Stat1 is a suppressor of ErbB2/Neu-mediated cellular transformation and mouse mammary gland tumor formation

The anti-tumor function of Stat1 as a regulator of innate immunity and tumor immune surveillance has been long studied and is well understood; however, less clear is its tumor-site specific role. Although Stat1 phosphorylated at tyrosine (Y) 701 and serine (S) 727 is essential for interferon (IFN) signalling, its function in signalling induced in breast cancer cells is not understood. Herein, we show that Stat1 Y701 phosphorylation is increased in human breast tumor cells with elevated levels of ErbB2/HER-2 and in cells transfected with ErbB2/Neu. However, pharmacological inhibition of ErbB2/HER-2 results in the inhibition of Stat1 Y701 phosphorylation indicating an atypical role of phosphorylated Stat1 in the inhibition of ErbB2/HER-2 signalling. Consistent with this notion, we found that Stat1 suppresses tumor development by an activated form of ErbB2/Neu in mouse embryonic fibroblasts in xenograft tumor assays; however, this anti-tumor function of Stat1 does not rely on Y701 and S727 phosphorylation. Experiments with transgenic mice demonstrated that Stat1 acts to suppress Neu-mediated breast tumorigenesis through immune regulatory and tumor-site specific mechanisms. Our data reveal a previous uncharacterized anti-tumor activity of Stat1 in ErbB2/Neu-mediated cell transformation and breast oncogenesis with possible implications in the diagnosis and treatment of ErbB2-positive breast cancers.     

Differential effects of CpG DNA on IFN-beta induction and STAT1 activation in murine macrophages versus dendritic cells: alternatively activated STAT1 negatively regulates TLR signaling in macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.     

Prion protein is secreted in soluble forms in the epididymal fluid and proteolytically processed and transported in seminal plasma

Interferon tau (IFNtau) is the pregnancy recognition signal produced by the conceptus trophectoderm and acts in a paracine manner on the ovine endometrium to increase expression of IFN-stimulated genes primarily in the stroma and deep glandular epithelium, including IFN regulatory factor-1 (IRF-1). The roles of Stat1, Stat2, and IRF-9 in IFNtau regulation of IRF-1 expression were determined using human stromal fibroblasts lacking specific IFN signaling components or complemented with specific Stat1 mutants. In parental (2fTGH) cells treated with IFNtau, Stat1alpha/beta was tyrosine phosphorylated by 15 min, and IRF-1 mRNA and protein increased from 0 to 6 h, was maximal at 6 h, and decreased to 24 h. In contrast, IFNtau did not affect IRF-1 expression in Stat1- and Stat2-deficient cells or in Stat1-deficient cells complemented with Stat1 Y701Q or Stat1 R602L mutants. In Stat1-deficient cells complemented with the Stat1 S727A mutant, Stat1alpha, or Stat1beta and treated with IFNtau, IRF-1 increased from 0 to 6 h, was maximal at 6 h, and decreased thereafter. In IRF-9-deficient cells stimulated with IFNtau, IRF-1 increased from 0 to 6 h but did not exhibit the sharp decline from 6 to 12 h observed in other cells. Collectively, results indicate that IFNtau effect on IRF-1 expression is primarily regulated by tyrosine-phosphorylated Stat1alpha or Stat1beta dimers, whereas the decline of IRF-1 after 6 h of IFNtau treatment is regulated by IRF-9.     

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.     

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.     

Mumps virus V protein antagonizes interferon without the complete degradation of STAT1

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.     

Stat1 mediates an auto-regulation of hsp90β gene in heat shock response

We have reported earlier that a heat shock element in the first intron of human hsp90β gene (iHSE) acts as an intronic enhancer to bind the heat shock factor (HSF1) and activates hsp90β gene under heat shock. Here, we show that, in addition to the HSF1, Stat1 phosphorylation is indispensable in the event. We show that Jak2, a Janus kinase specifically associated with the β subunit of IFNγ receptor, and PKCε? an isoform of the atypical PKC family, are the two dominant kinases responsible for the heat shock induced phosphorylation on Y701 and S727 of Stat1. However, the activation of these kinases under heat shock requires the association of chaperone proteins of the Hsp90 family, in particular, the Hsp90β under heat shock. Furthermore, Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex is likely recruited by HSF1 and Stat1 at the iHSE under heat shock. Brg1 further confers an open chromatin conformation at the promoter region that is pivotal to the heat shock induced fully activation of the hsp90β gene in Jurkat cells. This is a novel example of how multiple activation steps occur under heat shock, first on the kinases and then the Stat1 and the SWI/SNF chromatin remodeling complex that follows to conduct an auto-regulation based fully activation of the gene.