Twist1, a bHLH transcription factor, promotes breast tumor cell epithelial-mesenchymal transition (EMT), invasiveness and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (S68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This S68 is phosphorylated by p38, JNK and ERK1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in HEK293 and breast cancer cells. Prevention of S68 phosphorylation by an alanine (A) mutation (S68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of MAPKs by an active Ras protein or TGF-β treatment significantly increases S68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, while blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and un-induced S68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness upon stimulation with TGF-β or the active Ras as well as taxol resistance compared to same cells expressing the S68A-Twist1 mutant. Importantly, the levels of S68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor-negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1.

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR␥ induces hepatic steatosis, and liganded PPAR␥ promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR␥ function, transactivation, expression, and promoter activity. PPAR␥ transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB-and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix ( The cyclin-dependent kinase holoenzymes are a family of serine/threonine kinases that play a pivotal role in controlling progression through the cell cycle (38,47). Dysregulation of the cell cycle control apparatus is an almost uniform aberration in tumorigenesis (48). The cyclins encode regulatory subunits of the kinases which phosphorylate specific proteins, including the retinoblastoma (pRB) protein, to promote transition through specific cell cycle checkpoints (47, 57). Cyclin D1 plays a pivotal role in G 1 /S phase cell cycle progression in fibroblasts and is rate limiting in growth factor-or estrogen-induced mammary epithelial cell proliferation (29, 67). Cyclin D1 overexpression is found in Ͼ30% of human breast cancers, correlating with poor prognosis (23). Several different oncogenic signals induce cyclin D1 expression, including mutations of the Ras and Wnt/APC/␤-catenin pathway (2, 49). Mammary-targeted expression of cyclin D1 is sufficient for the induction of mammary adenocarcinoma, and cyclin D1 Ϫ/Ϫ mice are resistant to ErbB2-induced tumorigenesis (53,64).In addition to binding cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) and pRB, cyclin D1 forms physical associations with P/CAF (p300/CBP-associated factor), Myb, MyoD, and the cyclin D1 myb-like binding protein (DMP1) (16,20,31,39). Binding of cyclin D1 to the estrogen receptor alpha (ER␣) enhances ligand-independent reporter gene activity, and liganded androgen receptor reporter gene activity is inhibited by cyclin D1 (33, 39, 68). The in vivo or genetic evidence indicating a requirement for cyclin D1 in nuclear receptor function remained to be determined. The peroxisome proliferator-activator receptors, including PPAR␣, PPAR␥, and PPAR␦, are ligand-activated nuclear receptors (42). Their modular structure resembles those of other nuclear hormone receptors with N-terminal AF-1, a DNA binding domain, and a carboxyl-terminal ligand-binding domain (LBD). PPAR␥ was cloned as a transcription factor involved in fat cell differentiation and is required for the induction of adipocyte differentiation (41, 51). Adenoviral delivery of PPAR␥ to the livers of mice induces hepatic steatosis, consistent with an important role for PPAR␥ in hepatocellular lipid biosynthesis (65). The PPAR␥ ligands include eicosanoids, such as 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ 2 ), and synthetic ligands of the thiazolidinedione (TZD) class. PPAR␥ ...

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon-b (IFNb) is an antiviral cytokine induced by viral infection or following non-specific inflammatory challenges of the CNS. Because of the well-known anti-inflammatory properties of IFNb, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNb signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNbinduced b-chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNb, including nuclear factor-jB (NF-jB), activator protein-1 (AP-1) and Jak/Stat. IFNb induced IjBa degradation and NF-jB (p65:p50) DNA binding. Inhibition of NF-jB by either adenoviral transduction of a super repressor IjBa, or an antioxidant inhibitor of NF-jB reduced expression of the b-chemokines, regulated upon activation, normal T-cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1b. IFNb also induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose-dependently inhibited b-chemokine mRNA and protein expression. PD98059 did not inhibit NF-jB binding, demonstrating that ERK was not responsible for NF-jB activation. Two downstream targets of ERK were identified in microglia: AP-1 and Stat1. IFNb induced AP-1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNb induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNb in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.

The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling during Trypanosoma cruzi infection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (