Functional involvement of Daxx in gp130‐mediated cell growth and survival in BaF3 cells

Abstract

Death domain-associated protein (Daxx) is a multifunctional protein that modulates both cell death and transcription. Several recent studies have indicated that Daxx is a mediator of lymphocyte death and/or growth suppression, although the detailed mechanism is unclear. Previously, we reported that Daxx suppresses IL-6 family cytokine-induced gene expression by interacting with STAT3. STAT3 is important for the growth and survival of lymphocytes; therefore, we here examined the role of Daxx in the gp130/STAT3-dependent cell growth/survival signals. We found that Daxx suppresses the gp130/STAT3-dependent cell growth and that Daxx endogenously interacts with STAT3 and inhibits the DNA-binding activity of STAT3. Moreover, small-interfering RNA-mediated knockdown of Daxx enhanced the expression of STAT3-target genes and accelerated the STAT3-mediated cell cycle progression. In addition, knockdown of Daxx-attenuated lactate dehydrogenase leakage from cells, indicating that Daxx positively regulates cell death during gp130/STAT3-mediated cell proliferation. Notably, Daxx specifically suppressed the levels of Bcl2 mRNA and protein, even in cytokine-unstimulated cells, indicating that Daxx regulates Bcl2 expression independently of activated STAT3. These results suggest that Daxx suppresses gp130-mediated cell growth and survival by two independent mechanisms: inhibition of STAT3-induced transcription and down-regulation of Bcl2 expression.Key words: Cell death . Daxx . gp130 . IL-6 . STAT3 IntroductionDeath domain-associated protein (Daxx) is a multifunctional protein that modulates both cell death and transcription [1]. Daxx is present in most cell types and is mainly located in the nucleus. Because previous studies have demonstrated that Daxx can bear both proand anti-cell-death activities depending on the stimulus and the cell type [1,2], the precise role of Daxx, in particular its ability to promote or hinder cell death, remains controversial. Nevertheless, in lymphocytes, the following observations indicate that Daxx has procell death and/or growth-suppressing functions. First, Daxx is induced by type I interferon in pro-B cells and is required for interferon-induced apoptosis [3]. Second, in Con A-stimulated splenocytes, Daxx up-regulation and interaction with PML correlate with the induction of B-cell apoptosis [4]. Third, CD40-induced proliferation is profoundly reduced in transgenic B cells over-expressing Daxx [5]. Fourth, analysis of T-cell-specific transgenic mice expressing a dominant negative form of Daxx (Daxx-DN) [18][19][20], and the chromatin-modifying protein a-thalassemia syndrome protein [21,22], it seems possible that Daxx regulates cellular processes by regulating transcription of specific genes under different conditions. We have previously shown that Daxx suppresses STAT3-mediated transcriptional activity and has a role in regulating IL-6 family cytokine signaling and gene induction in several cancer cell lines [16].The IL-6 family of cytokines utilizes the membrane glycoprotein gp130 as a cri...

A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3

“…An interleukin (IL)-3-dependent murine pro-B cell line, BaF-G133, was maintained in RPMI 1640 medium supplemented with 10% FCS with 10% of WEHI-3B conditioned medium as a source of IL-3 (25,26). ARL3-knockdown HeLa cell lines (cell lines 2-4 and 2-10) were established by transduction with pGPU6/GFP/Neo vector (Shanghai GenePharm, Shanghai, China) bearing short hairpin RNA (shRNA) targeting ARL3 (5Ј-GCAGCTTGCATCTGAA-GACAT-3Ј) and then selected with G418 (1 mg/ml; Sigma) (27).…”

“…Cell Proliferation Assay-The numbers of viable BaF-G133 cells after the indicated treatments were measured using a WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay (cell counting kit-8; Wako Pure Chemicals) (26). Briefly, 10 l of WST-8 solution was added to the cells in each well and incubated for 2 h. The absorbances were measured at a test wavelength of 450 nm and a reference wavelength of 595 nm using a microplate reader (Bio-Rad).…”

“…4, A and B, G-CSF/ STAT3-mediated SOCS3 expression was also decreased in ARL3 siRNA-treated BaF-G133 cells. BaF-G133 cells have also been used to measure gp130/STAT3-mediated cell growth by G-CSF treatment (25,26,40). We therefore tested the effect of reduced ARL3 expression on G-CSF/gp130/STAT3-mediated BaF-G133 cell growth.…”

Advances in the study of the biological function of Daxx

“…What is more, Daxx 501 to 625-induced apoptosis is mediated through the ASK1-MEK-JNK/p38-Bim-Bax-dependent caspase pathway. Muromoto et al (2010) found out that knock down of Daxx attenuated lactate dehydrogenase leakage from cells, indicating that Daxx positively regulates cell death during gp130/STAT3-mediated cell proliferation.…”

“…Muromoto et al (2010) found out that Daxx suppresses the gp130/STAT3-dependent cell growth and that Daxx endogenously interacts with STAT3 and inhibits the DNAbinding activity of STAT3. Moreover, small-interfering RNA-mediated knockdown of Daxx enhanced the expression of STAT3-target genes and accelerated the STAT3-mediated cell cycle progression.…”