RESUMEN
G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.
Asunto(s)
Diferenciación Celular , Eritropoyesis , Hemina , Leucemia Eritroblástica Aguda , Co-Represor 1 de Receptor Nuclear , Humanos , Células K562 , Eritropoyesis/genética , Leucemia Eritroblástica Aguda/patología , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Co-Represor 1 de Receptor Nuclear/genética , Hemina/farmacología , Células Eritroides/metabolismo , Células Eritroides/citología , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Hemoglobinas/metabolismo , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización IntracelularRESUMEN
The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.
Asunto(s)
Factores de Transcripción de Tipo Kruppel , Proteína C , Animales , Ratones , Proteína C/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Diferenciación Celular/genética , Transporte de Proteínas , Células Eritroides/metabolismoRESUMEN
Pharmacologically inhibiting nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome activation results in potent therapeutic effects in a wide variety of preclinical inflammatory disease models. NLRP3 deubiquitination is essential for efficient NLRP3 inflammasome activity, but it remains unclear whether this process can be harnessed for therapeutic benefit. Here, we show that thiolutin (THL), an inhibitor of the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease, blocks NLRP3 inflammasome activation by canonical, noncanonical, alternative, and transcription-independent pathways at nanomolar concentrations. In addition, THL potently inhibited the activation of multiple NLRP3 mutants linked with cryopyrin-associated periodic syndromes (CAPS). Treatment with THL alleviated NLRP3-related diseases in mouse models of lipopolysaccharide-induced sepsis, monosodium urate-induced peritonitis, experimental autoimmune encephalomyelitis, CAPS, and methionine-choline-deficient diet-induced nonalcoholic fatty liver disease. Mechanistic studies revealed that THL inhibits the BRCC3-containing isopeptidase complex (BRISC)-mediated NLRP3 deubiquitination and activation. In addition, we show that holomycin, a natural methyl derivative of THL, displays an even higher inhibitory activity against NLRP3 inflammasome than THL. Our study validates that posttranslational modification of NLRP3 can be pharmacologically targeted to prevent or treat NLRP3-associated inflammatory diseases. Future clinical development of derivatives of THL may provide new therapies for NLRP3-related diseases.
Asunto(s)
Enzimas Desubicuitinizantes/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Sangre Fetal , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lactamas/farmacología , Lactamas/uso terapéutico , Lipopolisacáridos , Masculino , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/inmunología , Embarazo , Cultivo Primario de Células , Pirrolidinonas/farmacología , Pirrolidinonas/uso terapéutico , Células THP-1 , Ubiquitinación/efectos de los fármacosRESUMEN
Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.
Asunto(s)
Eritropoyesis/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Células Precursoras Eritroides/citología , Técnicas de Silenciamiento del Gen , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/química , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Trasplante Heterólogo , Ubiquitinación , Regulación hacia ArribaRESUMEN
OBJECTIVE: To investigate effect of the soluble epidermal growth factor receptor (sEGFR/sErbB1) level in the peripheral blood in development, invasiveness, apoplexy of each type of pituitary tumor. METHODS: The sEGFR level was determined in peripheral serum from 190 patients with pituitary diseases by enzyme linked immunosorbent assay. The sEGFR levels were measured in 10 pituitary Rathke's pouch, 18 pituitary hyperplasia, 161 pituitary adenomas including 30 microadenomas, 83 large adenomas, 48 giant adenomas, 1 pituitary carcinoma, and 28 healthy controls. RESULTS: In the patients with pituitary hyperplasia, microadenoma, large adenoma, giant adenoma, and pituitary carcinoma, the sEGFR level was 188.92 +/- 32.62, 209.83 +/- 19.01, 333.20 +/- 69.33, 405.85 +/- 37.38, and 617.45 fmol/mL independently. They were all significantly higher than patients with pituitary Rathke's pouch (156.78 +/- 18.24 fmol/mL, P < 0.001) and healthy control group (159.11 +/- 40.50 fmol/mL, P < 0.05). The sEGFR level in pituitary carcinoma was higher than pituitary adenoma. In patients with pituitary adenoma, the sEGFR level was positive correlated to the size of pituitary adenomas (r=0.998), the significant difference was observed for the sEGFR level in each group of the patients with pituitary adenomas (P < 0.001). Furthermore, in patients with pituitary ACTH-secreting microadenomas, the serum sEGFR levels in invasiveness (295.00 +/- 77.80 fmol/mL) was higher than that in non-invasiveness (210.60 +/- 16.4 fmol/mL, P < 0.05). In patients with pituitary ACTH-secreting, PRL-secreting, GH-secreting, and non-functioning large adenomas, the serum sEGFR levels in invasiveness (407.86 +/- 28.50, 399.25 +/- 30.10, 386.00 +/- 13.08, and 369.25 +/- 36.70 fmol/mL) was higher than that in non-invasiveness (335.25 +/- 63.49, 300.64 +/- 47.57, 297.00 +/- 61.93, and 269.30 +/- 25.68 fmol/mL) respectively (P < 0.05). In patients with invasive pituitary PRL-secreting, GH-secreting, and non-functioning giant adenomas, the serum sEGFR levels not significantly different in between invasiveness (417.50 +/- 35.94, 409.50 +/- 69.14, and 417.50 +/- 44.13 fmol/mL) and non-invasiveness (386.00 +/- 49.64, 417.50 +/- 44.03, and 409.51 +/- 35.17 fmol/mL) (P > 0.05). In patients with pituitary large adenomas, the sEGFR levels in pituitary apoplexy (377.48 +/- 39.18 fmol/mL) was higher than that in non-pituitary apoplexy (343.18 +/- 68.17 fmol/mL, P > 0.05). CONCLUSIONS: The increased level of peripheral serum sEGFR is concomitant with development, proliferous size of the adenomas in patients with pituitary adenomas. In addition, the elevated levels of serum sEGFR occur in pituitary apoplexy as clinical active tumors, and the non-invasive ACTH secreting adenomas. The sEGFR levels could be differentiated helpfully between pituitary adenomas and non-pituitary adenomas. These data suggest that serum sEGFR could be as a referable marker of the size and activation of proliferation in pituitary adenoma.