RESUMEN
Growth hormone (GH) binds to its specific receptor (GHR) at the surface of target cells activating multiple signaling pathways implicated in growth and metabolism. Dysregulation of GHRs leads to pathophysiological states that most commonly affect stature. We previously showed the association of a polymorphic (nâ¯=â¯15-37) GT microsatellite in the human GHR gene promoter with short stature in a sex-specific manner. In the present study we evaluated the functional relevance of this polymorphism in regulating GHR expression. Using luciferase reporter assays, we found that the GT repeat had a significant cis regulatory effect in response to HIF1α and a potential repressor role following C/EBPß stimulation. Using a digital PCR application to measure allelic imbalance (AI), we showed a high prevalence of AI (â¼76%) at the GHR locus in lymphoblastoid cell lines (LCLs), with a significantly higher degree of imbalance in LCLs derived from males. Examination of expression of GHR as well as other members of the GH-IGF1 axis in the LCLs revealed significant associations of GHR, IGF1 and BCL2 expression with GT genotype in a sex-specific manner. Our results suggest that this GT microsatellite exerts both cis and trans effects in a sex-specific context, revealing a new mechanism by which GHR gene expression is regulated.
Asunto(s)
Proteínas Portadoras/genética , Enanismo/genética , Repeticiones de Microsatélite , Regiones Promotoras Genéticas , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/genética , Caracteres SexualesRESUMEN
Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site's methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8-13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation.
Asunto(s)
Metilación de ADN , Células Epiteliales/metabolismo , Factor de Transcripción Ikaros/genética , Proteínas de Neoplasias/genética , Alelos , Secuencias de Aminoácidos , Azacitidina/química , Sitios de Unión , Línea Celular , Proteínas del Huevo/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Células HEK293 , Humanos , Células MCF-7 , Proteínas de la Membrana/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Human GH binds to its receptor (GHR) on target cells and activates multiple intracellular pathways, leading to changes in gene expression, differentiation, and metabolism. GHR deficiency is associated with growth and metabolic disorders whereas increased GHR expression has been reported in certain cancers, suggesting that the GHR gene requires tight controls. Several regulatory mechanisms have been found within its 5'-untranslated region (UTR) promoter and coding regions. However, the 3'-UTR has not been previously examined. MicroRNAs (miRNAs) are small (19-22 nucleotides) noncoding RNAs that downregulate gene expression mainly through targeting the 3'-UTR of mRNAs and enhancing their degradation or inhibiting translation. In the present study, we investigated whether miRNAs regulate GHR expression. To define putative miRNA binding sites in the GHR 3'-UTR, we used multiple in silico prediction tools, analyzed conservation across species and the presence of parallel sites in GH/IGF axis-related genes, and searched for reports linking miRNAs to GHR-related physiological or pathophysiological activities. To test prioritized sites, we cotransfected a wild-type GHR 3'-UTR luciferase reporter vector as well as miRNA binding site mutants into HEK293 cells with miRNA mimics. Furthermore, we tested whether the miRNAs altered endogenous GHR mRNA and protein levels in HEK293 cells and in 2 cancer cell lines (MCF7 and LNCaP). Our experiments have identified miRNA (miR)-129-5p, miR-142-3p, miR-202, and miR-16 as potent inhibitors of human GHR expression in normal (HEK293) and cancer (MCF7 and LNCaP) cells. This study paves the way for the development of miRNA inhibitors as therapeutic agents in GH/GHR-related pathophysiologies, including cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Regiones no Traducidas 3' , Sitios de Unión , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Clonación Molecular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Células MCF-7 , Mutagénesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismoRESUMEN
Mammalian sulfotransferases (EC 2.8.2) are involved in many important facets of steroid hormone activity and metabolism. In this study, Arabidopsis AtST4a and AtST1 were identified and characterized as brassinosteroid sulfotransferases that appear to be involved in different aspects of hormone regulation. The two proteins share 44% identity in amino acid sequence, and belong to different plant sulfotransferase families. AtST4a was specific for biologically active end products of the brassinosteroid pathway. The enzyme sulfated brassinosteroids with diverse side-chain structures, including 24-epibrassinosteroids and the naturally occurring (22R, 23R)-28-homobrassinosteroids. AtST4a belongs to a small subfamily of sulfotransferases having two other members, AtST4b and -c. Among the three recombinant enzymes, only AtST4a was catalytically active with brassinosteroids. Transcript expression of AtST4 subfamily members was largely specific to the root. AtST4b- and -c transcript levels were induced by treatment with trans-zeatin, while AtST4a was repressed under the same conditions, supporting a divergent function of AtST4a. Co-regulation of AtST4b and -c correlated with their location in tandem on chromosome 1. AtST1 was stereospecific for 24-epibrassinosteroids, with a substrate preference for the metabolic precursor 24-epicathasterone, and exhibited catalytic activity with hydroxysteroids and estrogens. To gain more insight into this dual activity with plant and mammalian steroids, enzymatic activities of human steroid sulfotransferases toward brassinosteroids were characterized. The dehydroepiandrosterone sulfotransferase SULT2A1 displayed catalytic activity with a selected set of 24-epibrassinolide precursors, including 24-epicathasterone, with specific activities comparable to that measured for the endogenous substrate dehydroepiandrosterone. The comparable activity profiles of AtST1 and SULT2A1 suggest a similar architecture of the acceptor-binding site between the two enzymes, and may potentially reflect a common ability to conjugate certain xenobiotics.