No abstract

STAT transcription factors are tyrosine phosphorylated upon cytokine stimulation and enter the nucleus to activate target genes. We show that Rac1 and a GTPase-activating protein, MgcRacGAP, bind directly to p-STAT5A and are required to promote its nuclear translocation. Using permeabilized cells, we find that nuclear translocation of purified p-STAT5A is dependent on the addition of GTP-bound Rac1, MgcRacGAP, importin α, and importin β. p-STAT3 also enters the nucleus via this transport machinery, and mutant STATs lacking the MgcRacGAP binding site do not enter the nucleus even after phosphorylation. We conclude that GTP-bound Rac1 and MgcRacGAP function as a nuclear transport chaperone for activated STATs.

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds. [Mol Cancer Ther 2005;4(5):693 -703]

Vomitoxin (VT) and other trichothecene mycotoxins mediate a broad range of immunotoxic effects via the induction of inflammation-associated genes in leukocytes. The purpose of this study was to test the hypothesis that VT induces cyclooxygenase-2 (COX-2) gene expression in macrophages and that this is regulated at the level of mitogen-activated protein kinases (MAPKs). Exposure of the murine macrophage cell line RAW 264.7 to 50-250 ng/ml VT for 24 h markedly enhanced the production of prostaglandin E(2) (PGE(2)), a major COX-2 metabolite. PGE(2) elevation was preceded by increases in COX-2 mRNA (2 h) and COX-2 protein (15 h) in VT-treated cells. VT induced rapid (15 min) and persistent (up to 240 min) phosphorylation of extracellular, signal regulated protein kinases 1 and 2 (ERK1/2) and p38 MAPK as well as a rapid (15 min) but transient (up to 60 min) phosphorylation of c-Jun N-terminal kinases 1 and 2 (JNK1/2). The ERK inhibitor PD98059 and p38 inhibitor SB203580 suppressed VT-induced PGE(2) and COX-2 protein expression, whereas impairment of JNK function by transient transfection with a dominant negative (dn) JNK vector had no effect on COX-2 protein expression. Relatedly, in cells transfected with a COX-2 promoter-luciferase construct, PD98059- and SB203580-, but not dnJNK-treatment, suppressed VT-induced luciferase transcription. VT also increased COX-2 mRNA stability, and this was inhibited by PD98059 but not by SB203580. Taken together, these results indicate that VT-induced PGE(2) production and COX-2 expression by elevating transcriptional activity and mRNA stability. Enhanced transcriptional activity was modulated by ERK and p38 signaling pathways, whereas mRNA stability was promoted exclusively by VT-activated p38 phosphorylation. These data provide insight into possible general mechanisms by which VT and other trichlothecenes upregulate proinflammatory genes and impart immunotoxicity.

No abstract

For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region -197 to -160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation-Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.

IntroductionThe signal transducer and activator of transcription (STAT) family members STAT1-4, STAT5A, STAT5B, and STAT6 are activated through phosphorylation by the Janus kinase (JAK) family upon cytokine stimulation. The phosphorylated STATs form homodimers or heterodimers and translocate into the nucleus, where they regulate expression of their target genes. [1][2][3][4] Among them, STAT3 is activated mainly by the interleukin-6 (IL-6) family cytokines including IL-6, oncostatin M, and leukemia inhibitory factor (LIF), and is implicated in a wide range of biologic processes, including nephrogenesis, gliogenesis, hepatogenesis, T-cell proliferation, inflammation, and oncogenesis. [5][6][7][8][9][10][11] The critical role of STAT3 in myeloid differentiation was demonstrated by the use of dominant negative mutants. [12][13][14] In contrast, in embryonic stem cells, STAT3 is required for self-renewal. [15][16][17][18][19] In addition, STAT3 is activated in a broad spectrum of human hematologic malignancies. 20 STAT3 can also be negatively regulated. Among the known inhibitors of STAT proteins are the suppressor of cytokine signaling (SOCS) proteins, 21 also known as Janus kinase binding (JAB) proteins 22 or STAT-induced STAT inhibitors (SSIs). 23 While SOCS proteins interact with JAKs and reduced their tyrosine kinase activity, 21-23 a STAT3 inhibitor protein inhibitor of activated STAT3 (PIAS3) directly binds to STAT3 and inhibits its activity. 24 A zinc finger protein Gfi-1 enhances STAT3 signaling by preventing this binding of PIAS3 to STAT3. 25 Several other molecules have been found to interact with activated STAT3. Among them, cellular Jun (c-Jun) forms a complex with STAT3 and activates the ␣ 2 -macroglobulin promoter that contains both STAT3-and c-Jun-binding sites. 26,27 Like many other transcription factors, STAT3 associates with a transcriptional cofactor, cAMP response element binding proteinbinding protein/p300 (CBP/p300), to form a transcriptional complex. 28 A protein called gene-associated with retinoid interferoninduced mortality (GRIM)-19 suppresses STAT3 activity through cytoplasmic retention of STAT3, whereas an endothelial cellderived zinc finger protein (EZI) enhances STAT3 activity through nuclear retention of STAT3. 29,30 There may be more such regulators in various steps of STAT3 action, such as translocation to the nucleus, induction of chromatin remodeling, and proteolysis.In a search for key molecules that prevent IL-6-induced terminal differentiation of murine myeloid leukemia M1 cells, we identified an antisense DNA for the full-length form of male germ cell Rac guanosine triphosphatase-activating protein (MgcRac-GAP) through functional cloning. 31 An N-terminus-truncated form of MgcRacGAP had been isolated and named male germ cell Rac GAP, as its expression was highest in testis. 32 Rac, Cdc42, and RhoA, the Rho family of small guanosine triphosphates (GTPases), play pleiotropic roles in a variety of cell functions such as transformation, migration, cytokinesis, and transcriptional...

No abstract