To explore the activation patterns of signal transducer and activator of transcription 3 (Stat3) in acute myeloid leukemia (AML), we examined whether the phosphorylation of tyrosine705 (Tyr705) and serine727 (Ser727) residues was abnormally regulated in cells from patients with AML. In 5 of 20 (25%) patients with AML, Stat3 was constitutively phosphorylated on Tyr705 and Ser727, which were not further up-regulated by treatment with IL-6. Furthermore, Stat3 was constitutively bound to the IRE response element in these cells as determined by electrophoretic mobility shift assay, and stimulation with IL-6 did not result in increased DNA binding. Interestingly, AML cells with constitutive Stat3 activation also secreted high levels of IL-6 protein. Treating these AML cells with anti-IL-6 resulted in restored IL-6-inducible Stat3 phosphorylation on both Tyr705 and Ser727 with low or undetectable basal phosphorylation levels in unstimulated cells. In contrast, treatment with anti-IL-1 did not result in altered Stat3 phosphorylation patterns. The constitutive IL-6 expression was associated with elevated levels of suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 mRNA expression, which were not down-regulated by anti-IL-6. These data indicate that the constitutive Stat3 activation in the investigated AML blasts is caused by high IL-6 secretion levels, thus stimulating the Jak/Stat pathway in an autocrine manner, a paracrine manner, or both. (Blood. 2000;95:3765-3770)
"To date, conflicting evidence exists concerning the kinase responsible for STAT3 (ser727) phosphorylation. Some members of the MAPK family, such as Protein kinase C, Jun N-terminal kinase, extracellular signal-regulated kinase1/2 (ERK1/2), p38, and mammalian target of rapamycin (mTOR), seem to be involved, but their implications remain unclear    . The apparent divergence of results may be due to the variation of cell systems and stimuli employed in the different studies. "
[Show abstract][Hide abstract] ABSTRACT: Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.
The Scientific World Journal 10/2013; 2013(2-3):259845. DOI:10.1155/2013/259845 · 1.73 Impact Factor
"In contrast to the tumor suppressor function of activated STAT1, STAT3 is mainly identified as an oncogene , . Constitutive tyrosine and serine phosphorylation of STAT3 has been demonstrated in many different malignancies, including AML , . Inhibition of STAT3 activation induces apoptosis in AML cell lines and primary samples . "
[Show abstract][Hide abstract] ABSTRACT: Dasatinib (BMS-354825) is a FDA-approved multitargeted kinase inhibitor of BCR/ABL and Src kinases. It is now used in the treatment of chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapies, including imatinib. Here we report a novel effect of dasatinib on inducing the differentiation of acute myeloid leukemia (AML) cells through MEK/ERK-dependent activation of signal transducer and activator of transcription 1 (STAT1). We found that dasatinib could induce the differentiation of AML cells as demonstrated by the expression of differentiation marker CD11b, G0/G1 phase arrest and decreased ratio of nucleus to cytoplasm. Of note, dasatinib induced robust phosphorylation of STAT1 both at Tyr701 and Ser727 as well as the redistribution of STAT1 from the cytoplasm to the nucleus, thus leading to the transcription of STAT1-targeted genes. Knocking down STAT1 expression by shRNA significantly attenuated dasatinib-induced differentiation, indicating an important role of STAT1 in myeloid maturation. We further found that dasatinib-induced activation of STAT1 was regulated by the MEK/ERK kinases. The phosporylation of MEK and ERK occurred rapidly upon dasatinib treatment and increased progressively as differentiation was induced. MEK inhibitors PD98059 and U0216 not only inhibited the phosphorylation of STAT1, but also abrogated dasatinib-induced myeloid differentiation, suggesting that MEK/ERK dependent phosphorylation of STAT1 might be indispensable for the differentiating effect of dasatinib in AML cells. Taken together, our study suggests that STAT1 is an important mediator in dasatinib-induced differentiation of AML cells, whose activation requires the activation of MEK/ERK cascades.
PLoS ONE 06/2013; 8(6):e66915. DOI:10.1371/journal.pone.0066915 · 3.23 Impact Factor
"STAT3 has two phosphorylation sites, Ser727 and Tyr705. Phosphorylation of these sites leads to activation of STAT3 (Schuringa et al., 2000). As expected, total STAT3 expression in the testis was not changed with any treatment (Fig. 2A and B). "
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that the mitochondria-derived cytoprotective peptide humanin (HN), when administered intratesticularly to rats, rescues germ cells from apoptosis secondary to testicular stress of hormonal deprivation induced by gonadotropin-releasing hormone antagonist (GnRH-A). To decipher the cellular mechanisms of HN action in the amelioration of GnRH-A-induced germ cell apoptosis, adult male rats received the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of GnRH-A on Day 1 and daily IT injection of saline; (iii) daily IT injection of synthetic HN; and (iv) GnRH-A injection on Day 1 and daily IT injection of HN (GnRH-A+HN). HN alone had no effect on germ cell apoptosis. GnRH-A increased germ cell apoptosis and BAX in the testicular mitochondrial fractions. Synthetic HN decreased germ cell apoptosis induced by GnRH-A and BAX in the mitochondria. We deduced that the cytoprotective action of synthetic HN on GnRH-A-induced germ cell apoptosis was mediated by attenuating p38 mitogen-activated protein kinase activity and increasing STAT3 phosphorylation. The effect of synthetic HN on the expression of endogenous rat HN in the testis was studied using rat HN specific antibody. GnRH-A treatment increased, but concomitant treatment with synthetic HN reduced endogenous rat HN expression in both cytosolic and mitochondrial fractions in testis. Co-immunoprecipitation experiments demonstrated that the increased rat HN was physically associated with BAX in the cytosolic testicular fractions after GnRH-A treatment. Double-immunofluorescence staining confirmed the co-localization of BAX and rat HN in the cytoplasm of Leydig cells and spermatocytes after GnRH-A treatment. We conclude that the cytoprotective effect of exogenously administered synthetic HN is mediated by interactions of endogenous rat HN with BAX in the cytoplasm preventing the entry of BAX to the mitochondria to govern the fate of germ cell survival or death during pro-apoptotic stress to the testis in rats.
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