RTP801 is a novel retinoic acid-responsive gene associated with myeloid differentiation.

OBJECTIVE: Retinoids are crucial in the regulation of fundamental cellular processes including terminal differentiation of both normal and malignant myeloid progenitors. The aim of this study was to identify and characterize retinoic acid (RA) target genes. METHODS AND RESULTS: RTP801 is a recently cloned stress response gene that acts as a negative regulator of the mTOR pathway. Here we identified RTP801 as a novel early RA target gene in myeloid cells. RTP801 mRNA levels are induced in acute myeloid leukemia (AML) cell lines during RA-dependent differentiation and are differentially expressed during maturation of normal CD34(+) cells. The myeloid-specific, differentiation-related transcription factor C/EBPepsilon also induces RTP801 expression. Overexpression of RTP801 in the U937 leukemic cells leads to growth inhibition and apoptosis. Conversely, silencing of endogenous RTP801 by shRNA reduces RA-induced differentiation of the U937 cells. Downregulation of RTP801 also abrogates hypoxia-induced inhibition of mTOR in those cells. CONCLUSION: Taken together, our data suggest that RTP801 is an important RA-regulated gene involved in myeloid differentiation, which could represent a therapeutic target in leukemia.

Detection of aberrant gene expression in CD34+ hematopoietic stem cells from patients with agnogenic myeloid metaplasia using oligonucleotide microarrays.

Agnogenic myeloid metaplasia (AMM) is a clonal stem cell disorder that leads to ineffective hematopoiesis, bone marrow fibrosis, and extramedullary hematopoiesis. The molecular mechanisms underlying the development of this syndrome are currently unknown. Therefore, the aim of this study was to characterize aberrant gene expression in CD34+ hematopoietic stem cells from patients with AMM. We used oligonucleotide microarrays to analyze gene expression profiles in CD34+ hematopoietic stem cells from patients with AMM compared with expression in CD34+ cells from healthy individuals. We identified 95 highly differentially expressed genes (48 upregulated and 47 down-regulated) that are potentially involved in regulating abnormal hematopoietic proliferation and differentiation and confirmed many of them by quantitative polymerase chain reaction. Using class membership prediction analysis, we identified 75 genes whose expression profiles can accurately differentiate AMM samples from the controls. Using these 75 genes, we were able to discriminate patients with AMM from the controls by hierarchical clustering (Spearman's confidence correlation). The predictive power of these genes was verified by applying the algorithm to an unknown test set containing expression data from eight additional CD34+ samples (four AMM, four control). Our results indicate that a subset of genes may be used to differentiate patients with AMM from healthy individuals. Furthermore, we identify 95 genes whose aberrant expression may be involved in AMM.

Protein partners of C/EBPepsilon.

CCAAT-enhancer binding protein-epsilon (C/EBPepsilon) is a nuclear transcription factor implicated in the regulation of terminal myeloid differentiation. Using a yeast two-hybrid screen, potential interaction partners of C/EBPepsilon involved in myeloid development were identified. C/EBPepsilon was found to associate with other C/EBP family members, including C/EBPepsilon and CHOP as well as other proteins that are known to contain a leucine-zipper protein interaction motif including CREB2, LDOC1, E6TP1, and AF-17. In addition, C/EBPepsilon demonstrated the potential for interaction with proteins that do not possess a leucine-zipper motif, including proteins that may be involved in sumoylation (protein inhibitor of activated STAT1 [PIAS1] and ubiquitin-conjugating enzyme E2I). As expected, the association of C/EBPepsilon with other C/EBP family members depends on the presence of a functional leucine-zipper motif. Mapping studies of C/EBPepsilon with PIAS1 (as an example of a nonleucine-zipper-containing protein) showed that C/EBPepsilon interacts with the amino-terminal domain of PIAS1. The function of C/EBPepsilon interacting proteins was further investigated. Co-expression of C/EBPepsilon with C/EBPdelta resulted in potent transactivation in a lactoferrin reporter system. A gel mobility shift assay suggests that C/EBPepsilon, C/EBPalpha, and C/EBPdelta proteins can bind as heterodimers to a C/EBP consensus DNA-binding site. As CHOP is known to represent a transcriptional repressor, the functional interaction between C/EBPepsilon and CHOP was investigated. Co-expression of C/EBPepsilon and c-Myb with CHOP caused marked transcriptional repression of target reporter genes. Our results suggest heterodimeric partners of C/EBPepsilon modulate the function of C/EBPepsilon in mediating gene transcription during myelopoiesis.

Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes.

Retinoic acid (RA) promotes granulocytic differentiation of normal hematopoietic cells and acute promyelocytic leukemia (APL) blasts by transcriptional modulation of myeloid regulatory genes. In this study, we have identified the C/EBP homologous protein (CHOP) as a novel retinoid-responsive gene using a polymerase chain reaction (PCR)-based cDNA subtraction method. All-trans retinoic acid (ATRA) induced a biphasic expression of CHOP mRNA in the NB4 and HL60 AML cell lines. Levels of CHOP expression increased within 1 hour of exposure to ATRA. ATRA expression became nearly absent between 6 and 24 hours, and a second phase of induction occurred after 48 hours. Retinoid-dependent regulation of CHOP expression was also observed in normal human neutrophils but not in peripheral blood mononuclear cells. In addition, retinoid-dependent regulation of CHOP expression was not observed in retinoid-nonresponsive cell lines HL60R and NB4-R2. CHOP expression was regulated at the transcriptional level and was independent of new protein synthesis. CHOP heterodimerized with C/EBPepsilon and negatively regulated the myeloid-specific gene lactoferrin. Furthermore, CHOP transcriptionally inhibited C/EBPalpha- and C/EBPepsilon-dependent induction of secondary granule gene expression. RA signaling in granulocytic differentiation involves regulated expression of CHOP and C/EBPepsilon in a coordinated fashion.

Restoration of C/EBPalpha expression in a BCR-ABL+ cell line induces terminal granulocytic differentiation.

The transcription factor C/EBPalpha plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBPalpha were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBPalpha. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBPepsilon protein but not a functional C/EBPalpha, -beta, and -gamma. Restoration of C/EBPalpha expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBPalpha, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBPalpha was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBPalpha. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBPalpha expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBPalpha in acute phase of CML.

Comparative analysis of genes regulated by PML/RAR alpha and PLZF/RAR alpha in response to retinoic acid using oligonucleotide arrays.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations involving retinoic acid receptor alpha (RAR alpha) and its fusion partners including promyelocytic leukemia (PML) and promyelocytic leukemia zinc finger (PLZF). Using oligonucleotide arrays, we examined changes in global gene expression mediated by the ectopic expression of either PML/RAR alpha (retinoid-sensitive) or PLZF/RAR alpha (retinoid-resistant) in U937 cells. Of more than 5000 genes analyzed, 16 genes were commonly up-regulated, and 57 genes were down-regulated by both fusion proteins suggesting their role in the APL phenotype. In our APL model, for example, TNFAIP2, TNFR2, ELF4, RAR gamma, and HoxA1 were down-regulated by both fusion proteins in the absence of retinoic acid (RA). RA strongly up-regulated these genes in PML/RAR alpha, but not in PLZF/RAR alpha expressing U937 cells. Expression studies in NB4, retinoid-resistant NB4-R2, normal human CD34+ cells, and APL patient samples strongly suggest their role in the regulation of granulocytic differentiation. Furthermore, combined treatment with tumor necrosis factor alpha (TNF alpha) and RA synergistically enhanced granulocytic differentiation in NB4 cells but not in NB4-R2 cells. Our data indicate that APL pathogenesis and retinoid-induced granulocytic differentiation of APL cells involve genes in the cell death pathway, and that cooperation between the RA and TNFalpha signaling pathways exists. Targeting both the retinoid-dependent differentiation and the cell death pathways may improve leukemic therapy, especially in retinoid-resistant acute myeloid leukemia.

Macrophage functional maturation and cytokine production are impaired in C/EBP epsilon-deficient mice.

Members of the CCAAT/enhancer-binding protein (C/EBP) family are involved in the regulation of cellular differentiation and function of many tissues. Unlike the other members of the family, C/EBP epsilon expression is restricted to granulocytes, macrophages, and lymphocytes. C/EBP epsilon is highly conserved between human and rodents and is essential for terminal granulopoiesis in both species. To study the role that C/EBP epsilon plays in macrophages, wild-type and C/EBP epsilon-deficient (-/-) murine macrophages obtained from thioglycollate-elicited peritoneal lavages and differentiated bone marrow cells were compared. Although macrophage development occurred in both types of mice, the C/EBP epsilon -/- cells had a lower expression of macrophage markers and a morphologic and ultrastructural appearance of immaturity. Phagocytic function, measured by calculating the percentage of internalized opsonized fluorescein isothiocyanate (FITC)-labeled yeast, was significantly impaired in the C/EBP epsilon -/- macrophages compared with their wild-type counterparts. Furthermore, the differential expression of 26 macrophage-specific genes between wild-type and C/EBP-/- mice was analyzed. A subset of genes involved in differentiation, immune, and inflammatory responses was found down-regulated in the C/EBP-/- macrophages. Taken together, this study implicates the C/EBP epsilon gene as an important transcription factor required for normal function and development of macrophages.