Abstract
IL-13 is a Th2 cytokine that plays a critical role in antibody production, parasite immunity, allergic disorders, and as a negative regulator of anti-tumor immunity. Curiously, microarray ...screens of activated Th1 clones demonstrated high levels of IL-13 RNA in spite of essentially no IL-13 protein production. Such findings were subsequently confirmed in primary cells cultured in Th1 conditions. We hypothesized that these observations might be due to regulation by miRNAs. Indeed, a highly conserved Let-7 site is predicted in the 3’ UTR of IL-13. Furthermore, several Let-7 family members are more highly expressed in activated Th1 cells when compared with activated Th2 cells. To test our hypothesis, we generated luciferase reporter constructs containing the native 3’ UTR of IL-13 (WT) or a 3’ UTR with the Let-7 site mutated (Mut). Overexpression of Let-7 mimetics inhibited the IL-13 WT but not the IL-13 Mut construct. In addition, the transfection of the mimetics or infection with Let-7g expressing retrovirus led to the inhibition of native IL-13 protein production. Alternatively, inhibitors of Let-7 family members or infection with a retrovirus encoding the Let-7 antagonist LIN28B led to enhanced IL-13 production. Overall our data suggest Let-7 miRNA plays a critical role in the regulation of IL-13 and that such molecules might be exploited to inhibit IL-13 in allergic diseases and to enhance anti-tumor immunity.
Abstract
Activating mutations in codon D816 of the tyrosine kinase receptor, c-KIT, are found in the majority of patients with systemic mastocytosis, an aggressive disease characterized by ...proliferation of abnormal mast cells. Constitutive activation of the KIT signaling pathway is critical to the transformed phenotype. The MITF transcription factor is essential for normal mast cell development. We found that MITF is highly expressed in malignant mast cells from bone marrow biopsies of patients with systemic mastocytosis with the D816V KIT mutation; however, it is not highly expressed in mast cells from bone marrow biopsies of patients with other hematologic conditions. We show that in both primary and transformed mast cell lines, KIT signaling markedly upregulates MITF protein expression. MITF mRNA levels do not change significantly with KIT signaling, suggesting post transcriptional regulation. We performed an array screen for KIT-regulated microRNAs in mast cells and found that miR-539 and miR-381 are downregulated by KIT signals; furthermore, they suppressed MITF expression through conserved sites in the MITF 3' UTR. We demonstrate the requirement of MITF for the transformed phenotype by inhibiting tumor growth in colony forming assays and in a murine xenograft model by knockdown of MITF with a shRNA-expressing lentivirus. This work demonstrates a novel regulatory pathway between two critical mast cell factors, KIT and MITF, mediated by microRNAs; dysregulation of this pathway may contribute to abnormal mast cell proliferation and malignant mast cell disease.
Polycythemia vera (PV), the most common myeloproliferative disorder, arises due to somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. A somatic JAK2 V617F point ...mutation is found in over 80% of PV patients; however, it is not clear if the JAK2 V617F is the disease initiating mutation, sincethere are PV JAK2 V617F negative patients who have monoclonal hematopoiesis and erythropoietin independent erythropoiesis;in individual PV families, there are PV subjects with and without the JAK2 V617F mutation; andanalysis of clonal PV populations reveals the presence of <50 and >50% mutated JAK2 cells (Nussenzweig' abstract this mtg), suggesting a mixed population of cells with regard to JAK2 status.In order to search for possible PV contributing molecular defect(s), we studied microRNAs (miRNAs) in a homogeneous population of in vitro expanded erythroid progenitors. MiRNAs are non-coding, small RNAs that regulate gene expression at the posttranscriptional level by direct mRNA cleavage, by translational repression, or by mRNA decay mediated by deadenylation. MiRNAs play an important regulatory role in various biological processes including human hematopoiesis. In vitro expanded erythroid progenitors were obtained from peripheral blood mononuclear cells of 5 PV patients (JAK2 V617F heterozygotes) and from 2 healthy donor controls. The cells were cultured in an erythroid-expansion medium for 21 days resulting in 70–80% homogenous erythroid cell population of identical differentiation stage. Gene expression profiling of miRNAs (Thomson, Nature Methods, 1:1, 2004) was performed using a custom microarray (Combimatrix) with 326 miRNA probes. Data were normalized by the global median method. The miRNAs with expression ratios greater than 1.5 or less than 0.5 were considered to be abnormal. Comparative analyses of controls versus PV samples revealed up-regulated expression of miR-let7c/f, miR-16, miR-451, miR-21, miR-27a, miR-26b and miR-320 and down-regulation of miR-150, miR-339 and miR-346 in PV. In addition, miR-27a, miR-26b and miR-320 were expressed only in PV. The putative targets of these miRNAs were predicted by TargetScan prediction algorithm. Up-regulated miR-let-7, miR-16 and miR-26b may modulate cyclin D2, which has an important role in G1/S transition and can be a target in the JAK2/STAT5 pathway (Walz, JBC , 281:18177, 2006). One of the putative targets of up-regulated miR-27a is EDRF1 (erythroid terminal differentiation related factor1), a positive regulator of erythroid differentiation. The BCL-6 gene is predicted to be the target of miR-339 and miR-346, and its activation blocks cellular differentiation. MiR-16 is known to be down-regulated in CLL, where it targets anti-apoptotic BCL-2; in contrast, we show that miR-16 is up-regulated in PV erythroid cells. We identified differentially expressed miRNAs in PV which target genes involved in the JAK/STAT pathway or genes that are modulated by JAK2 downstream molecules. This study indicates that miRNA dysregulation may play an important role in erythropoietic differentiation and proliferation in PV. Expression analyses of these miRNAs in a larger set of PV samples, using quantitative Real-Time-PCR, are in progress. Further, earlier erythroid and pluripotent hematopoietic progenitors are also being analyzed.
PDF