Peta A. Tilbrook

Lyn tyrosine kinase is essential for erythropoietin- induced differentiation of J2E erythroid cells

Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum. In contrast, a mutant J2E clone (J2E‐NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone. We have shown previously that intracellular signalling is disrupted in the J2E‐NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation. In this study we investigated the defect in J2E‐NR cells that is responsible for their inability to differentiate. Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E‐NR cells. On closer examination, both lyn mRNA and protein content were reduced >500‐fold. Consistent with a defect in lyn, amphotropic retroviral infection of J2E‐NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically. Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells. However, erythropoietin‐supported viability was unaffected by reducing lyn activity. The ability of two other erythropoietin‐responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn. Finally, co‐immunoprecipitation and yeast two‐hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex. These data indicate for the first time an essential role for lyn in erythropoietin‐initiated differentiation of J2E cells but not in the maintenance of cell viability.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61β subunit

We report here the isolation of a new member of the ADP‐ribosylation factor (ARF)‐like family (ARL‐6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage‐restricted expression, ARL‐6 mRNA increased with erythropoietin‐induced maturation of J2E cells, and decreased with interleukin 6‐induced differentiation of M1 monoblastoid cells. In tissues, ARL‐6 mRNA was most abundant in brain and kidney. While ARL‐6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP‐γS, like many members of the ARF/ARL family. Using the yeast two‐hybrid system, six molecules which interact with ARL‐6 were identified including SEC61β, a subunit of the heterotrimeric protein conducting channel SEC61p. Co‐immunoprecipitation of ARL‐6 confirmed a stable association between ARL‐6 and SEC61β in COS cells. These results demonstrate that ARL‐6, a novel member of the ADP‐ribosylation factor‐like family, interacts with the SEC61β subunit.

HLS7, a hemopoietic lineage switch gene homologous to the leukemia‐inducing gene MLF1

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid‐specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin‐ or chemically‐induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin‐responsive colonies in semi‐solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

New insights into the regulation of erythroid cells

The regulation of erythroid cells is complex and occurs at multiple levels. Erythroid precursors, once committed to this lineage, develop in association with specific macrophages within erythroblastic islands. While erythropoietin (Epo) is the principal regulator of erythroid progenitors, other cytokines and nuclear hormones also play an important role in the maturation of these cells. Signalling from the Epo‐receptor activates several pathways, including the JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Transcription factors such as GATA‐1, EKLF and NF‐E2 are crucial for progression along the erythroid maturation pathway; these, and a myriad of other transcription factors, must be expressed at the correct developmental stage for normal red blood cells to be formed. IUBMB Life, 56: 177‐184, 2004

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

The erythropoietin receptor.

The erythropoietin (epo) receptor is a member of the cytokine receptor family. It is expressed almost exclusively on erythroid precursor cells and controls the development of red blood cells. The epo receptor has no intrinsic kinase activity, but binds intracellular tyrosine kinases to elicit its signals. Alterations in the transmission of the signalling cascade lead to clinically abnormal red blood cell production.

Erythropoietin preconditioning in neuronal cultures : Signaling, protection from in vitro ischemia, and proteomic analysis

In this study we confirmed the presence of the erythropoietin (EPO) receptor on both cultured cortical neurons and PC12 cells and showed that EPO can induce changes in p38, ERK, and JNK signaling molecules in these cells. We induced EPO preconditioning in cortical neuronal cultures that protected neurons from a subsequent in vitro ischemic insult (transient oxygen‐glucose deprivation). To investigate downstream changes in protein expression in EPO‐preconditioned cortical neuronal cultures, we used two‐dimensional gel electrophoresis. Overall, EPO preconditioning resulted in protein up‐regulation, and, from 84 of the most differentially expressed proteins selected for identification, the proteins or tentative proteins were identified in 57 cases, representing 40 different proteins. Different protein spots representing the same or closely related protein(s) occurred for 13 of the identified proteins and are likely to represent posttranslational modifications or proteolytic fragments of the protein. Two proteins (78‐kD glucose‐regulated protein and tropomyosin, fibroblast isoform 1) were detected in control neuronal cultures, but not following EPO preconditioning treatment, whereas one protein (40S ribosomal protein SA) was detected only following EPO preconditioning. Most of the other proteins identified had not previously been associated with EPO preconditioning and will aid in the understanding of EPOs neuroprotective response and possibly the development of new therapeutic interventions to inhibit neuronal death in acute and chronic neurodegenerative diseases.

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn−/− mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn−/− erythroblasts, and the phenotype of Lyn−/− animals was remarkably similar to GATA-1low animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn−/− animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn−/− mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

Differential regulation of SOCS genes in normal and transformed erythroid cells

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells – erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1−/− mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases.

The J2E erythroid cell line, transformed by retroviral v-raf/v-myc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constituitive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.