RESUMO
Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.
Assuntos
Proteínas de Homeodomínio , Lúpus Eritematoso Sistêmico , Camundongos , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Alelos , Isoformas de Proteínas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , DNA , Ubiquitinas/genéticaRESUMO
BACKGROUND: Bone marrow (BM) is progressively filled with adipocytes during aging process. Thus, BM adipocytes-derived adiponectin (APN) affects the function of bone marrow-derived mesenchymal stem cells (BMSCs). However, little is known about the effect of APN on migration ability of BMSCs cultured under hypoxic conditions, which is similar to the BM microenvironment. RESULTS: We found that the population and migration ability of BMSCs from APN KO mice was higher than that of WT mice due to increased stability of hypoxia inducible factor 1α (HIF1α). Stem cell factor (SCF)-activated STAT3 stimulated the induction of HIF1α which further stimulated SCF production, indicating that the SCF/STAT3/HIF1α positive loop was highly activated in the absence of APN. It implies that APN negatively regulated this positive loop by stimulating HIF1α degradation via the inactivation of GSK3ß. Furthermore, APN KO BMSCs were highly migratory toward EL-4 lymphoma, and the interaction between CD44 in BMSCs and hyaluronic acid (HA) from EL-4 enhanced the migration of BMSCs. On the other hand, the migrated BMSCs recruited CD8+ T cells into the EL-4 tumor tissue, resulting in the retardation of tumor growth. Additionally, gradually increased APN in BM on the aging process affects migration and related functions of BMSCs, thus aged APN KO mice showed more significant suppression of EL-4 growth than young APN KO mice due to higher migration and recruitment of CD8+ T cells. CONCLUSION: APN deficiency enhances CD44-mediated migration ability of BMSCs in the hypoxic conditions by the SCF/STAT3/HIF1α positive loop and influences the migration ability of BMSCs for a longer time depending on the aging process. Video Abstract.
Assuntos
Adiponectina , Células-Tronco Mesenquimais , Animais , Camundongos , Medula Óssea/metabolismo , Células da Medula Óssea , Linfócitos T CD8-Positivos , Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
BACKGROUND: Lymphocyte antigen 6 complex locus K (LY6K), a glycosylphosphatidylinositol-anchored protein, plays a dynamic role in cancer metastasis. In the current study, we deciphered the effects of LY6K on transforming growth factor-ß (TGF-ß) and epidermal growth factor (EGF) signaling through clathrin- and caveolin-1 (CAV-1)-mediated endocytosis. METHODS: Analysis of the TCGA and GTEx dataset were performed to explore the expression and survival of LY6K in cancer patients. Short interfering RNA (siRNA) was used to knockdown the expression of LY6K in human cervical cancer patients. The effect of lack of LY6K on cell proliferation, migration, and invasion was performed, and RT-qPCR and immunoblotting were performed to identify LY6K-affected TGF-ß and EGF signaling pathways. Additionally, Immunofluorescence (IF) and transmission electron microscope (TEM) were performed to identify the role of LY6K in CAV-1- and Clathrin-mediated endocytosis. RESULTS: Lymphocyte antigen 6 complex locus K expression level is elevated in higher grade cervical cancer patients correlating with poor overall survival, progression-free survival, and disease-free survival. LY6K-depletion in HeLa and SiHa cancer cells suppressed EGF-induced proliferation and TGF-ß-enhanced migration and invasion. Both TGF-ß receptor-I (TßRI) and EGF receptor (EGFR) localized at the plasma membrane regardless of LY6K expression, and LY6K bound TßRI irrespective of the presence of TGF-ß; however, LY6K did not bind EGFR. LY6K-depleted cells showed impaired Smad2 phosphorylation upon TGF-ß treatment and lower proliferation rates following long-term treatment with EGF. We revealed the atypical movement of TßRI and EGFR from plasma membrane upon ligand stimulation in LY6K-depleted cells and an impaired movement of the endocytic proteins clathrin and CAV-1. CONCLUSIONS: Our study demonstrates the key role of LY6K in both clathrin- and CAV-1-mediated endocytic pathways regulated by TGF-ß and EGF, and it suggests a correlation between LY6K overexpression in cervical cancer cells and poor overall survival.
Assuntos
Fator de Crescimento Epidérmico , Neoplasias do Colo do Útero , Feminino , Humanos , Fator de Crescimento Transformador beta , Neoplasias do Colo do Útero/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Clatrina/metabolismo , Antígenos Ly , Proteínas Ligadas por GPIRESUMO
This corrects the article on p. 138 in vol. 24, PMID: 33818016.
RESUMO
PURPOSE: Melanoma-associated antigen C2 (MAGEC2) is an oncogene associated with various types of cancers. However, the biological function of MAGEC2 in circulating tumor cells remains unclear. In this study, we investigated the role of MAGEC2 using adapted suspension cells (ASCs), which were previously developed to study circulating tumor cells (CTCs). METHODS: Differential gene expression in adherent cells (ADs) and ASCs was examined using RNA-seq analysis. MAGEC2 expression was assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and ChIP-seq analysis. Depletion of MAGEC2 expression was performed using siRNA. MAGEC2-depleted ADs and ASCs were used to investigate changes in the proliferation rate and cell cycle. Then, the protein levels of signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3, and downstream of STAT3 were measured using control and MAGEC2-depleted ADs and ASCs. In ASCs, the direct effect of active STAT3 inhibition with Stattic, a STAT3 inhibitor, was assessed in terms of proliferation and apoptosis. Finally, an Annexin V/7-AAD assay was performed to determine the percentage of apoptotic cells in the Stattic-treated cells. RESULTS: MAGEC2 was highly expressed in ASCs when compared with ADs. Depletion of MAGEC2 reduced the proliferation rate and viability of ASCs. To elucidate the underlying mechanism, the level of STAT3 was examined owing to its oncogenic properties. Tyrosine-phosphorylated active STAT3 was highly expressed in ASCs and decreased in MAGEC2-depleted ASCs. Furthermore, on treating ASCs with Stattic, an active STAT3 inhibitor, the cells were markedly sensitive to intrinsic pathway-mediated apoptosis. CONCLUSIONS: High MAGEC2 expression may play an important role in the survival of ASCs by maintaining the expression of activated STAT3 to prevent apoptotic cell death.