RESUMO
In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).
Assuntos
Estudos de Associação Genética/métodos , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Mutação/genética , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Células Cultivadas , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to 2 patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients, rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.
Assuntos
Receptores de Trombopoetina , Trombocitopenia , Humanos , Receptores de Trombopoetina/genética , Trombocitopenia/etiologia , Fator de Transcrição STAT3/genética , Estudos Longitudinais , MutaçãoRESUMO
Disease relapse and treatment-induced immunotoxicity pose significant clinical challenges for patients with hematological cancers. Here, we reveal distinctive requirements for neutralizing TNF receptor ligands APRIL and BAFF and their receptor activity in MM and DLBCL, impacting protein translation and production in MM cells and modulating the translation efficiency of the ATM interactor (ATMIN/ACSIZ). Therapeutically, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as an inhibitor of APRIL and BAFF. While wild-type sBCMA-Fc effectively blocked APRIL signaling in MM, it lacked activity in DLBCL due to its weak BAFF binding. To expand the therapeutic utility of sBCMA-Fc, we engineered an affinity-enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) 4- and 500-fold stronger in binding to APRIL and BAFF, respectively. The mutant sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, we also demonstrated an adequate toxicity profile and on-target mechanism of action in nonhuman primate studies.
Assuntos
Linfoma Difuso de Grandes Células B , Mieloma Múltiplo , Animais , Fator Ativador de Células B/genética , Antígeno de Maturação de Linfócitos B/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/terapia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Transdução de Sinais , Proteína Transmembrana Ativadora e Interagente do CAML , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
We identify berberine (BBR), a compound isolated from a Chinese herb, as a new cholesterol-lowering drug. Oral administration of BBR in 32 hypercholesterolemic patients for 3 months reduced serum cholesterol by 29%, triglycerides by 35% and LDL-cholesterol by 25%. Treatment of hyperlipidemic hamsters with BBR reduced serum cholesterol by 40% and LDL-cholesterol by 42%, with a 3.5-fold increase in hepatic LDLR mRNA and a 2.6-fold increase in hepatic LDLR protein. Using human hepatoma cells, we show that BBR upregulates LDLR expression independent of sterol regulatory element binding proteins, but dependent on ERK activation. BBR elevates LDLR expression through a post-transcriptional mechanism that stabilizes the mRNA. Using a heterologous system with luciferase as a reporter, we further identify the 5' proximal section of the LDLR mRNA 3' untranslated region responsible for the regulatory effect of BBR. These findings show BBR as a new hypolipidemic drug with a mechanism of action different from that of statin drugs.
Assuntos
Anticolesterolemiantes/uso terapêutico , Berberina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Hipercolesterolemia/tratamento farmacológico , Receptores de LDL/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Berberina/química , Berberina/farmacologia , Northern Blotting , China , Colesterol/sangue , LDL-Colesterol/sangue , Cricetinae , Primers do DNA , Citometria de Fluxo , Humanos , Fígado/metabolismo , Plasmídeos/genética , Receptores de LDL/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangue , Células Tumorais CultivadasRESUMO
Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.
RESUMO
OBJECTIVE: In our previous studies that examined in vivo activities of oncostatin M (OM) in upregulation of hepatic LDL receptor (LDLR) expression, we observed reductions of LDL-cholesterol and triglyceride (TG) levels in OM-treated hyperlipidemic hamsters. Interestingly, the OM effect of lowering plasma TG was more pronounced than LDL-cholesterol reduction, suggesting additional LDLR-independent actions. Here, we investigated mechanisms underlying the direct TG-lowering effect of OM. METHODS AND RESULTS: We demonstrate that OM activates transcription of long-chain acyl-coenzymeA (CoA) synthetase isoforms 3 and 5 (ACSL3, ACSL5) in HepG2 cells through the extracellular signal-regulated kinase (ERK) signaling pathway. Increased acyl-CoA synthetase activities in OM-stimulated HepG2 cells and in livers of OM-treated hamsters are associated with decreased TG accumulation and increased fatty acid beta-oxidation. We further show that overexpression of ACSL3 or ACSL5 alone in the absence of OM led to fatty acid partitioning into beta-oxidation. Importantly, we demonstrate that transfection of siRNAs targeted to ACSL3 and ACSL5 abrogated the enhancing effect of OM on fatty acid oxidation in HepG2 cells. CONCLUSIONS: These new findings identify ACSL3 and ACSL5 as OM-regulated genes that function in fatty acid metabolism and suggest a novel cellular mechanism by which OM directly lowers the plasma TG in hyperlipidemic animals through stimulating the transcription of ACSL specific isoforms in the liver.
Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Hipertrigliceridemia/metabolismo , Fígado/metabolismo , Oncostatina M/metabolismo , Ativação Transcricional , Triglicerídeos/metabolismo , Animais , Linhagem Celular Tumoral , Colesterol na Dieta , Coenzima A Ligases/genética , Cricetinae , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácidos Graxos/sangue , Regulação Enzimológica da Expressão Gênica , Humanos , Hipertrigliceridemia/induzido quimicamente , Hipertrigliceridemia/enzimologia , Hipertrigliceridemia/genética , Metabolismo dos Lipídeos/genética , Fígado/enzimologia , Oxirredução , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Triglicerídeos/sangueRESUMO
OM (oncostatin M) activates the human LDLR [LDL (low-density lipoprotein) receptor] gene transcription in HepG2 cells through the SIRE (sterol-independent regulatory element) of LDLR promoter. The SIRE sequence consists of a C/EBP (CCAAT/enhancer-binding protein)-binding site and a CRE (cAMP-response element). Our previous studies [Zhang, Ahlborn, Li, Kraemer and Liu (2002) J. Lipid Res. 43, 1477-1485; Zhang, Lin, Abidi, Thiel and Liu (2003) J. Biol. Chem. 278, 44246-44254] have demonstrated that OM transiently induces EGR-1 (early growth response gene product 1) expression and EGR-1 activates LDLR transcription primarily through a protein-protein interaction with C/EBPbeta, which serves as a co-activator of EGR-1. In the present study, we examined the direct role of C/EBPbeta as a transactivator in OM-regulated LDLR gene transcription independent of EGR-1. We show that OM induces C/EBPbeta expression with kinetics slower than EGR-1 induction. A significant increase in C/EBPbeta protein level is detected by 2 h of OM treatment and remains elevated for 24 h. Chromatin immunoprecipitation assays demonstrate that the amount of C/EBPbeta bound to the LDLR SIRE sequence is increased 2.8-fold of control by 2 h of OM treatment, reached the highest level of 8-fold by 4 h, and slowly declined thereafter. To further examine the requirement of C/EBPbeta in OM-stimulated LDLR expression, we developed a His-tagged dominant-negative mutant of C/EBPbeta (His-C/EBPbeta-P4; where P4 is plasmid 4 in our mutation series), consisting of the DNA-binding and leucine zipper domains of C/EBPbeta (amino acids 246-345). Expression of His-C/EBPbeta-P4 in HepG2 cells significantly diminishes the OM-induced increase in LDLR promoter activity and the elevation of endogenous LDLR mRNA expression. Taken together, these new findings identify C/EBPbeta as an OM-induced transactivator in LDLR gene transcription and provide a better understanding of the molecular mechanism underlying the sterol-independent regulation of LDLR expression.
Assuntos
Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/genética , Citocinas/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Receptores de LDL/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/genética , Humanos , Cinética , Neoplasias Hepáticas/genética , Mutagênese Sítio-Dirigida , Oncostatina M , Regiões Promotoras Genéticas , Receptores de LDL/efeitos dos fármacos , Receptores de LDL/genética , Elementos Reguladores de Transcrição , Esteróis , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , Células Tumorais CultivadasRESUMO
OBJECTIVE: Our recent studies identified berberine (BBR) as a novel cholesterol-lowering drug that upregulates low-density lipoprotein (LDL) receptor expression through mRNA stabilization. Here, we investigated mechanisms underlying regulatory effects of BBR on LDL receptor (LDLR) messenger. METHODS AND RESULTS: We show that the extracellular signal-regulated kinase (ERK) signaling pathway is used primarily by BBR to attenuate the decay of LDLR mRNA in HepG2 cells. Using different reporter constructs, we demonstrate that BBR affects LDLR mRNA stability entirely through 3' untranslated region (UTR) in an ERK-dependent manner, and this stabilizing effect is more prominent in liver-derived cells than nonhepatic cell lines. In contrast to BBR, the mRNA stabilizing effect of bile acid chenodeoxycholic acid is mediated through the LDLR coding sequence, whereas the 5'UTR, 3'UTR, and the coding sequence of LDLR mRNA are all implicated in the action of phorbol 12-myristate 13-acetate. By performing UV cross-linking and SDS-PAGE, we identify 2 cytoplasmic proteins of 52 and 42 kDa that specifically bind to the LDLR 3'UTR in BBR-inducible and ERK-dependent manners. CONCLUSIONS: These new findings demonstrate that the BBR-induced stabilization of LDLR mRNA is mediated by the ERK signaling pathway through interactions of cis-regulatory sequences of 3'UTR and mRNA binding proteins that are downstream effectors of this signaling cascade.
Assuntos
Berberina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Medicina Herbária , Hipercolesterolemia/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores de LDL/genética , Regiões 3' não Traduzidas/fisiologia , Ácidos e Sais Biliares/farmacologia , Carcinógenos/farmacologia , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Citomegalovirus/genética , Humanos , Hipercolesterolemia/metabolismo , Técnicas In Vitro , Neoplasias Hepáticas , Sistema de Sinalização das MAP Quinases/fisiologia , Plasmídeos , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR) has been implicated in multiple types of human cancers, including multiple myeloma (MM). Through an in silico drug discovery approach based on protein-compound virtual docking, we identified the anthracycline antibiotic doxorubicin as an in vitro and in vivo inhibitor of XBP1 activation, a previously unknown activity for this widely utilized cancer chemotherapeutic drug. Through a series of mechanistic and phenotypic studies, we showed that this novel activity of doxorubicin was not due to inhibition of topoisomerase II (Topo II). Consistent with its inhibitory activity on the IRE1α-XBP1 branch of the UPR, doxorubicin displayed more potent cytotoxicity against MM cell lines than other cancer cell lines that have lower basal IRE1α-XBP1 activity. In addition, doxorubicin significantly inhibited XBP1 activation in CD138(+) tumor cells isolated from MM patients. Our findings suggest that the UPR-modulating activity of doxorubicin may be utilized clinically to target IRE1α-XBP1-dependent tumors such as MM.
Assuntos
Doxorrubicina/farmacologia , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/química , Etoposídeo/química , Etoposídeo/farmacologia , Humanos , Splicing de RNA/genética , Inibidores da Topoisomerase/farmacologiaRESUMO
Previous studies identified the putative SCAP ligands including compound GW707 as a new class of up-regulators of LDL receptor (LDLR) transcription by activation of the sterol-regulatory element binding proteins (SREBP). These compounds increase LDLR expression in hepatoma cells in vitro and lower plasma LDL-c in hamsters. However, it is unknown, what signaling pathways are utilized by these agents that lead to the activation of LDLR transcription. Here, we report that the ERK signaling cascade is critically involved in GW707-mediated induction of LDLR expression. We show that: a) blocking ERK activation with U0126, the inhibitor of ERK upstream kinase MEK, completely abolishes the inducing effects of GW707 on LDLR promoter activity, LDLR mRNA expression, and DiI-LDL uptake in HepG2 cells; b) treating HepG2 cells with GW707 induces a dose-dependent conversion of SREBP-2 from the 125 kDa precursor form to the 68 kDa activated form and U0126 does not inhibit this cleavage process, but U0126 significantly reduces the total amount of SREBP-2 protein in GW707-treated cells without affecting the expression levels of other proteins involving in SREBP processing; and c) inhibition of ERK signaling pathway has no effects on the promoter activity or mRNA expression of SREBP-2. Collectively, these new findings establish an important role of ERK signaling pathway in SCAP ligand-induced transcription of LDLR and imply that the protein synthesis or turnover rate of SREBP-2 may be regulated by ERK.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Receptores de LDL/genética , Esteroides/farmacologia , Ativação Transcricional , Butadienos/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Nitrilas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Esteroides/antagonistas & inibidores , Esteroides/metabolismoRESUMO
Excessive oxidative stress and associated macromolecular damage are considered to be key features of aging, and appear to contribute to the age-related decline in steroid hormone production in adrenal and testicular Leydig cells. The current studies were initiated to examine the potential mechanism by which excessive oxidative stress during aging attenuates the functional expression of the oxidant-responsive transcription factor Activator protein-1. Chronic oxidative stress was induced in vivo by maintaining groups of rats on a diet deficient in vitamin E for 6 months. Plasma, liver, and adrenal tissues from vitamin E-deficient animals had negligible levels of this vitamin and showed high susceptibility to in vitro lipid peroxidation. Synthesis and secretion of corticosterone in response to corticotropin (ACTH), dibutyryl-cAMP, or 20alpha-hydroxycholesterol in vitro was significantly reduced in adrenocortical cells prepared cells from rats deficient in vitamin E. AP-1 DNA-binding activity was diminished approximately 55 % in adrenal extracts from vitamin E-deficient rats with no corresponding change in the binding activity of SP-1. The vitamin E deficiency-mediated loss of AP-1 activity was not due to an alteration in the dimeric composition of constituent proteins, but rather to a general down-regulation of steady-state levels of members of the Fos and Jun families of proteins. Interestingly, vitamin E deficiency also reduced the expression of the redox-regulated Ref-1 protein. Collectively these data demonstrate that chronic oxidative stress specifically down-regulates essential components of the AP-1 transcription factor complex, and suggest that aberrancies in AP-1 expression may adversely affect processes crucial for intracellular cholesterol transport and steroid hormone production.
Assuntos
Glândulas Suprarrenais/metabolismo , Estresse Oxidativo/fisiologia , Esteroides/biossíntese , Fator de Transcrição AP-1/metabolismo , Deficiência de Vitamina E/complicações , Animais , Transporte Biológico , Colesterol/metabolismo , DNA/metabolismo , Dimerização , Masculino , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-jun/análise , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/análise , alfa-Tocoferol/análiseRESUMO
The 3'untranslated region (UTR) of human LDL receptor (LDLR) mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover and mediates the stabilization induced by berberine (BBR). However, the identities of the specific RNA binding proteins involved in the regulation of LDLR mRNA stability at the steady state level or upon BBR treatment are unknown. By conducting small interfering RNA library screenings, biotinylated RNA pull-down, mass spectrometry analysis, and functional assays, we now identify heterogeneous nuclear ribonucleoprotein D (hnRNP D), hnRNP I, and KH-type splicing regulatory protein (KSRP) as key modulators of LDLR mRNA stability in liver cells. We show that hnRNP D, I, and KSRP interact with AREs of the LDLR 3'UTR with sequence specificity. Silencing the expression of these proteins increased LDLR mRNA and protein levels. We further demonstrate that BBR-induced mRNA stabilization involves hnRNP I and KSRP, as their cellular depletions abolished the BBR effect and BBR treatment reduced the binding of hnRNP I and KSRP to the LDLR mRNA 3'UTR. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE binding proteins, including hnRNP D, hnRNP I, and KSRP. Our results suggest that interference with the ability of destabilizing ARE binding proteins to interact with LDLR-ARE motifs is likely a mechanism for regulating LDLR expression by compounds such as BBR and perhaps others.
Assuntos
Sequência de Bases , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de LDL/genética , Regiões 3' não Traduzidas , Berberina/metabolismo , Linhagem Celular , Genes Reporter , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de LDL/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis approximately 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging.
Assuntos
Corticosteroides/deficiência , Envelhecimento/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Córtex Suprarrenal/enzimologia , Corticosteroides/biossíntese , Fatores Etários , Animais , Ativação Enzimática , Inibidores Enzimáticos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sequestradores de Radicais Livres , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Peroxidação de Lipídeos , Sistema de Sinalização das MAP Quinases , Masculino , Metaloporfirinas , Oxidantes , Fosforilação , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H(2)O(2) or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable superoxide-dismutase/catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide- and H(2)O(2)-mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt(2)cAMP and Bt(2)cAMP+hHDL(3)-stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPKalpha isoform, along with relatively low-level expression of p38 MAPKgamma. By contrast, little or no expression was detected for p38 MAPKbeta and p38 MAPKdelta isoforms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPKalpha or dnp38 MAPKbeta construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPKgamma or dnp38 MAPKdelta construct. These results indicate that activated p38 MAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.
Assuntos
20-alfa-Di-Hidroprogesterona/biossíntese , Glândulas Suprarrenais/metabolismo , Sistema de Sinalização das MAP Quinases , Estresse Oxidativo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Linhagem Celular Tumoral , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacologia , MAP Quinase Quinase 3/fisiologia , MAP Quinase Quinase 6/fisiologia , Camundongos , Fosfoproteínas/genética , Fosforilação , Superóxidos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Our previous studies have identified berberine (BBR), an alkaloid isolated from the Chinese herb huanglian, as a unique cholesterol-lowering drug that upregulates hepatic low density lipoprotein receptor (LDLR) expression through a mechanism of mRNA stabilization. Here, we demonstrate that the root extract of goldenseal, a BBR-containing medicinal plant, is highly effective in upregulation of liver LDLR expression in HepG2 cells and in reducing plasma cholesterol and low density lipoprotein cholesterol (LDL-c) in hyperlipidemic hamsters, with greater activities than the pure compound BBR. By conducting bioassay-driven semipurifications, we demonstrate that the higher potency of goldenseal is achieved through concerted actions of multiple bioactive compounds in addition to BBR. We identify canadine (CND) and two other constituents of goldenseal as new upregulators of LDLR expression. We further show that the activity of BBR on LDLR expression is attenuated by multiple drug resistance-1 (MDR1)-mediated efflux from liver cells, whereas CND is resistant to MDR1. This finding defines a molecular mechanism for the higher activity of CND than BBR. We also provide substantial evidence to show that goldenseal contains natural MDR1 antagonist(s) that accentuate the upregulatory effect of BBR on LDLR mRNA expression. These new findings identify goldenseal as a natural LDL-c-lowering agent, and our studies provide a molecular basis for the mechanisms of action.
Assuntos
LDL-Colesterol/sangue , Hydrastis/química , Plantas Medicinais/química , Animais , Berberina/farmacologia , Linhagem Celular , Cricetinae , Medicina Herbária , Humanos , Fígado/efeitos dos fármacos , Masculino , Estrutura Molecular , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , RNA Interferente PequenoRESUMO
Our previous studies have demonstrated the activity of oncostatin M (OM) in stimulating the transcription of the human LDL receptor (LDLR) gene in HepG2 cells through a sterol-independent regulatory mechanism. The current studies were designed to determine whether this in vitro property of OM could be recapitulated in vivo to increase LDLR expression in cholesterol-loaded livers and consequently decrease plasma levels of LDL-cholesterol (LDL-C) and total cholesterol (TC) using hypercholesterolemic hamsters as an experimental model. We show that administration of human recombinant OM for 7 days in hamsters fed a high-fat diet significantly reduced plasma levels of TC, LDL-C, and triglyceride in dose- and time-dependent manners. This lipid-lowering effect was associated with increased hepatic LDLR mRNA expression, as determined by quantitative real-time RT-PCR. Additionally, hepatic fat storage and cholesterol content in the hypercholesterolemic animals were substantially reduced by OM treatment. As a consequence, the increased aminotransferase levels in the high-fat diet-fed hamsters were normalized nearly to baseline values. These results not only corroborate the in vitro finding of OM in the regulation of LDLR but also, for the first time, demonstrate that OM has a strong lipid-lowering effect under in vivo conditions in which the levels of circulating LDL-C are high and liver LDLR transcription is repressed.
Assuntos
Regulação da Expressão Gênica , Lipídeos/sangue , Peptídeos/fisiologia , Animais , Linhagem Celular , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Cricetinae , Feminino , Humanos , Cinética , Metabolismo dos Lipídeos , Fígado/metabolismo , Oncostatina M , Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transaminases/metabolismo , Triglicerídeos/metabolismoRESUMO
The sterol-independent regulatory element (SIRE) of the LDL receptor (LDLR) promoter mediates oncostatin M (OM)-induced transcription of the LDLR gene through a cholesterol-independent pathway. Our prior studies have detected specific associations of the zinc finger transcription factor Egr1 with the SIRE sequence in OM-stimulated HepG2 cells. Because the SIRE motif is composed of a c/EBP binding site and a cAMP response element, both of which are quite divergent from the classical GC-rich Egr1 recognition sequences, we hypothesized that Egr1 may regulate LDLR transcription through interacting with members of the c/EBP and CREB families. Here, we show that treating HepG2 cells with OM specifically leads to prominent increases of the levels of c/EBPbeta and Egr1 bound to the LDLR promoter in vivo. In vitro, the binding of Egr1 to the SIRE sequence is weak, but is strikingly enhanced in the presence of HepG2 nuclear extract. Mammalian two-hybrid assays demonstrate that the N-terminal transactivation domain of Egr1 specifically interacts with c/EBPbeta but not with c/EBPalpha or CREB. The OM treatment further enhances this interaction, resulting in a large increase in the Egr1 transactivating activity. The direct protein to protein contact between Egr1 and c/EBPbeta is also demonstrated by co-immunoprecipitation experiments. Furthermore, we show that a mutation of the phosphorylation motif of c/EBPbeta diminished the OM-stimulated interaction of Egr1 and c/EBPbeta. Taken together, we provide strong evidence that Egr1 regulates LDLR transcription via a novel mechanism of protein-protein interaction with c/EBPbeta.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces , Receptores de LDL/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma Hepatocelular , Núcleo Celular/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Interações Medicamentosas , Proteína 1 de Resposta de Crescimento Precoce , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas de Imunoadsorção , Neoplasias Hepáticas , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oncostatina M , Peptídeos/farmacologia , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão , Elementos de Resposta/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Asbestos and its carcinogenic properties have been extensively documented. Asbestos exposure induces diverse cellular events associated with lung injury. Previously, we have shown that treatment with chrysotile shows significant alteration in phase I and phase II drug metabolizing enzyme system. In this study we have examined some potential mechanisms by which garlic treatment attenuates chrysotile-mediated pulmonary toxicity in rat. Female Wistar rats received an intratracheal instillation of 5 mg chrysotile (0.5 mL saline) as well as intragastric garlic treatment (1% body weight (v/w); 6 days per week). Effect of garlic treatment was evaluated after 1, 15, 30, 90, and 180 days by assaying aryl hydrocarbon hydroxylase (AHH), glutathione (GSH), glutathione S-transferase (GST), and production of thiobarbituric acid reactive substances (TBARS) in rat lung microsome. The results showed that AHH and TBARS formation were significantly reduced at day 90 and day 180 in chrysotile treated garlic cofed rats; GSH recovered 15 days later to the near normal level and GST elevated significantly after treatment of garlic as compared to chrysotile alone treated rat lung microsome. The data obtained shows that inhibition of AHH activity and induction of GST activity could be contributing factor in chrysotile-mediated pulmonary toxicity in garlic cofed rats. However, recovery of GSH and inhibition of TBARS formation by garlic and its constituent(s) showed that garlic may give protection by altering the drug metabolizing enzyme system.