response to a bout of endurance exercise in physically active and sedentary older adults.

Age-related declines in cardiorespiratory fitness and physical function are mitigated by regular endurance exercise in older adults. This may be due, in part, to changes in the transcriptional program of skeletal muscle following repeated bouts of exercise. However, the impact of chronic exercise training on the transcriptional response to an acute bout of endurance exercise has not been clearly determined. Here, we characterized baseline differences in muscle transcriptome and exercise-induced response in older adults who were active/endurance trained or sedentary. RNA-sequencing was performed on vastus lateralis biopsy specimens obtained before, immediately after, and 3 h following a bout of endurance exercise (40 min of cycling at 60%-70% of heart rate reserve). Using a recently developed bioinformatics approach, we found that transcript signatures related to type I myofibers, mitochondria, and endothelial cells were higher in active/endurance-trained adults and were associated with key phenotypic features including V̇o2peak, ATPmax, and muscle fiber proportion. Immune cell signatures were elevated in the sedentary group and linked to visceral and intermuscular adipose tissue mass. Following acute exercise, we observed distinct temporal transcriptional signatures that were largely similar among groups. Enrichment analysis revealed catabolic processes were uniquely enriched in the sedentary group at the 3-h postexercise timepoint. In summary, this study revealed key transcriptional signatures that distinguished active and sedentary adults, which were associated with difference in oxidative capacity and depot-specific adiposity. The acute response signatures were consistent with beneficial effects of endurance exercise to improve muscle health in older adults irrespective of exercise history and adiposity.NEW & NOTEWORTHY Muscle transcript signatures associated with oxidative capacity and immune cells underlie important phenotypic and clinical characteristics of older adults who are endurance trained or sedentary. Despite divergent phenotypes, the temporal transcriptional signatures in response to an acute bout of endurance exercise were largely similar among groups. These data provide new insight into the transcriptional programs of aging muscle and the beneficial effects of endurance exercise to promote healthy aging in older adults.

A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery.

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.

Heterogeneous origins and functions of mouse skeletal muscle-resident macrophages.

Tissue-resident macrophages can originate from embryonic or adult hematopoiesis. They play important roles in a wide range of biological processes including tissue remodeling during organogenesis, organ homeostasis, repair following injury, and immune response to pathogens. Although the origins and tissue-specific functions of resident macrophages have been extensively studied in many other tissues, they are not well characterized in skeletal muscle. In the present study, we have characterized the ontogeny of skeletal muscle-resident macrophages by lineage tracing and bone marrow transplant experiments. We demonstrate that skeletal muscle-resident macrophages originate from both embryonic hematopoietic progenitors located within the yolk sac and fetal liver as well as definitive hematopoietic stem cells located within the bone marrow of adult mice. Single-cell-based transcriptome analyses revealed that skeletal muscle-resident macrophages are distinctive from resident macrophages in other tissues as they express a distinct complement of transcription factors and are composed of functionally diverse subsets correlating to their origins. Functionally, skeletal muscle-resident macrophages appear to maintain tissue homeostasis and promote muscle growth and regeneration.

Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1.

Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

LßT2 and αT3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LßT2 cells are more mature gonadotrope precursors than αT3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in αT3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In LßT2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for αT3-1 and a female sex for LßT2 cells. Among LßT2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, LßT2a and LßT2b. The two lines differed in morphological appearance, with LßT2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. LßT2a showed higher basal levels of Egr1, Fos, and Lhb but lower Fos induction. Fshb induction reached significance only in LßT2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

In vitro modeling of hepatocellular carcinoma molecular subtypes for anti-cancer drug assessment.

Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and S2, determined by our previous transcriptome meta-analysis of multiple clinical hepatocellular carcinoma cohorts, are presented in a panel of hepatoma cell lines widely used by the research community. Interestingly, cell line that resembles gene expression pattern of S3 subtype, representing less aggressive tumors, was not identified in the panel. MYC pathway-activated S2-like cell lines showed higher sensitivity to a small molecule BET bromodomain inhibitor, (+)-JQ1, which has anti-MYC activity. These results support the use of hepatoma cell lines as models to evaluate molecular subtype-specific drug response, which is expected to lead to development of tailored, precision care of the patients with hepatocellular carcinoma.

Combination of Gene Expression Signature and Model for End-Stage Liver Disease Score Predicts Survival of Patients With Severe Alcoholic Hepatitis.

BACKGROUND & AIMS: Patients with severe alcoholic hepatitis (AH) have a high risk of death within 90 days. Corticosteroids, which can cause severe adverse events, are the only treatment that increases short-term survival. It is a challenge to predict outcomes of patients with severe AH. Therefore, we developed a scoring system to predict patient survival, integrating baseline molecular and clinical variables. METHODS: We obtained fixed liver biopsy samples from 71 consecutive patients diagnosed with severe AH and treated with corticosteroids from July 2006 through December 2013 in Brussels, Belgium (derivation cohort). Gene expression patterns were analyzed by microarrays and clinical data were collected for 180 days. We identified gene expression signatures and clinical data that are associated with survival without liver transplantation at 90 and 180 days after initiation of corticosteroid therapy. Findings were validated using liver biopsies from 48 consecutive patients with severe AH treated with corticosteroids, collected from March 2010 through February 2015 at hospitals in Belgium and Switzerland (validation cohort 1) and in liver biopsies from 20 patients (9 received corticosteroid treatment), collected from January 2012 through May 2015 in the United States (validation cohort 2). RESULTS: We integrated data on expression patterns of 123 genes and the model for end-stage liver disease (MELD) scores to assign patients to groups with poor survival (29% survived 90 days and 26% survived 180 days) and good survival (76% survived 90 days and 65% survived 180 days) (P < .001) in the derivation cohort. We named this assignment system the gene signature-MELD (gs-MELD) score. In validation cohort 1, the gs-MELD score discriminated patients with poor survival (43% survived 90 days) from those with good survival (96% survived 90 days) (P < .001). The gs-MELD score also discriminated between patients with a poor survival at 180 days (34% survived) and a good survival at 180 days (84% survived) (P < .001). The time-dependent area under the receiver operator characteristic curve for the score was 0.86 (95% confidence interval 0.73-0.99) for survival at 90 days, and 0.83 (95% confidence interval 0.71-0.96) for survival at 180 days. This score outperformed other clinical models to predict survival of patients with severe AH in validation cohort 1. In validation cohort 2, the gs-MELD discriminated patients with a poor survival at 90 days (12% survived) from those with a good survival at 90 days (100%) (P < .001). CONCLUSIONS: We integrated data on baseline liver gene expression pattern and the MELD score to create the gs-MELD scoring system, which identifies patients with severe AH, treated or not with corticosteroids, most and least likely to survive for 90 and 180 days.

Molecular Liver Cancer Prevention in Cirrhosis by Organ Transcriptome Analysis and Lysophosphatidic Acid Pathway Inhibition.

Cirrhosis is a milieu that develops hepatocellular carcinoma (HCC), the second most lethal cancer worldwide. HCC prediction and prevention in cirrhosis are key unmet medical needs. Here we have established an HCC risk gene signature applicable to all major HCC etiologies: hepatitis B/C, alcohol, and non-alcoholic steatohepatitis. A transcriptome meta-analysis of >500 human cirrhotics revealed global regulatory gene modules driving HCC risk and the lysophosphatidic acid pathway as a central chemoprevention target. Pharmacological inhibition of the pathway in vivo reduced tumors and reversed the gene signature, which was verified in organotypic ex vivo culture of patient-derived fibrotic liver tissues. These results demonstrate the utility of clinical organ transcriptome to enable a strategy, namely, reverse-engineering precision cancer prevention.

Clinicopathological indices to predict hepatocellular carcinoma molecular classification.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

A genomic and clinical prognostic index for hepatitis C-related early-stage cirrhosis that predicts clinical deterioration.

OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100,000/mm(3)), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.

MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells.

MicroRNA-137 (miR-137) has been shown to play an important role in the differentiation of neural stem cells. Embryonic stem (ES) cells have the potential to differentiate into different cell types including neurons; however, the contribution of miR-137 in the maintenance and differentiation of ES cells remains unknown. Here, we show that miR-137 is mainly expressed in ES cells at the mitotic phase of the cell cycle and highly upregulated during differentiation. We identify that ES cell transcription factors, Klf4 and Tbx3, are downstream targets of miR-137, and we show that endogenous miR-137 represses the 3' untranslated regions of Klf4 and Tbx3. Transfection of ES cells with mature miR-137 RNA duplexes led to a significant reduction in cell proliferation and the expression of Klf4, Tbx3, and other self-renewal genes. Furthermore, we demonstrate that increased miR-137 expression accelerates differentiation of ES cells in vitro. Loss of miR-137 during ES cell differentiation significantly impeded neuronal gene expression and morphogenesis. Taken together, our results suggest that miR-137 regulates ES cell proliferation and differentiation by repressing the expression of downstream targets, including Klf4 and Tbx3.

Localization of BRCA1 protein in breast cancer tissue and cell lines with mutations.

BACKGROUND: The breast and ovarian cancer susceptibility gene (BRCA1) encodes a tumor suppressor. The BRCA1 protein is found primarily in cell nuclei and plays an important role in the DNA damage response and transcriptional regulation. Deficiencies in DNA repair capabilities have been associated with higher histopathological grade and worse prognosis in breast cancer. METHODS: In order to investigate the subcellular distribution of BRCA1 in tumor tissue we randomly selected 22 breast carcinomas and tested BRCA1 protein localization in frozen and contiguous formalin-fixed, paraffin embedded (FFPE) tissue, using pressure cooker antigen-retrieval and the MS110 antibody staining. To assess the impact of BRCA1 germline mutations on protein localization, we retrospectively tested 16 of the tumor specimens to determine whether they contained the common Ashkenazi Jewish founder mutations in BRCA1 (185delAG, 5382insC), and BRCA2 (6174delT). We also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC). RESULTS: In FFPE tissue, with MS110 antibody staining, we frequently found reduced BRCA1 nuclear staining in breast tumor tissue compared to normal tissue, and less BRCA1 staining with higher histological grade in the tumors. However, in the frozen sections, BRCA1 antibody staining showed punctate, intra-nuclear granules in varying numbers of tumor, lactating, and normal cells. Two mutation carriers were identified and were confirmed by gene sequencing. We have also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC) and found altered sub-nuclear and nucleolar localization patterns consistent with a functional impact of the mutation on protein localization. CONCLUSIONS: The data presented here support a role for BRCA1 in the pathogenesis of sporadic and inherited breast cancers. The use of well-characterized reagents may lead to further insights into the function of BRCA1 and possibly the further development of targeted therapeutics.

Involvement of histone demethylase LSD1 in short-time-scale gene expression changes during cell cycle progression in embryonic stem cells.

The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G(1)/S/G(2) phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression.

Differential modulation of Akt/glycogen synthase kinase-3beta pathway regulates apoptotic and cytoprotective signaling responses.

We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3beta (GSK-3beta). Ropinirole-induced protection was associated with phosphorylation of GSK-3beta (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3beta (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3beta and reduced levels of beta-catenin, a transcriptional activator that is regulated by GSK-3beta. Knock-out of GSK-3beta expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3beta, increases GSK-3beta activity, and promotes an interaction with beta-catenin and its degradation. Inhibition of GSK-3beta activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3beta downstream of Akt plays a critical role in cell death and survival in these models.

p53 mediates nontranscriptional cell death in dopaminergic cells in response to proteasome inhibition.

Proteasome dysfunction has been demonstrated in Parkinson disease (PD), and proteasome inhibitors have been shown to induce degeneration of dopaminergic neurons in vitro and in vivo. The mechanism whereby proteasome dysfunction leads to dopaminergic cell death, however, is unknown. In this study, we show that proteasome inhibition in both PC12 cells and dopaminergic neurons derived from embryonic stem cells is associated with mitochondrial membrane permeabilization, activation of caspase-3, and nuclear changes consistent with apoptosis. Prior to the emergence of apoptotic features, we found that proteasome inhibition induced increased levels of phosphorylated p53. Inhibition of p53 by pifithrin-alpha or by RNA interference prevented mitochondrial membrane permeabilization and cytotoxicity. There was no increase in p53 mRNA in proteasome-inhibited cells, suggesting that p53 was increased in a transcription-independent manner. Further, there was no increase in Puma or Bax mRNA and p53 co-immunoprecipitated with Bcl-xL and Mdm2. These findings suggest that p53 mediates cell death by way of a direct mitochondrial effect in this model. We also observed increased levels of phosphorylated p53 in dopamine neurons of the substantia nigra pars compacta of mice following systemic administration of a proteasome inhibitor. These changes preceded degeneration of dopaminergic neurons. Increased phosphorylated p53 was also demonstrated in the substantia nigra pars compacta of post-mortem PD brains. These results suggest that abnormalities in p53 signaling play a role in dopaminergic cell death induced by proteasome inhibition and may be relevant to neurodegeneration in PD.

Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog.

The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur.

Early single cell bifurcation of pro- and antiapoptotic states during oxidative stress.

In a population of cells undergoing oxidative stress, an individual cell either succumbs to apoptotic cell death or maintains homeostasis and survives. Exposure of PC-12-D(2)R cells to 200 microm hydrogen peroxide (H(2)O(2)) induces apoptosis in about half of cells after 24 h. After 1-h exposure to 200 microm H(2)O(2), both antiapoptotic extracellular regulated kinase (ERK) phosphorylation and pro-apoptotic Ser-15-p53 phosphorylation are observed. Microarray and real-time PCR assays of gene expression after H(2)O(2) exposure identified several transcripts, including egr1, that are rapidly induced downstream of ERK. Single cell analysis of egr1 induction and of phospho-ERK and phospho-p53 formation revealed the presence of two distinct cellular programs. Whereas the proportion of cells activating ERK versus p53 at 1 h depended on H(2)O(2) concentration, individual cells showed exclusively either phospho-p53 formation or activation of ERK and egr1 induction. Exposure to H(2)O(2) for 1 h also elicited these two non-overlapping cellular responses in both dopaminergic SN4741 cells and differentiated postmitotic PC-12-D(2)R cells. Repressing p53 with pifithrin-alpha or small interfering RNA increased ERK phosphorylation by H(2)O(2), indicating that p53-dependent suppression of ERK activity may contribute to the bi-stable single cell responses observed. By 24 h, the subset of cells in which ERK activity was suppressed exhibit caspase 3 activation and the nuclear condensation characteristic of apoptosis. These studies suggest that the individual cell rapidly and stochastically processes the oxidative stress stimulus, leading to an all-or-none cytoprotective or pro-apoptotic signaling response.