RESUMEN
Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.
Asunto(s)
Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Gonadotrofos/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transactivadores/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteínas del Ojo/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Gonadotrofos/efectos de los fármacos , Proteínas de Homeodominio/genética , Masculino , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Factores de Transcripción Paired Box/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esteroides/farmacología , Transactivadores/genética , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Proteína Homeobox SIX3RESUMEN
Posttranslational modifications regulate physiology either by directly modulating protein function or by impacting immune recognition of self-proteins. Citrullination is a posttranslational modification formed by the conversion of arginine residues into the citrulline amino acid by protein arginine deiminase (PAD) family members. We have identified mast cells as a major source of the PAD2 enzyme. Activation of the P2X7 purinergic receptor (P2X7) by the inflammatory "danger" signal ATP induces PAD2 activity and robust protein citrullination. P2X7-mediated activation of PAD2 is sensitive to p38 MAPK and protein kinase C inhibitors, and PAD2 regulates the expression of the TNFR2, Adamts-9, and Rab6b transcripts in mast cells. Further, the PAD2 enzyme and its citrullinated substrate proteins are released from mast cells on activation with ATP. PAD2 expression is closely linked with inflammation in rheumatoid arthritis (RA) synovial tissue, and PAD2 and citrullinated proteins are found in the synovial fluid of RA patients. In addition, RA is associated with the development of autoantibodies to citrullinated self-proteins. Our results suggest that P2X7 activation of mast cells may play a role in inflammation by providing PAD2 and PAD2 substrates access to the extracellular space.
Asunto(s)
Adenosina Trifosfato/fisiología , Citrulina/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Proteína-Arginina N-Metiltransferasas/fisiología , Receptores Purinérgicos P2X7/fisiología , Animales , Línea Celular Tumoral , Células Cultivadas , Espacio Extracelular/enzimología , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Mastocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Purinérgicos P2X7/metabolismo , Especificidad por Sustrato/inmunologíaRESUMEN
Coronin-1A is a WD repeat protein family member, highly expressed in all hematopoietic lineages, and acts as a regulator of F-actin dynamics and Ca2+ signaling. In Coro1a(Lmb3) mice results in inactivation of the protein and leads to disease resistance in a model of lupus erythematosus. In Coro1a(-/-) and Coro1a(Lmb3) mice, peripheral T cells exhibit impairments in survival, migration, activation, and Ca2+ flux. In this study, we show that in vitro-differentiated mast cells from Coro1a(Lmb3) mice are viable, developed normally, and are fully functional in assays of degranulation, cytokine secretion, and chemotactic migration, despite increased F-actin levels. In Coro1a(Lmb3) mast cells, Ca2+ flux in response to physiological FcεRI stimulation is unaffected. Finally, Coro1a(Lmb3) mice showed similar in vivo mast cell responses as the WT mice. Coronin-1B and Coronin-1C expression levels were not increased in Coro1a(Lmb3) mast cells but were higher in mast cells than in CD4 T cells or B cells in WT mice. We conclude that Coronin-1A activity is not required for mast cell function.
