Shu Xing

Identification of an Acquired JAK2 Mutation in Polycythemia Vera

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C→ T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val617 to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2(V617F), showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs.

Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growth.

JAK2V617F, a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva™) is a potent inhibitor of JAK2V617F activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2V617F-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2V617F-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2V617F-positive PV and other myeloproliferative disorders.

Potent and selective inhibition of T-cell protein tyrosine phosphatase (TCPTP) by a dinuclear copper(II) complex

A dinuclear Cu(II) complex, [Cu(2)(μ-IDA)(phen)(3)(NO(3))]NO(3)·4H(2)O (phen = 1,10-phenanthroline, H(2)IDA = iminodiacetic acid), was found to potently and selectively inhibit T-cell protein tyrosine phosphatase, and lead to the anti-proliferation and apoptosis of C6 glioma cells.

Magnolia officinalis Extract Contains Potent Inhibitors against PTP1B and Attenuates Hyperglycemia in db/db Mice

Protein tyrosine phosphatase 1B (PTP1B) is an established therapeutic target for type 2 diabetes mellitus (T2DM) and obesity. The aim of this study was to investigate the inhibitory activity of Magnolia officinalis extract (ME) on PTP1B and its anti-T2DM effects. Inhibition assays and inhibition kinetics of ME were performed in vitro. 3T3-L1 adipocytes and C2C12 myotubes were stimulated with ME to explore its bioavailability in cell level. The in vivo studies were performed on db/db mice to probe its anti-T2DM effects. In the present study, ME inhibited PTP1B in a reversible competitive manner and displayed good selectivity against PTPs in vitro. Furthermore, ME enhanced tyrosine phosphorylation levels of cellular proteins, especially the insulin-induced tyrosine phosphorylations of insulin receptor β-subunit (IRβ) and ERK1/2 in a dose-dependent manner in stimulated 3T3-L1 adipocytes and C2C12 myotubes. Meanwhile, ME enhanced insulin-stimulated GLUT4 translocation. More importantly, there was a significant decrease in fasting plasma glucose level of db/db diabetic mice treated orally with 0.5 g/kg ME for 4 weeks. These findings indicated that improvement of insulin sensitivity and hypoglycemic effects of ME may be attributed to the inhibition of PTP1B. Thereby, we pioneered the inhibitory potential of ME targeted on PTP1B as anti-T2DM drug discovery.

Relevance of JAK2V617F positivity to hematological diseases - survey of samples from a clinical genetics laboratory

BackgroundJAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value.MethodsIn the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy.ResultsWe found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities.ConclusionsOur study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.

Tea contains potent inhibitors of tyrosine phosphatase PTP1B

Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 value of 0.4-4 g dry tea leaves per liter of water. Black tea shows the strongest inhibition activities, followed by oolong and then by green tea. Biochemical fractionations demonstrated that the major effective components in tea corresponded to oxidized polyphenolic compounds. This was further verified by the fact that tea catechins became potent inhibitors of PTP1B upon oxidation catalyzed by tyrosinases. When applied to cultured cells, tea extracts induced tyrosine phosphorylation of cellular proteins. Our study suggests that some beneficial effects of tea may be attributed to the inhibition of PTP1B.

Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases

Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI‐TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono‐phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.

Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0.

Identification of common differentially‑expressed miRNAs in ovarian cancer cells and their exosomes compared with normal ovarian surface epithelial cell cells

The aim of the present study was to identify common microRNAs (miRNAs) in ovarian cancer (OC) cells and their exosomes using microarray data (accession number GSE76449) available from the Gene Expression Omnibus database, including exosomal samples from 3 OC cell lines, 1 normal ovarian surface epithelial cell line and their original cell samples. Differentially-expressed miRNAs (DE-miRNAs) were identified using the Linear Models for Microarray data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2 database. The potential functions of the target genes of the DE-miRNAs were analyzed using the Database for Annotation, Visualization and Integrated Discovery tool. The association between crucial miRNAs and target genes, and their clinical associations, were validated using The Cancer Genome Atlas data. As a result, 12 upregulated and 12 downregulated DE-miRNAs were shared by the 3 OC cell lines compared with normal controls in the exosomal samples, while 5 upregulated and 65 downregulated DE-miRNAs were shared between the original cells. Among them, 9 downregulated DE-miRNAs were shared between exosomal and original cells. The target genes of 4 common DE-miRNAs between exosomal and original cells (miR-127-3p, miR-339-5p, miR-409-3p and miR-654-3p) were predicted. Functional enrichment analysis indicated that these target genes may be involved in the Wnt signaling pathway (miR-409-3p-CTBP1 and miR-339-5p-CHD8) and Proteoglycans in cancer (miR-127-3p-PPP1CA). The negative associations between these 3 miRNAs and target genes were confirmed by a Pearsons correlation analysis. miR-127 was negatively associated with tumor grade. In conclusion, our results describe a set of miRNAs involved in OC development, in exosomal and non-exosomal manners, by regulating their target genes. They may be potential targets for treatment of OC.