Kenjirou Kamezaki

Roles of Stat3 and ERK in G-CSF signaling

G‐CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil‐granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G‐CSF–induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G‐CSF–induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3‐deficient mice. In hematopoietic cells from Stat3‐deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G‐CSF stimulation. Stat3‐null bone marrow cells displayed a significant activation of extra‐cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G‐CSF stimulation. Furthermore, the augmented proliferation of Stat3‐deficient bone marrow cells in response to G‐CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G‐CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G‐CSF.

Intracellular signal transduction of interferon on the suppression of haematopoietic progenitor cell growth

Summary. Interferon (IFN)‐α and IFN‐γ suppress the growth of haematopoietic progenitor cells. IFN‐α activates Janus kinase‐1 (Jak1) and Tyrosine kinase‐2 (Tyk2), followed by the phosphorylation of the signal transducers and activators of transcription, Stat1 and Stat2. IFN‐γ activates Jak1 and Jak2, followed by the activation of Stat1. Activated Stats bind the promoter regions of IFN‐inducible genes. We evaluated the role of Tyk2 and Stat1 in the IFN‐mediated inhibition of haematopoietic progenitor cell growth. While IFN‐α (1000 U/ml) suppressed the number of granulocyte‐macrophage colony‐forming units (CFU‐GM) or erythroid burst‐forming units (BFU‐E) from wild‐type mouse bone marrow cells, this suppression was partially inhibited by a deficiency in Tyk2 and completely inhibited by a deficiency in Stat1. High levels of IFN‐α (10 000 U/ml) suppressed the CFU‐GM or BFU‐E obtained from Stat1‐deficient mice, but did not suppress this growth in cells from Tyk2‐deficient mice. Stat1 was phosphorylated by IFN‐α in Tyk2‐deficient cells, although the level of phosphorylation was weaker than that observed in wild type mice. Thus, the inhibitory signal on haematopoietic progenitor cells mediated by IFN‐α may be transduced by two signalling pathways, one regulated by Tyk2 and the other dependent on Stat1. IFN‐γ also suppressed the number of CFU‐GM or BFU‐E, and this pathway was mediated by IFN‐γ in a Stat1‐dependent manner, independently of Tyk2.

The effect of anabolic steroids on anemia in myelofibrosis with myeloid metaplasia : retrospective analysis of 39 patients in Japan

Between 1999 and 2005,285 patients received new diagnoses of myelofibrosis with myeloid metaplasia (MMM) in Japan. Anemic symptoms were present in 162 patients, and hemoglobin (Hb) concentrations were <10 g/dL in 197 patients. Fifty-five MMM patients were treated with anabolic steroids, and their effect on anemia during MMM was evaluated in 39 patients. A “good” response was defined as an Hb increase of ≥1.5 g/dL, cessation of transfusion dependence, and an Hb concentration of >10 g/dL maintained for at least 8 weeks. A “minimum” response was defined as an Hb increase of ≥1.5 g/dL and transfusion independence for at least 8 weeks. Both good and minimum responses were considered “favorable.” Favorable responses were achieved in 17 patients (44%, 8 good and 9 minimum responses). None of the pretreatment variables, such as the lack of transfusion dependence, a higher Hb concentration at the start of treatment, or the absence of cytogenetic abnormalities, were associated with a response to anabolic steroid therapy. Adverse events associated with anabolic steroid therapy were moderate and transient. Two patients required definitive withdrawal of treatment. Thus, anabolic steroids are well tolerated and effective for the treatment of anemia in a subset of MMM patients.

The lipocalin 24p3, which is an essential molecule in IL-3 withdrawal-induced apoptosis, is not involved in the G-CSF withdrawal-induced apoptosis.

Abstract: Many hematopoietic cells undergo apoptosis when deprived of specific cytokines. Lipocalin 24p3, reported to be induced in hematopoietic cells by interleukin 3 (IL‐3) depletion, induces hematopoietic cell apoptosis despite the presence of IL‐3. As granulocyte colony stimulating factor (G‐CSF) depletion also induces the apoptosis of G‐CSF‐dependent cell line cells, we examined the effect of 24p3 on the apoptotic function induced by G‐CSF depletion. 24p3 was induced by the depletion of IL‐3, but not G‐CSF, in cytokine‐dependent cell lines. Although 24p3 suppressed growth induced by IL‐3, it did not influence G‐CSF‐dependent cell growth. These observations show that 24p3 is not involved in the G‐CSF withdrawal‐induced apoptosis, although it is essential in IL‐3 withdrawal‐induced apoptosis.

Tyrosine Kinase 2 Interacts with and Phosphorylates Receptor for Activated C Kinase-1, a WD Motif-Containing Protein

Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cγ, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-αβ receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138–203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

A Novel Mutation in the Juxtamembrane Intracellular Sequence of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Gene in a Patient with Severe Congenital Neutropenia Augments G-CSF Proliferation Activity but Not through the MAP Kinase Cascade

We analyzed the structure of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a 6-year-old female patient with severe congenital neutropenia (SCN) who experienced severe recurrent infections since 1 month of age. There is no family history of any similar disease. When the patient was 4 months old, she began receiving treatment with recombinant human G-CSF that resulted in a small increase in the neutrophil count sufficient for the prevention and treatment of bacterial infection. An analysis of complementary DNA for the patient’s G-CSF receptor revealed a 3-base pair deletion in the juxtamembrane intracellular sequence. This deletion at the beginning of exon 16 was thought to be caused by alternative splicing; analysis of the DNA revealed a G-to-A point mutation of the final nucleotide of intron 15. To evaluate the functional activity of the G-CSF receptor with this 3-base pair deletion of the juxtamembrane region, we transfected this G-CSF receptor mutant into an interleukin 3-dependent cell line, BAF/3. BAF/3 cells expressing the mutant G-CSF receptor showed augmented proliferation activity in response to G-CSF compared with cells having the wild-type G-CSF receptor. Although the proliferation signal of G-CSF in normal hematopoiesis is transduced through the activation of MAP kinases, this G-CSF receptor mutant showed decreased activation of ERK1/2 in response to G-CSF compared with the wild type, but the transduced signal for Stat3 activation by G-CSF was of the same magnitude as that of the wild-type G-CSF receptor. This result means that the augmented proliferation activity in response to G-CSF that we observed in cells having the G-CSF receptor gene with the 3-base pair deletion is transduced through an intracellular signaling pathway other than MAP kinase. Because SCN patients with a mutation in the G-CSF receptor frequently develop leukemia, this 3-base pair deletion in the juxtamembrane sequence of the G-CSF receptor gene in this patient may be one step in the course of leukemic transformation.