The biological effects of C/EBPalpha in K562 cells depend on the potency of the N-terminal regulatory region, not on specificity of the DNA binding domain.
ABSTRACT: The transcription factor C/EBP? is more potent than C/EBP? in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological effects, modulation of gene expression, and binding to C/EBP?-regulated promoters by wild-type and chimeric C/EBP?/C/EBP? proteins. Wild-type and N-C/EBP?+ C/EBP?-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBP? and N-C/EBP?+C/EBP?-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBP? and N-C/EBP?+C/EBP?-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBP? or C/EBP? inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBP?. Gene expression profiles induced by C/EBP? resembled those modulated by N-C/EBP?+C/EBP?-DBD, whereas C/EBP? induced a pattern similar to that of N-C/EBP?+C/EBP?-DBD. C/EBP? activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBP?-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
Project description:The CCAAT/enhancer binding protein ? (C/EBP?) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBP? to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBP? in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBP? mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBP?-ER chimeric proteins. We show here that S21D C/EBP?-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBP?-ER, while S21A C/EBP?-ER was less efficient. Furthermore, wild-type C/EBP? suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBP? mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBP? in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBP? immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBP? due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBP? in K562 cells.
Project description:CCAAT/enhancer-binding protein alpha (C/EBP?) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBP? for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBP?, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBP? protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42?kDa CCAAT-enhancer-binding protein alpha (p42C/EBP?) and mutant isoform 30?kDa CCAAT-enhancer-binding protein alpha (p30C/EBP?), this may explain perturbed p42C/EBP?/p30C/EBP? ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBP? inducible K562- p42C/EBP?-estrogen receptor (ER) cells inhibits ?-estradiol (E2)-induced C/EBP? degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBP? in C/EBP? stably transfected cells (?-estradiol inducible K562 cells stably expressing p42C/EBP?-ER (K562-C/EBP?-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBP?-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBP? protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBP? often experienced in myeloid leukemia.
Project description:Ectopic expression of CAAT/enhancer binding protein ? (C/EBP?) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBP?-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBP? in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBP? interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBP?. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBP? required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells.
Project description:The c-Myb transcription factor coordinates proliferation and differentiation of hematopoietic precursor cells. Myb has three consecutive N-terminal SANT-type repeat domains (R1, R2, R3), two of which (R2, R3) form the DNA-binding domain (DBD). Three amino acid substitutions in R2 alter the way Myb regulates genes and determine the leukemogenicity of the retrovirally transduced v-Myb oncogene. The molecular mechanism of how these mutations unleash the leukemogenic potential of Myb is unknown. Here we demonstrate that the c-Myb-DBD binds to the N-terminal histone tails of H3 and H3.3. C-Myb binding facilitates histone tail acetylation, which is mandatory during activation of prevalent differentiation genes in conjunction with CCAAT enhancer-binding proteins (C/EBP). Leukemogenic mutations in v-Myb eliminate the interaction with H3 and acetylation of H3 tails and abolish activation of endogenous differentiation genes. In primary v-myb-transformed myeloblasts, pharmacologic enhancement of H3 acetylation restored activation of differentiation genes and induced cell differentiation. Our data link a novel chromatin function of c-Myb with lineage-specific expression of differentiation genes and relate the loss of this function with the leukemic conversion of Myb.
Project description:Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.
Project description:Protoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.We have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBP? by western blot and quantitative real time PCR analyses.PP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBP?, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.We hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.These results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBP?.
Project description:Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5.
Project description:Here, it was determined that chronic lymphocytic leukemia (CLL) cells express the ? subunit, but not the ? subunit, of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF2R). GM-CSFR? was detected on the surface, in the cytosol, and in the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFR? antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFR? promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFR? promoter, then stimulated with IL6 to activate STAT3 and to identify STAT3-binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL6-stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3-siRNA or CLL cells with STAT3-shRNA significantly downregulated GM-CSFR? mRNA and protein levels. RNA transcripts, involved in regulating cell survival pathways, and the proteins KAP1 (TRIM28) and ISG15 coimmunoprecipitated with GM-CSFR?. GM-CSFR?-bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFR?-bound ISG15 inhibited the NF-?B pathway. Nevertheless, overexpression of GM-CSFR? protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFR? knockdown induced apoptosis in CLL cells, suggesting that GM-CSFR? provides a ligand-independent survival advantage.Constitutively, activation of STAT3 induces the expression of GM-CSFR? that protects CLL cells from apoptosis, suggesting that inhibition of STAT3 or GM-CSFR? may benefit patients with CLL.
Project description:Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. Here, we used a quantitative phospho-tyrosine analysis to generate a comprehensive signaling map of G-CSF induced tyrosine phosphorylation in the normal versus mutated (proximal: T618I and truncated: Q741x) G-CSFRs. Unbiased clustering and kinase enrichment analysis identified rapid induction of phospho-proteins associated with endocytosis by the wild type G-CSFR only; while G-CSFR mutants showed abnormal kinetics of canonical Stat3, Stat5, and Mapk phosphorylation, and aberrant activation of Bruton's Tyrosine Kinase (Btk). Mutant-G-CSFR-expressing cells displayed enhanced sensitivity (3-5-fold lower IC50) for ibrutinib-based chemical inhibition of Btk. Primary murine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant receptor and retrovirally transduced human CD34+ umbilical cord blood cells expressing mutant receptors displayed enhanced sensitivity to Ibrutinib. A significantly lower clonogenic potential was displayed by both murine and human primary cells expressing mutated receptors upon ibrutinib treatment. Finally, a dramatic synergy was observed between ibrutinib and ruxolinitib at lower dose of the individual drug. Altogether, these data demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, and suggest repositioning Ibrutinib for therapy of myeloid leukemia bearing CSF3R mutations. Phospho-tyrosine data are available via ProteomeXchange with identifier PXD009662.
Project description:The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of (125)I-labelled IL-6 to (GR-FNIII)gp130, or of (125)I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, (125)I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.