Serine phosphorylation of STAT3 is essential for Mcl-1 expression and macrophage survival

Abstract

The Bcl-2 family member Mcl-1 is essential for macrophage survival. However, the mechanisms that contribute to the expression of Mcl-1 in these cells have not been fully characterized. The present study focused on the role of signal transducer and activator of transcription 3 (STAT3) in regulation of Mcl-1 in macrophages. Sodium salicylate (NaSal) treatment induced apoptotic cell death in primary human macrophages in a dose-and time-dependent fashion. Incubation with NaSal resulted in the loss of mitochondrial transmembrane potential, the release of cytochrome c and second mitochondria-derived activator of caspase/ direct IAP binding protein with low pH of isoelectric point (pI) from the mitochondria, and the activation of caspases 9 and 3. Western blot analysis and reverse transcription-polymerase chain reaction demonstrated that NaSal down-regulated the expression of Mcl-1. Electrophoretic mobility shift assay and Western blot analysis for phosphorylated STAT3 demonstrated that STAT3 was constitutively activated in macrophages and that this IntroductionMcl-1 was first cloned from ML-1 cells, a human myeloid leukemia cell line, as an early-induction gene. 1,2 Structurally and functionally, Mcl-1 belongs to the prosurvival Bcl-2 family. 3 Mice transgenic for Mcl-1 demonstrated the enhanced viability of a wide range of hematopoietic cell types, including B and T lymphocytes, and CD 11b-positive myeloid cells including monocytes, macrophages, and polymorphonuclear leukocytes (PMNs) at both immature and mature stages of differentiation. 4 Mcl-1 is essential for the survival of certain hematopoietic cells because apoptosis is rapidly triggered by antisense (AS) depletion of Mcl-1 in primary human macrophages, in differentiating U937 cells, a human monocytic cell line, and in human polymorphonuclear cells (PMNs). [5][6][7] The cell type-specific mechanisms that contribute to the regulation of Mcl-1 are controversial. PI3K/Akt signaling pathway mediated Mcl-1 expression in interleukin-3 (IL-3)-induced TF-1 leukemia cells 8 and IL-6-stimulated human hepatoma cell line Hep3B. 9 In contrast, in human myeloma cells, the IL-6-induced expression of Mcl-1 was mediated through the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway. 10 Recent observations employing human PMNs and leukemic large granular lymphocytes (LGLs) demonstrated that the JAK/STAT3 pathway was responsible for the regulation of Mcl-1 expression. 11,12 Our recently published observations demonstrated that the PI3K/Akt-1 pathway was critical for the expression of Mcl-1 in primary human macrophages. 7 Functional analysis of the murine Mcl-1 promoter revealed a STAT binding SIE (serum inducible element) at position Ϫ86 to Ϫ93 and a CRE (cAMP response element) at Ϫ70 to Ϫ76, which contributed to IL-3 stimulation of Mcl-1 gene expression. 8 Putative SIE and CRE sites have also been identified in human Mcl-1 promoter region, 13 however, the mechanisms by which they may contribute to the regulation of Mcl-1 expression remain to be ...

Bacterial N-acylhomoserine lactone-induced apoptosis in breast carcinoma cells correlated with down-modulation of STAT3

“…For example, the high level of Bcl XL expressed in the Fas-resistant myeloma cell line U266 is reduced upon STAT3 inactivation, whereupon the cells undergo apoptosis (Catlett-Falcone et al, 1999). More recently, STAT3 has been implicated in the expression of Mcl-1, another Bcl-2 prosurvival family member, and members of the inhibitors of apoptosis (IAP) family in human macrophages and neutrophils, respectively (Hasegawa et al, 2003;Liu et al, 2003). While loss of Mcl-1 is associated with cytochrome c release from mitochondria and the activation of procaspase 9, IAPs are reported to interfere with the activation of procaspases 8 and 9 (Deveraux et al, 1998).…”

Increased macrophage activation mediated through toll‐like receptors in rheumatoid arthritis

“…The primary human macrophages used in this study were differentiated in vitro for 7 days from monocytes, which were purified by elutriation from the PB of healthy donors, as previously described (32)(33)(34)(35)(36)(37). Purified monocytes were suspended in medium without fetal bovine serum (FBS) and allowed to adhere to cell culture dishes for 1 hour.…”

“…Purified monocytes were suspended in medium without fetal bovine serum (FBS) and allowed to adhere to cell culture dishes for 1 hour. Afterward, medium was changed to RPMI 1640 supplemented with 20% FBS, 100 units penicillin, 100 g/ml streptomycin, and 1 g/ml polymyxin B. Adherent cells were allowed to differentiate into normal control macrophages, as previously described (32)(33)(34)(35)(36)(37).…”

Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation

“…It is generally believed that serine phosphorylation of STAT3 modulates its maximum activity, perhaps by affecting the recruitment of co-factors (14,40), whereas Tyr 705 phosphorylation is required for the activation. However, recent studies have suggested that STAT3 can be activated through Ser 727 phosphorylation in the absence of Tyr 705 phosphoryla- tion, involving, for instance, Notch signaling in neuronal stem cells (41) or constitutive activation of STAT3 in macrophages (42). Most recently, Ser 727 -phosphorylated and Tyr 705 -dephosphorylated STAT3 is shown to promote prostate tumorigenesis (43).…”