RESUMO
Patients with end-stage liver disease exhibit progressive skeletal muscle atrophy, highlighting a negative crosstalk between the injured liver and muscle. Our study was to determine whether TGFß ligands function as the mediators. Acute or chronic liver injury was induced by a single or repeated administration of carbon tetrachloride. Skeletal muscle injury and repair was induced by intramuscular injection of cardiotoxin. Activin type IIB receptor (ActRIIB) ligands and growth differentiation factor 8 (Gdf8) were neutralized with ActRIIB-Fc fusion protein and a Gdf8-specific antibody, respectively. We found that acute hepatic injury induced rapid and adverse responses in muscle, which was blunted by neutralizing ActRIIB ligands. Chronic liver injury caused muscle atrophy and repair defects, which were prevented or reversed by inactivating ActRIIB ligands. Furthermore, we found that pericentral hepatocytes produce excessive Gdf8 in injured mouse liver and cirrhotic human liver. Specific inactivation of Gdf8 prevented liver injury-induced muscle atrophy, similar to neutralization of ActRIIB ligands. Inhibition of Gdf8 also reversed muscle atrophy in a treatment paradigm following chronic liver injury. Direct injection of exogenous Gdf8 protein into muscle along with acute focal muscle injury recapitulated similar dysregulated muscle regeneration as that observed with liver injury. The results indicate that injured liver negatively communicate with the muscle largely via Gdf8. Unexpectedly, inactivation of Gdf8 simultaneously ameliorated liver fibrosis in mice following chronic liver injury. In vitro, Gdf8 induced human hepatic stellate (LX-2) cells to form a septa-like structure and stimulated expression of profibrotic factors. Our findings identified Gdf8 as a novel hepatomyokine contributing to injured liver-muscle negative crosstalk along with liver injury progression.
RESUMO
Activins are a subgroup of the TGFß superfamily of growth and differentiation factors, dimeric in nature and consisting of two inhibin beta subunits linked via a disulfide bridge. Canonical activin signaling occurs through Smad2/3, with negative feedback initiated by Smad6/7 following signal transduction, which binds activin type I receptor preventing phosphorylation of Smad2/3 and activation of downstream signaling. In addition to Smad6/7, other inhibitors of activin signaling have been identified as well, including inhibins (dimers of an inhibin alpha and beta subunit), BAMBI, Cripto, follistatin, and follistatin-like 3 (fstl3). To date, activins A, B, AB, C, and E have been identified and isolated in mammals, with activin A and B having the most characterization of biological activity. Activin A has been implicated as a regulator of several important functions of liver biology, including hepatocyte proliferation and apoptosis, ECM production, and liver regeneration; the role of other subunits of activin in liver physiology are less understood. There is mounting data to suggest a link between dysregulation of activins contributing to various hepatic diseases such as inflammation, fibrosis, and hepatocellular carcinoma, and emerging studies demonstrating the protective and regenerative effects of inhibiting activins in mouse models of liver disease. Due to their importance in liver biology, activins demonstrate utility as a therapeutic target for the treatment of hepatic diseases such as cirrhosis, NASH, NAFLD, and HCC; further research regarding activins may provide diagnostic or therapeutic opportunity for those suffering from various liver diseases.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Folistatina , Ativinas/fisiologia , Receptores de Ativinas , MamíferosRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown. METHODS: Hepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development. RESULTS: Hepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH. CONCLUSIONS: Despite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset. IMPACT AND IMPLICATIONS: Liver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer.
Assuntos
Carcinoma Hepatocelular , Ferroptose , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Hepatopatia Gordurosa não Alcoólica/complicações , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/complicações , Fator 4 Ativador da Transcrição/metabolismo , Cistina/metabolismo , Inflamação/complicações , Carcinogênese , Glutamatos , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismoRESUMO
The role of activin B, a transforming growth factor ß (TGFß) superfamily cytokine, in liver health and disease is largely unknown. We aimed to investigate whether activin B modulates liver fibrogenesis. Liver and serum activin B, along with its analog activin A, were analyzed in patients with liver fibrosis from different etiologies and in mouse acute and chronic liver injury models. Activin B, activin A, or both was immunologically neutralized in mice with progressive or established carbon tetrachloride (CCl4 )-induced liver fibrosis. Hepatic and circulating activin B was increased in human patients with liver fibrosis caused by several liver diseases. In mice, hepatic and circulating activin B exhibited persistent elevation following the onset of several types of liver injury, whereas activin A displayed transient increases. The results revealed a close correlation of activin B with liver injury regardless of etiology and species. Injured hepatocytes produced excessive activin B. Neutralizing activin B largely prevented, as well as improved, CCl4 -induced liver fibrosis, which was augmented by co-neutralizing activin A. Mechanistically, activin B mediated the activation of c-Jun-N-terminal kinase (JNK), the induction of inducible nitric oxide synthase (iNOS) expression, and the maintenance of poly (ADP-ribose) polymerase 1 (PARP1) expression in injured livers. Moreover, activin B directly induced a profibrotic expression profile in hepatic stellate cells (HSCs) and stimulated these cells to form a septa structure. Conclusions: We demonstrate that activin B, cooperating with activin A, mediates the activation or expression of JNK, iNOS, and PARP1 and the activation of HSCs, driving the initiation and progression of liver fibrosis.
Assuntos
Tetracloreto de Carbono , Ribose , Ativinas , Difosfato de Adenosina/efeitos adversos , Animais , Tetracloreto de Carbono/toxicidade , Humanos , Cirrose Hepática/induzido quimicamente , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Ribose/efeitos adversos , Fator de Crescimento Transformador beta/efeitos adversosRESUMO
BACKGROUND: Type 2 diabetes and obesity are often seen concurrently with skeletal muscle wasting, leading to further derangements in function and metabolism. Muscle wasting remains an unmet need for metabolic disease, and new approaches are warranted. The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin releasing factor receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance, glucose metabolism, and muscle mass. The aim of this study was to investigate the effects of modified UCN2 peptides as a pharmaceutical therapy to counteract the loss of skeletal muscle mass associated with obesity and casting immobilization. METHODS: High-fat-fed mice (C57Bl/6J; 26 weeks old) and ob/ob mice (11 weeks old) were injected daily with a PEGylated (Compound A) and non-PEGylated (Compound B) modified human UCN2 at 0.3 mg/kg subcutaneously for 14 days. A separate group of chow-fed C57Bl/6J mice (12 weeks old) was subjected to hindlimb cast immobilization and, after 1 week, received daily injections with Compound A. In vivo functional tests were performed to measure protein synthesis rates and skeletal muscle function. Ex vivo functional and molecular tests were performed to measure contractile force and signal transduction of catabolic and anabolic pathways in skeletal muscle. RESULTS: Skeletal muscles (extensor digitorum longus, soleus, and tibialis anterior) from high-fat-fed mice treated with Compound A were ~14% heavier than muscles from vehicle-treated mice. Chronic treatment with modified UCN2 peptides altered the expression of structural genes and transcription factors in skeletal muscle in high-fat diet-induced obesity including down-regulation of Trim63 and up-regulation of Nr4a2 and Igf1 (P < 0.05 vs. vehicle). Signal transduction via both catabolic and anabolic pathways was increased in tibialis anterior muscle, with increased phosphorylation of ribosomal protein S6 at Ser235/236 , FOXO1 at Ser256 , and ULK1 at Ser317 , suggesting that UCN2 treatment modulates protein synthesis and degradation pathways (P < 0.05 vs. vehicle). Acutely, a single injection of Compound A in drug-naïve mice had no effect on the rate of protein synthesis in skeletal muscle, as measured via the surface sensing of translation method, while the expression of Nr4a3 and Ppargc1a4 was increased (P < 0.05 vs. vehicle). Compound A treatment prevented the loss of force production from disuse due to casting. Compound B treatment increased time to fatigue during ex vivo contractions of fast-twitch extensor digitorum longus muscle. Compound A and B treatment increased lean mass and rates of skeletal muscle protein synthesis in ob/ob mice. CONCLUSIONS: Modified human UCN2 is a pharmacological candidate for the prevention of the loss of skeletal muscle mass associated with obesity and immobilization.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético , Obesidade/tratamento farmacológico , Obesidade/etiologia , Peptídeos , UrocortinasRESUMO
The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration.Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identified temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.
Assuntos
Androgênios/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Regeneração , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Linhagem Celular , Folistatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Ligantes , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/lesões , Ratos , Receptores Androgênicos/metabolismo , Receptores CXCR4/genética , Receptores de Glucocorticoides/genética , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Follistatin 315 heparan sulfate-binding deficient mutant human IgG4 Fc fusion (FST-ΔHBS-Fc) is a follistatin (FST) based Fc fusion protein currently being developed as a novel therapy for several potential indications, including muscle wasting. Previous assessments of the pharmacokinetics and therapeutic activity of FST-ΔHBS-Fc have shown a close association of the exposure-response relationship. The current work builds upon these initial studies by investigating the glycosylation characteristics of FST-ΔHBS-Fc after recombinant expression and its impact on the pharmacokinetics in mice and Cynomolgus monkeys. The data presented indicate that FST-ΔHBS-Fc is heterogeneously glycosylated at the three putative sites in FST when recombinantly expressed in stably transfected Chinese hamster ovary cells. Such carbohydrate heterogeneity, especially with regards to sialic acid incorporation, directly results in sugar-dependent clearance in both mice and Cynomolgus monkeys. Examination of the pharmacokinetics of FST-ΔHBS-Fc molecules containing variable sialic acid content in asialoglycoprotein receptor 1 (ASPGR-1) knockout mice supports the receptor's role as part of the clearance mechanism of the molecules. Based on the evaluation of several variably sialylated lots of material in pharmacokinetic assessments, we define specifications for average sialic acid incorporation into FST-ΔHBS-Fc that result in limited sugar-mediated clearance. Taken together, these studies highlight the importance of establishing an early understanding of the glycosylation/pharmacokinetic relationships of FST-ΔHBS-Fc, which will provide a basis for future application toward optimal systemic drug delivery and dosing strategies.
Assuntos
Terapia Biológica/tendências , Folistatina/química , Folistatina/farmacocinética , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Camundongos SCIDRESUMO
Activin A, a member of the transforming growth factor-ß superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.
Assuntos
Ativinas/antagonistas & inibidores , Contração Muscular/fisiologia , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Animais , Eletroporação , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Regeneração/fisiologia , TranscriptomaRESUMO
Follistatin (FST) is a member of the tissue growth factor ß family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.
Assuntos
Folistatina/farmacologia , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Músculo Esquelético/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Regeneração , Animais , Folistatina/genética , Camundongos , Músculo Esquelético/fisiologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genéticaRESUMO
In this issue of Cell Metabolism, Mounier et al. (2013) show that AMPKα1 is a crucial contributor to the regeneration of damaged muscle tissues, acting in macrophages at the nexus between proinflammatory debris removal and resolution of muscle tissue inflammation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , Músculo Esquelético/metabolismo , Fagocitose/imunologia , Animais , MasculinoRESUMO
Human follistatin is a regulatory glycoprotein with widespread biologic functions, including antiinflammatory activities, wound-healing properties, and muscle-stimulating effects. The role of follistatin in a wide range of biologic activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease-modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic/pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315 (FST315). Our data indicate that the intrinsic PK/PD properties of native FST315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Here, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing FST315 to a murine IgG(1) Fc and removing the intrinsic heparan sulfate-binding activity of follistatin. The engineered variant molecule had ~100- and ~1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native FST315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy toward the development of a follistatin therapeutic with potential use in patients affected with skeletal muscle diseases.
Assuntos
Folistatina/farmacologia , Folistatina/farmacocinética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/farmacocinética , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Folistatina/genética , Células HEK293 , Meia-Vida , Heparina/metabolismo , Humanos , Imunoglobulina G/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Sefarose/metabolismoRESUMO
An estrogen receptor (ER) ß ligand (LY3201) with a preference for ERß over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17ß is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD(+) terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP(+) astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERß was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERß agonist results in changes opposite to those reported in estradiol-17ß-treated mice and suggests that ERα and ERß play opposing roles in the brain.
Assuntos
Benzopiranos/farmacologia , Encéfalo/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Receptor beta de Estrogênio/efeitos dos fármacos , Transdução de Sinais , Ácido gama-Aminobutírico/metabolismo , Animais , Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Receptor beta de Estrogênio/metabolismo , Ligantes , Camundongos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The LHX3 transcription factor is required for pituitary and nervous system development in mammals. Mutations in the human gene are associated with hormone-deficiency diseases. The gene generates two mRNAs, hLHX3a and hLHX3b, which encode three proteins with different properties. Here, the cis elements and trans-acting factors that regulate the basal transcription of the two mRNAs are characterized. A comparative approach was taken featuring analysis of seven mammalian Lhx3 genes, with a focus on the human gene. Two conserved, TATA-less, GC-rich promoters that are used to transcribe the mRNAs precede exons 1a and 1b of hLHX3. Transcription start sites were mapped for both promoters. Deletion experiments showed most activity for reporter genes containing the basal promoters in the context of -2.0 kb of hLHX3a and 1.8 kb of intron 1a (hLHX3b). Transfection, site-directed mutation, electrophoretic mobility shift, Southwestern blot, and chromatin immunoprecipitation approaches were used to characterize the interaction of transcription factors with conserved elements in the promoters. Specificity protein 1 is a regulator of both promoters through interaction with GC boxes. In addition, a distal element within intron 1a that is recognized by nuclear factor I is critical for hLHX3b promoter function. We conclude that dual promoters allow regulated production of two hLHX3 mRNAs.
Assuntos
Proteínas de Homeodomínio/genética , Fatores de Transcrição NFI/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , DNA Complementar/genética , Humanos , Rim , Proteínas com Homeodomínio LIM , Mamíferos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de TranscriçãoRESUMO
The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by approximately 5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERbeta but not ERalpha was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERbeta physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERbeta-regulated genes.
Assuntos
Resistência a Múltiplos Medicamentos/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteínas de Ligação a RNA/biossíntese , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptor beta de Estrogênio/metabolismo , Etoposídeo/farmacocinética , Etoposídeo/farmacologia , Feminino , Humanos , Mitoxantrona/farmacocinética , Mitoxantrona/farmacologia , Neoplasias Ovarianas/genética , Pirrolidinas/farmacologia , Splicing de RNA , Fatores de Processamento de RNA , RNA Catalítico/genética , RNA Catalítico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Tamoxifeno/farmacologia , Tiofenos/farmacologia , TransfecçãoRESUMO
LHX3 is a LIM homeodomain transcription factor with established roles in pituitary and nervous system development. Mutations in the human LHX3 gene are associated with severe hormone deficiency diseases. Previous studies have shown that the human LHX3 gene produces at least three protein isoforms: LHX3a, LHX3b, and M2-LHX3. In gene activation assays, LHX3a and M2-LHX3 are significantly more active than LHX3b because the actions of LHX3b are repressed by an inhibitory domain in its amino terminus. In this report, we investigate the molecular characteristics that result in reduced transcriptional capacity of LHX3b by determining the optimal DNA binding preference of LHX3b. Site selection experiments using purified human LHX3b reveal that it selects AT-rich sequences that contain ATTA/TAAT motifs. The pool of sequences selected by LHX3b is similar to that selected by LHX3a but does not conform to as strict a consensus. Further, the LHX3b-selected sites are bound more avidly by LHX3a and M2-LHX3 suggesting that LHX3b does not act by recognizing LHX3b-specific binding sites in target genes. We conclude that the amino terminal repression domain of LHX3b mostly acts to reduce the transcriptional potency of LHX3 by inhibiting the DNA binding affinity of the homeodomain, with some reduction in DNA binding specificity.
Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , DNA/química , Regulação da Expressão Gênica , Genes Reporter/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Luciferases/análise , Luciferases/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Guggulsterone (GS) is the active substance in guggulipid, an extract of the guggul tree, Commiphora mukul, used to treat a variety of disorders in humans, including dyslipidemia, obesity, and inflammation. The activity of GS has been suggested to be mediated by antagonism of the receptor for bile acids, the farnesoid X receptor (FXR). Here, we demonstrate that both stereoisomers of the plant sterol, (E)- and (Z)-GS, bind to the steroid receptors at a much higher affinity than to FXR. Both stereoisomers bind to the mineralocorticoid receptor (MR) with a Ki value of approximately 35 nM, which is greater than 100 times more potent than their affinity for FXR. Both (E)- and (Z)-GS also displayed high affinity for other steroid receptors, including the androgen (AR), glucocorticoid (GR), and progesterone receptors (PR) with Ki values ranging from 224 to 315 nM. In cell-based functional cotransfection assays, GSs behaved as antagonists of AR, GR, and MR, but as agonists of PR. Agonist activity was also demonstrated with estrogen receptor (ER) alpha; however, the potency was very low (EC50 > 5000 nM). In addition, GS displayed activity in functional assays in cell lines expressing endogenous AR, GR, ER, and PR. These data suggest that the variety of pharmacological effects exhibited by GS may be mediated by targeting several steroid receptors.
Assuntos
Hipolipemiantes/farmacologia , Pregnenodionas/farmacologia , Pregnenodionas/farmacocinética , Receptores de Esteroides/metabolismo , Linhagem Celular , Humanos , Cinética , Ligantes , Fitoterapia , Extratos Vegetais/farmacologia , Ensaio Radioligante , TransfecçãoRESUMO
We cloned and characterized the ISL2 and LHX2 LIM-homeodomain transcription factors of the Mexican salamander, or axolotl, Ambystoma mexicanum. Using a degenerate PCR approach, partial cDNAs representing five LIM-homeodomain genes were cloned, indicating conservation of this class of transcription factors in urodeles. Full-length cDNAs for Isl2 and Lhx2 were identified and sequenced. The predicted ISL2 and LHX2 proteins are well conserved, especially in the LIM and DNA-binding domains. The Isl2 and Lhx2 genes are expressed at all examined stages of embryogenesis and display tissue-restricted expression patterns in adults. In functional tests, axolotl LHX2 was inactive compared to homologous mammalian factors and adopted unusual DNA/protein complexes. However, axolotl ISL2 bound and induced transcription from the rat insulin gene. These experiments demonstrate conservation of key developmental regulatory proteins in salamanders and will allow future studies of their potential roles in the molecular regulation of tissue regeneration in such species.
Assuntos
Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Proteínas com Homeodomínio LIM , Luciferases/metabolismo , Camundongos , Reação em Cadeia da Polimerase , Biossíntese de ProteínasRESUMO
The mammalian anterior pituitary gland is a compound endocrine organ that regulates reproductive development and fitness, growth, metabolic homeostasis, the response to stress, and lactation, by actions on target organs such as the gonads, the liver, the thyroid, the adrenals, and the mammary gland. The protein and peptide hormones that control these physiological parameters are secreted by specialized pituitary cell types that derive from a common origin in the early ectoderm. Collectively, the broad physiological importance of the pituitary gland, its intriguing organogenesis, and the clinical and agricultural significance of its actions, have established pituitary development as an excellent model system for the study of the gene-regulatory cascades that guide vertebrate cell determination and differentiation. We review the transcriptional pathways that regulate the commitment of the individual pituitary cell lineages and that subsequently modulate trophic hormone gene activity in the differentiated cells of the mature gland.