RESUMO
Various small molecule pharmacologic agents with different known functions produce similar outcomes in diverse Mendelian and complex disorders, suggesting that they may induce common cellular effects. These molecules include histone deacetylase inhibitors, 4-phenylbutyrate (4PBA) and trichostatin A, and two small molecules without direct histone deacetylase inhibitor activity, hydroxyurea (HU) and sulforaphane. In some cases, the therapeutic effects of histone deacetylase inhibitors have been attributed to an increase in expression of genes related to the disease-causing gene. However, here we show that the pharmacological induction of mitochondrial biogenesis was necessary for the potentially therapeutic effects of 4PBA or HU in two distinct disease models, X-linked adrenoleukodystrophy and sickle cell disease. We hypothesized that a common cellular response to these four molecules is induction of mitochondrial biogenesis and peroxisome proliferation and activation of the stress proteome, or adaptive cell survival response. Treatment of human fibroblasts with these four agents induced mitochondrial and peroxisomal biogenesis as monitored by flow cytometry, immunofluorescence and/or western analyses. In treated normal human fibroblasts, all four agents induced the adaptive cell survival response: heat shock, unfolded protein, autophagic and antioxidant responses and the c-jun N-terminal kinase pathway, at the transcriptional and translational levels. Thus, activation of the evolutionarily conserved stress proteome and mitochondrial biogenesis may be a common cellular response to such small molecule therapy and a common basis of therapeutic action in various diseases. Modulation of this novel therapeutic target could broaden the range of treatable diseases without directly targeting the causative genetic abnormalities.
Assuntos
Adrenoleucodistrofia/tratamento farmacológico , Tratamento Farmacológico , Ácidos Hidroxâmicos/uso terapêutico , Hidroxiureia/uso terapêutico , Fenilbutiratos/uso terapêutico , Proteoma/metabolismo , Tiocianatos/uso terapêutico , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Adrenoleucodistrofia/fisiopatologia , Linhagem Celular , Humanos , Isotiocianatos , Renovação Mitocondrial/efeitos dos fármacos , Proteoma/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , SulfóxidosRESUMO
Fetal hemoglobin (HbF) level has emerged as an important prognostic factor in sickle-cell disease (SCD) and can be measured by the proportion of HbF-containing erythrocytes (F-cells). Recently, BCL11A (zinc-finger protein) was identified as a regulator of HbF, and the strongest association signals were observed either directly for rs766432 or for correlated single-nucleotide polymorphisms (SNPs). To identify additional independently associated genetic variants, we performed a genome-wide association study (GWAS) on the proportion of F-cells in individuals of African ancestry with SCD from the Silent Infarct Transfusion (SIT) Trial cohort. Our study not only confirms the association of rs766432 (P-value <3.32 × 10(-13)), but also identifies an independent novel intronic SNP, rs7606173, associated with F-cells (P-value <1.81 × 10(-15)). The F-cell variances explained independently by these two SNPs are â¼13% (rs7606173) and â¼11% (rs766432), whereas, together they explain â¼16%. Additionally, in men, we identify a novel locus on chromosome 17, glucagon-like peptide-2 receptor (GLP2R), associated with F-cell regulation (rs12103880; P-value <3.41 × 10(-8)). GLP2R encodes a G protein-coupled receptor and involved in proliferative and anti-apoptotic cellular responses. These findings highlight the importance of denser genetic screens and suggest further exploration of the BCL11A and GLP2R loci to gain additional insight into HbF/F-cell regulation.
Assuntos
Anemia Falciforme/genética , População Negra/genética , Proteínas de Transporte/genética , Cromossomos Humanos Par 17/genética , Eritrócitos/metabolismo , Hemoglobina Fetal/análise , Proteínas Nucleares/genética , Teorema de Bayes , Estudos de Coortes , Contagem de Eritrócitos , Estudo de Associação Genômica Ampla , Genótipo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Haplótipos/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Receptores de Glucagon/genética , Proteínas RepressorasRESUMO
OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.
Assuntos
Antígenos CD34 , Antidrepanocíticos/farmacologia , Azacitidina/análogos & derivados , Butiratos/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Hemoglobina Fetal/biossíntese , Hidroxiureia/farmacologia , Monócitos/metabolismo , Adenilil Ciclases/metabolismo , Azacitidina/farmacologia , Colforsina/farmacologia , Decitabina , Células Eritroides/citologia , Células Eritroides/metabolismo , Humanos , Células K562 , Monócitos/citologia , RNA Mensageiro/biossínteseRESUMO
The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential. We used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to screen for HbS-binding RNA aptamers modified with nuclease-resistant 2'-fluoropyrimidines. Polymerization assays were employed to identify aptamers with polymerization-inhibitory properties. Two noncompeting aptamers, DE3A and OX3B, were found to bind hemoglobin, significantly increase the delay time, and reduce the rate of polymerization of HbS. These modifiable, nuclease-resistant aptamers are potential new therapeutic agents for SCD.