W Kruijer

Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts

In the present study, we examined the underlying mechanism, which causes the constitutive tyrosine phosphorylation of signal transducer and activator of transcription 5 (STAT5) in acute myeloid leukemia (AML) blasts. Constitutive STAT5 phosphorylation was observed in 18 of 26 (69%) patients with AML. The constitutive STAT5 phosphorylation was caused by different mechanisms. In the majority of the investigated cases (71% (12 of 17)) constitutive STAT5 phosphorylation was associated with autophosphorylation of the type III receptor tyrosine kinase Flt3. In 47% (eight of 17) of these cases autophosphorylation of Flt3 coincided with tandem duplications of the Flt3 gene, resulting in constitutive phosphorylation of the receptor, while 24% (four of 17) of the cases demonstrated STAT5 phosphorylation and Flt3 autophosphorylation without mutations. In addition, a subset of AML cases (29% (five of 17)) had no autophosphorylation of the Flt3 receptor, but demonstrated constitutive STAT5 phosphorylation, which was partly due to autocrine growth factor production. All AML cases with high STAT5 and Flt3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Addition of the receptor tyrosine III kinase inhibitor AG1296 strongly inhibited STAT5 phosphorylation and enhanced the percentage of apoptotic cells without modulating the Bcl-xl protein levels. These data indicate that in the majority of AML cases the constitutive STAT5 phosphorylation is caused by Flt3 phosphorylation mostly due to mutations in the receptors and associated with a low degree of spontaneous apoptosis.

The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway.

In the present study we investigated a possible role for the p38 mitogen‐activated protein (MAP) kinase pathway in mediating nuclear factor‐kappa B (NF‐κB) transcriptional activity in the erythroleukaemic cell line TF‐1. TF‐1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF‐κB and GAL4p65‐regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK specific inhibitor SB203580 (1u2003μM) or overexpression of kinase‐deficient mutants of MKK3 or MKK6 did not affect OA‐enhanced NF‐κB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10u2003μM SB203580 enhanced rather than inhibited NF‐κB‐mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit. The SB203580‐mediated increase in NF‐κB transcriptional activity was associated with enhanced phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 and c‐Jun N‐terminal kinase (JNK), but not p38 kinase. Overexpression of kinase‐deficient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant‐negative Raf‐1 abrogated SB203580‐enhanced NF‐κB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing effects of SB203580 on NF‐κB‐mediated gene transcription. This study demonstrates that the p38 MAP kinase pathway is not involved in the OA‐induced activation of NF‐κB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF‐κB transcriptional activity.

Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction

The cytokine interleukin‐6 (IL‐6) rapidly activates a latent cytoplasmic transcription factor, acute‐phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate‐early gene induction by IL‐6 and, as we show here, stimulation of the promoters of the genes for α 2‐macroglobulin, Jun‐B, and intercellular adhesion molecule‐1 (ICAM‐1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL‐6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL‐6 signal transduction leading to target gene induction.

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-κB, might determine the final cellular response.

An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation

In this study, we examined the involvement of the phosphatidylinositol 3-kinase (PI3-K) and p70S6 kinase signal transduction pathway in the interleukin-1(IL-1)-mediated proliferation and cytokine production by normal and leukemic myeloid cells. Total AML blast populations, early progenitor (CD34(+)/CD36(-)) cells, and more differentiated (CD34(-)/CD36(+)) cells were treated with the PI3-K inhibitor Ly294002 and p70S6K inhibitor rapamycin. The effects on proliferation, IL-6 protein secretion, and intracellular signaling cascades were determined and compared with normal CD34(+) cells and monocytes. The function of the PI3-K pathway was dependent on the differentiation state of the AML cell population. In immature blasts, the IL-1-induced proliferation was strongly inhibited by Ly294002 and rapamycin, without a distinct effect on IL-6 protein production. In contrast, in mature monocytic blast cells inhibition of the PI3-K signaling route had a stimulatory effect on IL-6 protein secretion. Interestingly, these findings were not specifically linked to the malignant counterpart but were also observed with normal CD34(+) sorted cells vs mature monocytes. Evidence is provided that the Ly294002-induced increase in IL-6 protein secretion is linked to the cAMP dependent signaling pathway and not to changes in the phosphorylation of ERK or p38. However, although the enhanced IL-6 protein secretion is cAMP dependent, it was not found to be mediated by protein kinase A (PKA) or by the GTP-ase Rap1. This study indicates that inhibition of the PI3-K signaling pathway has an inhibitory effect on cell proliferation but a stimulatory effect on IL-6 expression mediated by a cAMP-dependent but PKA-independent route.

Downregulation of IL-6-induced STAT3 tyrosine phosphorylation by TGF-β1 is mediated by caspase-dependent and -independent processes

To explore the possible cross-talk between the IL-6 and TGF-β1 pathways in AML blast cells, the effect of TGF-β1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-β1 pretreatment was observed in four out of 40 AML cases (10%), although all of the AML cases responded to TGF-β1 by means of SMAD3 translocation. The reduced IL-6-mediated STAT3 tyrosine phosphorylation after pre-treatment with TGF-β1 was associated with apoptosis and coincided with the degradation of certain cellular proteins, including JAK1 and -2 and Tyk2, without affecting the ERK expression and phosphorylation. Furthermore, treatment of AML blasts with the cytostatic agent VP16, as an alternative way to induce apoptosis, resulted in a similar degree of degradation of JAK kinases and concomitant reduction of IL-6-mediated STAT3 tyrosine phosphorylation. Although degradation of JAK kinases could be rescued by incubating the cells with the pan-caspase inhibitor Z-VAD-fmk, the attenuating effect of TGF-β1 treatment on the STAT3 tyrosine phosphorylation was still partly present. It was shown that in AML cells cultured in the presence of Z-VAD-fmk, TGF-β1 pretreatment resulted in a reduction of JAK1 phosphorylation upon IL-6 stimulation. Expression of SOCS1 and -3 could be ruled out as a possible cause of reduced JAK1 phosphorylation levels in the investigated AML case.

TAK1 activation of the mouse JunB promoter is mediated through a CCAAT box and NF-Y

The JunB gene is activated by many stimuli including transforming growth factor β (TGFβ) family members and interleukin‐6 (IL‐6). Here the effect of TGFβ activated kinase 1 (TAK1), a mitogen activated protein kinase kinase kinase (MAPKKK) implicated in TGFβ, bone morphogenetic protein (BMP) and interleukin‐1 (IL‐1) signaling, on JunB promoter activity was investigated. Promoter analysis led to the identification of a CCAAT motif in the JunB gene, essential for activation by TAK1. Transfer of this CCAAT element to a heterologous minimal promoter conferred TAK1‐responsiveness. The CCAAT‐binding transcription factor, nuclear factor Y (NF‐Y), activated the JunB promoter and a dominant negative NF‐YA construct inhibited TAK1 activation of JunB. Our results demonstrate that JunB gene activation by TAK1 is mediated by the CCAAT‐binding factor NF‐Y.