ABSTRACT
Complement hyperactivation, angiopathic thrombosis and protein-losing enteropathy (CHAPLE disease) is a lethal disease caused by genetic loss of the complement regulatory protein CD55, leading to overactivation of complement and innate immunity together with immunodeficiency due to immunoglobulin wasting in the intestine. We report in vivo human data accumulated using the complement C5 inhibitor eculizumab for the medical treatment of patients with CHAPLE disease. We observed cessation of gastrointestinal pathology together with restoration of normal immunity and metabolism. We found that patients rapidly renormalized immunoglobulin concentrations and other serum proteins as revealed by aptamer profiling, re-established a healthy gut microbiome, discontinued immunoglobulin replacement and other treatments and exhibited catch-up growth. Thus, we show that blockade of C5 by eculizumab effectively re-establishes regulation of the innate immune complement system to substantially reduce the pathophysiological manifestations of CD55 deficiency in humans.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Complement Activation/drug effects , Complement C5/antagonists & inhibitors , Complement Inactivating Agents/therapeutic use , Energy Metabolism/drug effects , Hypoproteinemia/drug therapy , Immunity, Innate/drug effects , Protein-Losing Enteropathies/drug therapy , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/pharmacokinetics , Biomarkers/blood , CD55 Antigens/deficiency , CD55 Antigens/genetics , Complement C5/metabolism , Complement Inactivating Agents/adverse effects , Complement Inactivating Agents/pharmacokinetics , Genetic Predisposition to Disease , Humans , Hypoproteinemia/genetics , Hypoproteinemia/immunology , Hypoproteinemia/metabolism , Mutation , Phenotype , Protein-Losing Enteropathies/genetics , Protein-Losing Enteropathies/immunology , Protein-Losing Enteropathies/metabolism , Treatment OutcomeABSTRACT
Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.
Subject(s)
Biomarkers , Drug Development , Metabolism, Inborn Errors , Humans , Biomarkers/metabolism , Metabolism, Inborn Errors/drug therapy , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Drug Development/methods , Enzyme Assays/methods , Animals , Enzyme Replacement Therapy/methodsABSTRACT
The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and ß-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.
Subject(s)
Proprotein Convertases/chemistry , Receptors, LDL/chemistry , Receptors, LDL/metabolism , Serine Endopeptidases/chemistry , Down-Regulation , Humans , Hydrogen-Ion Concentration , Models, Molecular , Proprotein Convertase 9 , Proprotein Convertases/metabolism , Protein Binding , Protein Structure, Tertiary , Proteolysis , Serine Endopeptidases/metabolism , Surface Plasmon ResonanceABSTRACT
Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.
Subject(s)
Cholesterol, LDL/blood , Immunization , Proprotein Convertases/immunology , Serine Endopeptidases/immunology , Animals , Antibodies/immunology , Female , HEK293 Cells , Humans , Liver/metabolism , Mice , Proprotein Convertase 9 , Proprotein Convertases/metabolism , Receptors, LDL/metabolism , Serine Endopeptidases/metabolismABSTRACT
The objective of this study was to assess the relationship between factor XI (FXI) deficiency and the risks of bleeding and cardiovascular (CV) events. We conducted a retrospective cohort study using data from Maccabi Healthcare Services (MHS). We identified adults with FXI deficiency (severe: <15%, partial: 15 to <50%, any deficiency: <50%) that had been tested for FXI between 2007 and 2018 and matched to patients from the general MHS population. We estimated 10-year risks of outcomes using the Kaplan-Meier approach. Using Cox proportional hazards regression, we compared outcomes among patients with versus without FXI deficiency. Less than 10% of patients tested for FXI activity had activity levels <50% (mean age: 39 years; 72.2% females). Compared with the general population, patients with any FXI deficiency were at higher risk of severe bleeding (adjusted hazard ratio [aHR]: 2.56, 95% confidence interval [CI]: 1.13-5.81; 10-year risk: 1.90%, 95% CI: 0.50-3.20% vs. 0.90%, 95% CI: 0.50-1.30%) and clinically relevant nonsevere bleeding (CRNSB) (aHR: 1.45, 95% CI: 1.08-1.97; 10-year risk: 11.60%, 95% CI: 8.30-14.80% vs. 9.20%, 95% CI: 8.00-10.40%). Severe FXI deficiency was associated with a greater risk of CRNSB. While few CV events (N = 2) and venous thromboembolisms (VTE) (N = 1) were observed in the FXI overall deficient group, there was a nonsignificant negative association between any FXI deficiency and CV events (aHR: 0.55; 95% CI: 0.13-2.36) and VTEs (aHR: 0.45; 95% CI: 0.06-3.47). Overall FXI deficiency was associated with an increased risk of severe bleeding and CRNSB. Further research is warranted to explore the lower risk of CV and VTE among patients with FXI deficiency compared with the general population.
Subject(s)
Factor XI Deficiency , Venous Thromboembolism , Venous Thrombosis , Adult , Factor XI , Factor XI Deficiency/complications , Female , Hemorrhage/complications , Hemorrhage/epidemiology , Humans , Male , Retrospective Studies , Venous Thromboembolism/complications , Venous Thromboembolism/diagnosis , Venous Thromboembolism/epidemiology , Venous Thrombosis/complicationsABSTRACT
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Subject(s)
Cholesterol, LDL/blood , Immunoglobulin Fab Fragments/pharmacology , Receptors, LDL/chemistry , Serine Endopeptidases/immunology , Animals , Antibodies, Monoclonal/metabolism , Binding Sites , Cholesterol Ester Transfer Proteins/metabolism , Fluoroimmunoassay , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Macaca mulatta , Male , Mice , Mice, Transgenic , Proprotein Convertase 9 , Proprotein Convertases , Receptors, LDL/metabolism , Serine Endopeptidases/chemistryABSTRACT
PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.
Subject(s)
Antibodies, Monoclonal/pharmacology , Immunoglobulin Fab Fragments/pharmacology , Lipoproteins, LDL/metabolism , Receptors, LDL/metabolism , Serine Endopeptidases/metabolism , Amino Acid Substitution , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Hep G2 Cells , Humans , Hypercholesterolemia/drug therapy , Hypercholesterolemia/genetics , Hypercholesterolemia/immunology , Hypercholesterolemia/metabolism , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Lipoproteins, LDL/genetics , Lipoproteins, LDL/immunology , Mutagenesis, Site-Directed , Proprotein Convertase 9 , Proprotein Convertases , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, LDL/genetics , Receptors, LDL/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/immunologyABSTRACT
Insulin resistance and metabolic syndrome are rapidly expanding public health problems. Acting through the PI3K/Akt pathway, insulin and insulin-like growth factor-1 (IGF-1) inactivate FoxO transcription factors, a class of highly conserved proteins important in numerous physiological functions. However, even as FoxO is a downstream target of insulin, FoxO factors also control upstream signaling elements governing insulin sensitivity and glucose metabolism. Here, we report that sustained activation of either FoxO1 or FoxO3 in cardiac myocytes increases basal levels of Akt phosphorylation and kinase activity. FoxO-activated Akt directly interacts with and phosphorylates FoxO, providing feedback inhibition. We reported previously that FoxO factors attenuate cardiomyocyte calcineurin (PP2B) activity. We now show that calcineurin forms a complex with Akt and inhibition of calcineurin enhances Akt phosphorylation. In addition, FoxO activity suppresses protein phosphatase 2A (PP2A) and disrupts Akt-PP2A and Akt-calcineurin interactions. Repression of Akt-PP2A/B interactions and phosphatase activities contributes, at least in part, to FoxO-dependent increases in Akt phosphorylation and kinase activity. Resveratrol, an activator of Sirt1, increases the transcriptional activity of FoxO1 and triggers Akt phosphorylation in heart. Importantly, FoxO-mediated increases in Akt activity diminish insulin signaling, as manifested by reduced Akt phosphorylation, reduced membrane translocation of Glut4, and decreased insulin-triggered glucose uptake. Also, inactivation of the gene coding for FoxO3 enhances insulin-dependent Akt phosphorylation. Taken together, this study demonstrates that changes in FoxO activity have a dose-responsive repressive effect on insulin signaling in cardiomyocytes through inhibition of protein phosphatases, which leads to altered Akt activation, reduced insulin sensitivity, and impaired glucose metabolism.
Subject(s)
Forkhead Transcription Factors/metabolism , Insulin Resistance , Myocytes, Cardiac/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Animals , Calcineurin/metabolism , Forkhead Transcription Factors/pharmacology , Heart , Insulin/pharmacology , Mice , Myocytes, Cardiac/drug effects , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Phosphatase 2/metabolism , RatsABSTRACT
Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.
Subject(s)
Biomarkers, Pharmacological/blood , Blood Chemical Analysis , Blood Proteins/analysis , Animals , Anticoagulants/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Predictive Value of Tests , Reproducibility of ResultsABSTRACT
BACKGROUND: Cellular hypertrophy requires coordinated regulation of progrowth and antigrowth mechanisms. In cultured neonatal cardiomyocytes, Foxo transcription factors trigger an atrophy-related gene program that counters hypertrophic growth. However, downstream molecular events are not yet well defined. METHODS AND RESULTS: Here, we report that expression of either Foxo1 or Foxo3 in cardiomyocytes attenuates calcineurin phosphatase activity and inhibits agonist-induced hypertrophic growth. Consistent with these results, Foxo proteins decrease calcineurin phosphatase activity and repress both basal and hypertrophic agonist-induced expression of MCIP1.4, a direct downstream target of the calcineurin/NFAT pathway. Furthermore, hearts from Foxo3-null mice exhibit increased MCIP1.4 abundance and a hypertrophic phenotype with normal systolic function at baseline. Together, these results suggest that Foxo proteins repress cardiac growth at least in part through inhibition of the calcineurin/NFAT pathway. Given that hypertrophic growth of the heart occurs in multiple contexts, our findings also suggest that certain hypertrophic signals are capable of overriding the antigrowth program induced by Foxo. Consistent with this, multiple hypertrophic agonists triggered inactivation of Foxo proteins in cardiomyocytes through a mechanism requiring the PI3K/Akt pathway. In addition, both Foxo1 and Foxo3 are phosphorylated and consequently inactivated in hearts undergoing hypertrophic growth induced by hemodynamic stress. CONCLUSIONS: This study suggests that inhibition of the calcineurin/NFAT signaling cascade by Foxo and release of this repressive action by the PI3K/Akt pathway are important mechanisms whereby Foxo factors govern cell growth in the heart.
Subject(s)
Cardiomegaly/physiopathology , Forkhead Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Angiotensin II/pharmacology , Animals , Calcineurin/physiology , Calcium-Binding Proteins , Cardiomegaly/genetics , Cardiomegaly/pathology , Cells, Cultured , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Gene Expression Regulation/physiology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Nerve Tissue Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/drug effects , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.
Subject(s)
Biological Assay/methods , Programmed Cell Death 1 Receptor/analysis , Recombinant Proteins/analysis , Antibodies, Monoclonal/administration & dosage , Case-Control Studies , HEK293 Cells , Humans , Limit of Detection , Neoplasms/blood , NivolumabABSTRACT
The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.
Subject(s)
Antibodies, Neutralizing/immunology , Biological Assay , Biomarkers/analysis , Biopharmaceutics/organization & administration , Biotechnology/organization & administration , HumansABSTRACT
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Anticholesteremic Agents/therapeutic use , Cholesterol, LDL/blood , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Immunization, Passive , Metabolic Syndrome/therapy , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/immunology , Serine Endopeptidases/immunology , Simvastatin/therapeutic use , Sterol Regulatory Element Binding Proteins/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antibody Affinity , Anticholesteremic Agents/administration & dosage , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cholesterol Ester Transfer Proteins/genetics , Cholesterol Ester Transfer Proteins/metabolism , Gene Expression Profiling , Hep G2 Cells/drug effects , Hep G2 Cells/metabolism , Hepatocytes/metabolism , Humans , Lipid Metabolism/genetics , Liver/drug effects , Liver/metabolism , Macaca mulatta , Metabolic Syndrome/drug therapy , Metabolic Syndrome/genetics , Mice , Mice, Transgenic , Proprotein Convertase 9 , Proprotein Convertases/biosynthesis , Proprotein Convertases/genetics , RNA, Messenger/metabolism , Receptors, LDL/biosynthesis , Receptors, LDL/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Simvastatin/administration & dosageABSTRACT
In response to changes in workload, the heart grows or shrinks. Indeed, the myocardium is capable of robust and rapid structural remodeling. In the setting of normal, physiological demand, the heart responds with hypertrophic growth of individual cardiac myocytes, a process that serves to maintain cardiac output and minimize wall stress. However, disease-related stresses, such as hypertension or myocardial infarction, provoke a series of changes that culminate in heart failure and/or sudden death. At the other end of the spectrum, cardiac unloading, such as occurs with prolonged bed rest or weightlessness, causes the heart to shrink. In recent years, considerable strides have been made in deciphering the molecular and cellular events governing pro- and anti-growth events in the heart. Prominent among these mechanisms are those mediated by FoxO (Forkhead box-containing protein, O subfamily) transcription factors. In many cell types, these proteins are critical regulators of cell size, viability, and metabolism, and their importance in the heart is just emerging. Also in recent years, evidence has emerged for a pivotal role for autophagy, an evolutionarily conserved pathway of lysosomal degradation of damaged proteins and organelles, in cardiac growth and remodeling. Indeed, evidence for activated autophagy has been detected in virtually every form of myocardial disease. Now, it is clear that FoxO is an upstream regulator of both autophagy and the ubiquitin-proteasome system. Here, we discuss recent advances in our understanding of cardiomyocyte autophagy, its governance by FoxO, and the roles each of these plays in cardiac remodeling.
Subject(s)
Autophagy , Forkhead Transcription Factors/metabolism , Heart Failure/metabolism , Ventricular Remodeling , Cardiac Output , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Hypertrophy, Right Ventricular/metabolism , Myocytes, Cardiac/metabolism , Proteasome Endopeptidase Complex/metabolism , Risk Factors , Ubiquitin-Protein Ligases/metabolismABSTRACT
Adenylyl cyclase (AC) type VIII has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. In the present study, we directly examined the role for the transcription factor CREB (cAMP response element binding protein) in regulating ACVIII expression by cloning a 5.2 kilobase region upstream of the translation start site of the mouse ACVIII gene. Analysis of this fragment revealed consensus elements for several transcription factors, including a canonical cAMP response element (CRE) in close proximity to the transcription initiation region. Next, ACVIII promoter activity was studied in two neural-derived cell lines and in primary cultures of rat striatal neurons. Activation of the cAMP pathway by forskolin treatment increased promoter activity, and a series of deletion and point mutants demonstrated that this activation is mediated specifically via the canonical CRE site. Gel shift assays confirmed that this site can bind CREB and several CREB family proteins. Further, activation of the ACVIII promoter by forskolin was potentiated by expression of a constitutively active form of CREB, CREB-VP16, whereas it was inhibited by expression of a dominant-negative form of CREB, A-CREB. Finally, over-expression of CREB in vivo, by viral-mediated gene transfer, induced ACVIII promoter activity in the brains of ACVIII-LacZ transgenic mice. These results suggest that the ACVIII gene is regulated by CREB in vitro and in vivo and that this regulation may contribute to CREB-dependent neural plasticity.
Subject(s)
Adenylyl Cyclases/genetics , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP/genetics , Promoter Regions, Genetic , Adenylyl Cyclases/metabolism , Animals , Base Sequence , Brain/metabolism , Colforsin/pharmacology , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation , Neuronal Plasticity , Neurons/drug effects , Neurons/physiology , PC12 Cells , RatsABSTRACT
The present study explored a possible role for RGS (regulators of G protein signalling) proteins in the long term actions of morphine in the locus coeruleus (LC), a brainstem region implicated in opiate physical dependence and withdrawal. Morphine influences LC neurons through activation of micro -opioid receptors, which, being Gi/o-linked, would be expected to be modulated by RGS proteins. We focused on several RGS subtypes that are known to be expressed in this brain region. Levels of mRNAs encoding RGS2, -3, -4, -5, -7, -8 and -11 are unchanged following chronic morphine, but RGS2 and -4 mRNA levels are increased 2-3-fold 6 h following precipitation of opiate withdrawal. The increases in RGS2 and -4 mRNA peak after 6 h of withdrawal and return to control levels by 24 h. Immunoblot analysis of RGS4 revealed a striking divergence between mRNA and protein responses in LC: protein levels are elevated twofold following chronic morphine and decrease to control values by 6 h of withdrawal. In contrast, levels of RGS7 and -11 proteins, the only other subtypes for which antibodies are available, were not altered by these treatments. Intracellular application of wild-type RGS4, but not a GTPase accelerating-deficient mutant of RGS4, into LC neurons diminished electrophysiological responses to morphine. The observed subtype- and time-specific regulation of RGS4 protein and mRNA, and the diminished morphine-induced currents in the presence of elevated RGS4 protein levels, indicate that morphine induction of RGS4 could contribute to aspects of opiate tolerance and dependence displayed by LC neurons.