Complex Roles of Stat1 in Regulating Gene Expression

Chilakamarti V Ramana1, Moitreyee Chatterjee-Kishore1, Hannah Nguyen1 and George R Stark*,1

1Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, OH 44195 USA

Stat1 is a fascinating and complex protein with multiple, by which Stat1 is activated in response to IFNs and yet contrasting transcriptional functions. Upon activa- the nature of its interaction with other proteins and tion, it drives the expression of many genes but also speci®c gene targets are determined by the type of suppresses the transcription of others. These opposing IFN. The pathway leading to activation by Type II characteristics also apply to its role in facilitating IFN begins when IFNg binds to its receptor IFNGR, crosstalk between signal transduction pathways, as it catalyzing activation of Jak1 and Jak2 through auto- participates in both synergistic activation and inhibition and trans-phosphorylation. Already bound to the of gene expression. Stat1 is a functional transcription cytoplasmic domains of the IFNg receptor subunits factor even in the absence of inducer-mediated activation, (Bach et al., 1996; Kotenko et al., 1995; Sakatsume et participating in the constitutive expression of some al., 1995; Kaplan et al., 1996), the activated Jaks genes. This review summarizes the well studied involve- phosphorylate the receptor on speci®c tyrosine ment of Stat1 in IFN-dependent and growth factor- residues, including Y440 of IFNGR1, creating a dependent signaling and then describes the roles of Stat1 docking site for the recruitment of Stat1 via its Src- in positive, negative and constitutive regulation of gene homology 2 (SH2) domain (Greenlund et al., 1994, expression as well as its participation in crosstalk 1995; Igarashi et al., 1994). Other Stat family between signal transduction pathways. Oncogene members are activated by various cytokines through (2000) 19, 2619 ± 2627. similar recruitment mechanisms, making the IFNg signal transduction pathway a generic model for Stat- Keywords: constitutive transcription; negative regula- mediated cytokine signaling. tion; interferons; growth factors; crosstalk Receptor-bound Stat1 is activated through phosphorylation of Y701 by the Jaks, leading to the formation of homodimers by reciprocal phosphotyrosine-SH2 domain interactions (Schindler et al., 1992; Signal transducers and activators of transcription Shuai et al., 1992, 1993). The Stat1 homodimers then (Stats) comprise a family of transcription factors translocate to the nucleus by a mechanism that discovered through their key involvement in interferon depends on the GTPase activity of Ran/TC4 (Sekimoto (IFN) signaling. Their activation and mechanisms of et al., 1997), where they bind to the promoters of action represent the ®rst examples of direct signaling to IFNg-responsive genes via gamma activated sequence the nucleus by extracellular polypeptide ligands with- (GAS) elements (Darnell et al., 1994; Schindler and out the involvement of second messengers, tyrosine Darnell, 1995). Recent crystallographic studies reveal phosphorylation of transcription factors, and transcrip- that Stat1 homodimers interact with DNA over a tion factors that make direct contact with both 15 bp region, with relatively few contacts between the membrane-bound receptors and DNA (reviewed by Stat1 side chains and the DNA bases (Chen et al., Darnell et al., 1994; Schindler and Darnell, 1995; 1998). The DNA binding speci®city of Stat1 homo- Leaman et al., 1996a; Stark et al., 1998). Since the dimers may be aected additionally by their interac- initial discovery of Stats 1 and 2, the family has been tions with other transcription factors (Bluyssen et al., expanded to seven members. These play vital roles in 1995; Look et al., 1995). For most IFNg-inducible signaling pathways induced by many dierent cyto- genes, Stat1 binding results in the activation of kines and growth factors that regulate cell growth and expression. The transcriptional activity of Stat1 is dierentiation, the immune response, antiviral activity, enhanced at some point in the activation pathway by and homeostasis. This review focuses on the diverse the phosphorylation in the cytoplasm of serine 727 functions of Stat1. through a process that depends on Jak2 and the Pyk2 tyrosine kinase (Wen et al., 1995; Zhu et al., 1997; Takaoka et al., 1999; Kovarik et al., 1998). Stat1 and IFN-dependent signaling As shown in Figure 1, the activation of Stat1 by Type I IFN (IFNa/b) is initiated by ligand binding to IFN-dependent signaling involves members of four the IFNAR receptor, resulting in activation through protein families: (1) the Type I and II IFN receptors; auto- and trans-phosphorylation of Jak1 and Tyk2 (2) the receptor-associated Janus protein tyrosine (Colamonici et al., 1994; Gauzzi et al., 1996). Activated kinases (Jaks); (3) the Stats; and (4) members of the Jak1 and Tyk2 catalyze the sequential phosphorylation interferon regulatory factor (IRF) family of transcrip- of Y466 of IFNAR1 (Krishnan et al., 1996), Y660 of tion factors. As depicted in Figure 1, the mechanisms Stat2, and Y701 of Stat1. Heterodimerization of activated Stat1 and Stat2 is coordinated with their dissociation from the receptor, followed by binding of *Correspondence: GR Stark the heterodimer to p48, a member of the IRF family of Functions of Stat1 CV Ramana et al 2620 cytokines such as growth hormone, epidermal growth factor and interleukin-10, all of which activate Stat1 and Stat3 in vitro, are not substantially aected in Stat1-null mice (Meraz et al., 1996; Durbin et al., 1996). However, recent work of Lee et al. (2000) reveals that the lack of Stat1 does aect lymphocyte survival and proliferation in mice independently of the role of Stat1 in the response to IFNg. In summary, these studies con®rm the paramount role of Stat1 in IFN-dependent signaling and the related physiology.

Roles of Stat1 in signaling in response to growth factors and cytokines

Stat1 has been implicated as a mediator of biological responses to a variety of growth factors and cytokines, based on ligand-dependent tyrosine phosphorylation and activation, although these observations alone do not prove that Stat1 is essential for signal transduction. Jak1, Stat1 and Stat3 are phosphorylated in response to epidermal growth factor (EGF). However, Jak1 is not required either for Stat activation or for the Figure 1 Activation of Stat1 in IFNg and IFNa signaling. IFNg signaling involves ligand-induced oligomerization of the IFNg induction of the c-fos gene (Leaman et al., 1996b). receptor subunits (IFNGR1 and IFNGR2) leading to the Furthermore, Stat activation in response to EGF in phosphorylation and activation of Jak1, Jak2, IFNGR1 and Jak2- or Tyk2-null cells is normal. In cells transfected Stat1. Stat1 homodimers translocate to the nucleus and bind to with a kinase-dead mutant EGF receptor, there is no GAS elements. The IFNa/b-mediated association of IFNAR1 and Stat activation in response to EGF and c-fos is not 2 stimulates the phosphorylation and activation of Tyk2, Jak1, IFNAR1, Stat1 and Stat2. Stat1-2 heterodimers, in conjunction induced, indicating that the kinase activity of the with p48, form the trimeric complex ISGF3, which translocates to receptor is required, directly or indirectly, for these two the nucleus and binds to ISRE elements. Alternatively, Stat1 responses. Furthermore, transient transfections in Hela homodimers and Stat1-2 heterodimers translocate to the nucleus cells using a c-fos promoter lacking the Stat regulatory and bind to GAS elements element (SIE), indicate that this element may play a minor role in the induction of c-fos by EGF in these cells (Leaman et al., 1996b). Similarly, in Stat1-null transcription factors, to form the ISGF3 complex. mice, the induction of c-fos by EGF is not altered in ISGF3 translocates into the nucleus by an unknown the liver (Meraz et al., 1996). mechanism and binds to interferon-stimulated response PDGF-induced phosphorylation of Jaks and Stats is elements (ISREs) in the promoters of IFNa/b-inducible independent of any single Jak but does require receptor genes. Stat1 homodimers and Stat1-2 heterodimers not tyrosine kinase activity (Vignais et al., 1996). Mutagen- associated with p48, also formed in response to IFNa/ esis studies revealed that Stat activation by the PDGF b, bind to GAS elements to activate gene expression. receptor requires the juxtamembrane phosphorylation For example, in the case of the IRF1 gene, IFNa- sites but not Src tyrosine kinase activation. The induced Stat1-2 heterodimers stimulate expression by activation of MAP kinase and induction of c-fos is binding to a GAS element without p48 (Li et al., 1996). correlated with Stat but not Src activation by the However, based on studies in p48-null human cells, PDGF receptor (Sachsenmaier et al., 1999). Cell-free ISGF3 is by far the more important factor, activating studies have further shown that the activation of Stat1 most IFNa/b-inducible genes (John et al., 1991; requires it to interact with the PDGF receptor directly, Bluyssen et al., 1995). The levels of p48 and, therefore, whereas Stat3 activation additionally requires Jak the relative abundance of the various Stat1 complexes proteins (Vignais and Gilman, 1999). vary widely among dierent cell types. The binding of growth hormone leads to dimerization Analysis of the ISRE-ISGF3 interactions in the 6 ± of its receptor, accompanied by the phosphorylation and 16 and 9 ± 27 promoters demonstrate that ISGF3 activation of Jak1, Jak2, Stat1, Stat3 and Stat5 (Han et interacts with DNA over a 35 bp region (Dale et al., al., 1996). Jak2 but not Jak1 is required for the growth 1989; Imam et al., 1990). DNA contacts are made by hormone-dependent phosphorylation events that are Stat1 and p48, while Stat2 serves as the main provider required to couple the receptor to Stat-dependent of transcriptional activation (Qureshi et al., 1995; signaling pathways and also to pathways involving the Bluyssen and Levy, 1997). The speci®city of the Stat1 SH2-containing adaptor protein Shc (Han et al., 1996). DNA binding site is determined by residues between Similiarly, Jak2 is absolutely required for prolactin 400 and 500 (Horvath et al., 1995). p48 and Stat1 (PRL)-dependent phosphorylation of its receptor, the associate through the C-terminus of p48 and the N- activation of Stats, and the induction of b-lactoglobulin terminus of Stat1, and substitution of lysine 161 by gene expression (Han et al., 1997). Although Stat1 alanine in Stat1 abrogates the interaction as well as the homodimers do form in response to PRL, no defect in response to IFNa (Horvath et al., 1996). PRL-dependent signaling is apparent when Stat1 is Stat1-de®cient mice fail to elicit biological responses missing, suggesting that Stat5, which is strongly to IFNa or IFNg. Interestingly, signaling by other activated in response to PRL, is primarily responsible

Oncogene Functions of Stat1 CV Ramana et al 2621 for driving the expression of PRL-responsive genes (Han tional activation in response to Stat1 is likely to require et al., 1997). In interleukin-6- and granulocyte colony the recruitment of co-activators and cooperation with stimulating factor (G-CSF)-dependent signal transduc- general transcription factors and the core transcription, Jak1 is required for the activation of Stats 1 and 3. tional machinery (Figure 2). Recent studies on the b- Jak2, although activated, cannot mediate the activation IFN enhanceosome reveal that transcriptional activa- of Stat1 and Stat3 eciently in the absence of Jak1 tors cooperate to enhance the rate at which pre- (Guschin et al., 1995; Shimoda et al., 1997). Although initiation complexes assemble at the promoter (Yie et these studies and the phenotypes of Stat1-null mice al., 1999). The development of similar in vitro suggest that Stat1 may not play an essential role in transcription systems will help to elucidate the details response to growth factors or cytokines other than the of transcriptional regulation by Stat1. IFNs, a more subtle role in modulating gene expression An additional level of complexity in Stat1 signaling in a tissue-speci®c manner has now been demonstrated is revealed by the presence of tandem GAS sites in the (Lee et al., 2000). promoters of some IFN-inducible genes. Stat1 forms tetramers on such sites in a process that requires its amino-terminal region (Vinkemeier et al., 1996; Xu et Stat1 and crossover signaling al., 1996). Cooperative binding of ISGF3 has also been observed on the tandem ISREs of the promoter of the Endothelial cells express low levels of the protein IFNa-responsive 6-16 gene (Li et al., 1998). The N- tyrosine phosphatase Shp1 and IFNs a/b stimulate more terminal regions of Stat1 and Stat2 are both important Stat1 homodimer formation and transactivation through for the cooperative binding of ISGF3 to tandem GAS sites than IFNg (Min et al., 1998). Overexpression ISREs. The cooperative DNA binding properties of of Shp1 in endothelial cells abrogated IFNa/b signaling both Stat1 and ISGF3 are likely to contribute to through a GAS site, suggesting that the level of Shp1 can transcriptional activation of additional IFN-responsive regulate a crossover between the IFNa/b and IFNg genes that have tandem binding sites. signaling pathways (Min et al., 1998). The stimulation of NIH3T3 ®broblasts by PDGF results in the phosphorylation and nuclear translocation of Stat1, but IFNg- Negative transcriptional regulation by Stat1 regulated genes are not induced (Silvennoinen et al., 1993). However, growth factor stimulation of a mutant The transcription of several genes is repressed by IFNg PDGF receptor expressing only the Ras-GAP binding (Table 1). Major targets of negative regulation are site (Y771) promotes the induction of IFNg-responsive genes encoding proteins involved in regulating the genes rather than immediate-early genes (Fambrough et extracellular matrix (matrix metalloproteinases al., 1999). Shp2 is a positive regulator of PDGF (MMPs), perlecan, bullous pemphigoid antigen-1 signaling, a negative regulator of Jak-Stat signaling and (BPAG1), cell cycle genes (c-myc, cyclin D, cyclin A) is a candidate for altering the signal relay enzymes and several thyroid-speci®c genes (thyroglobulin, involved in crossover signaling. thyroidal peroxidase, and the thyrotropin receptor). Members of the MMP family of zinc-dependent neutral peptidases, including collagenases, gelatinases and Positive transcriptional regulation by Stat1 stromelysins that are involved in degrading the extracellular matrix, the invasion and metastasis of Two to ®ve hundred genes are activated in response to malignant cells, and tumor-induced angiogenesis (Wes- the IFNs (Boehm et al., 1998; Der et al., 1998), termarck and Kahari, 1999). Perlecan is a heparin including transcription factors such as IRF1, which sulfate proteoglycan found in basement membranes sustain the transcriptional response. Stat1 binds to the and on cell surfaces (Iozzo et al., 1994). IFNg-mediated transcriptional co-activators CBP, p300, pCIP, MCM- transcriptional suppression of the perlecan gene is 5, Nmi and BRCA1 (Zhang et al., 1996, 1998; Korzus Stat1-dependent and requires a distal promoter region et al., 1998; Zhu et al., 1999; Ouchi et al., 2000). CBP containing multiple GAS elements (Sharma and Iozzo, and p300 are related proteins that serve as coactivators for many transcription factors (Shikama et al., 1997). CBP/p300 serves important functions as a histone acetyl transferase, relaxing chromatin near transcription start sites, and as a platform for several important associated proteins. The association of Stat1 with CBP/p300 requires both the carboxyl- and amino- terminal regions of Stat1 (Zhang et al., 1996). In contrast, Stat2 interacts with CBP/p300 via its carboxyl-terminal region, also required for the formation of ISGF-3 (Bhattacharya et al., 1996). CBP/p300 interacts with pCIP, and both are involved in Figure 2 Promoter elements and transcription factors involved in Stat1-dependent gene activation. The promoter regions of transcriptional activation by Stat1 (Korzus et al., IFNg-responsive genes contain GAS elements that bind to Stat1 1998). The phosphorylation of serine 727 of Stat1 is and an assembly of coactivator complexes containing CBP/p300, required for it to interact with MCM-5 and BRCA1 pCIP, Nmi, BRCA1, MCM5 and other transcription factors. and for maximal transcriptional activity. Stat1 also Stat1 also interacts with constitutive transcription factors such as cooperates with both general (SP1) and inducible SP1. Functional interaction of Stat1-coactivator complexes with the constitutive and core transcription machinery (such as the transcription factors (AP1, NFkB) (Look et al., 1995; TATA box binding protein and associated factors) are also Korzus et al., 1997; Pine, 1997). Therefore, transcrip- necessary to initiate transcription

Oncogene Functions of Stat1 CV Ramana et al 2622 Table 1 Examples of Stat1-dependent transcriptional regulation Type of regulation Genes References

Positive 4200 IFN-stimulated genes Boehm et al., 1998 Der et al., 1998

Negative Bullous pemphigoid antigen Tamai et al., 1995 Perlecan Sharma and Iozzo, 1998 Matrix metalloproteinase1 Ala-Aho et al., 2000 Matrix metalloproteinase2 Qin et al., 1998 Matrix metalloproteinase13 Ala-Aho et al., 2000 Stromelysin Lewis et al., 1999 Pro-alpha collagen Yuan et al., 1999 Thyroglobulin Kung and Lau, 1998 Thyroid peroxidase Ashizawa et al., 1989 Thyrotropin receptor Ohe et al., 1996 Thyroid transcription factor Miyazaki et al., 1999 Angiotensin receptor a Ikeda et al., 1999 c-Myc Melamed et al., 1993 Ramana et al., 2000 Cyclin D Tiefenbrun et al., 1996 CDC25A Tiefenbrun et al., 1996 Cyclin A Sibinga et al., 1999

Constitutive Caspases 1, 2 and 3 Kumar et al., 1997 LMP2 Chatterjee-Kishore et al., 1998 MHC Lee et al., 2000

1998). In contrast, AP1-family transcription factors are Stat1-dependent signaling. The receptor-associated involved in the transcriptional inhibition of stromelysin protein tyrosine phosphatases Shp1 and Shp2 partici- expression by IFNg (Lewis et al., 1999). Suppression of pate in the negative regulation of Jak-Stat1 signaling thyroid-speci®c gene expression by IFNg may involve by inactivating the IFN receptors and Jaks through down-regulation of thyroid transcription factor (TTF2) dephosphorylation (David et al., 1993; Haque et al., expression and DNA binding activity (Miyazaki et al., 1997). The tyrosine phosphorylation and DNA binding 1999). IFNg suppresses c-myc expression in wild-type of Stat1 are enhanced in IFN-treated Shp2-null but not in Stat1-null mouse embryo ®broblasts ®broblasts (You et al., 1999). A nuclear tyrosine (Ramana et al., 2000). A consensus GAS element in phosphatase has been implicated in inactivating the c-myc promoter is necessary but not sucient for activated Stat1 (Haspel and Darnell, 1999). SOCS1, a suppression. Furthermore, phosphorylation of both the speci®c inhibitor of Stat1 signaling, can bind directly to tyrosine and serine residues of Stat1 is required to Jaks to inhibit their tyrosine kinase activities (Starr et suppress c-myc (Ramana et al., 2000) and inhibitors of al., 1997; Endo et al., 1997). Proteolytic degradation of Jak2 and Mek-1 reverse IFNg-mediated transcriptional Stat1 dimers following ubiquitination is another suppression (Ikeda et al., 1999; Ala-aho et al., 2000). mechanism by which Stat1 is negatively regulated The minimal promoter regions required for transcrip- (Kim and Maniatis, 1996). Ecient termination of tional inhibition of BPAG1 and cyclinA expression cytokine-induced activation is important for the contain multiple cis-acting elements but no GAS regulation of Stat1-dependent genes that are normally element (Tamai et al., 1995; Sibinga et al., 1999). It silent or expressed at low levels in untreated cells. is likely that, in these cases, suppression involves the modi®cation of coactivators or general transcription factors within the complexes that drive transcription. Involvement of Stat1 in crosstalk between signal Transcriptional suppression in eukaryotes is mediated transduction pathways by the interaction of repressors with silencer or negative regulatory elements. Several transcription Since multiple signal transduction pathways are factors have dual functions, being able to activate or activated by cytokines secreted during acute physiolo- repress transcription, depending upon the contexts of gical responses to bacterial or viral infections, in¯am- speci®c promoters by recruiting coactivators or co- mation, and healing, crosstalk between dierent repressors (Ogbourne and Antalis, 1998; Torchia et al., pathways is needed for coordination. Stat1 is involved 1998). The identi®cation and characterization of the both in the synergistic transcriptional activation of corepressors that interact with Stat1 should provide some genes and in functional antagonism of expression novel insights into mechanisms of transcriptional of others in response to multiple signals. As shown in suppression by IFNg. Figure 3, synergistic activation of the IRF1 and ICAM1 (intercellular adhesion molecule-1) genes by IFNg in combination with the proin¯ammatory Termination of Stat1-dependent signaling cytokine tumor necrosis factor (TNF) occurs through functional cooperation between IFNg-activated Stat1 Stat1 is rapidly and transiently activated in response to dimers and TNF-activated NFkB, each bound to its ligand stimulation. Pulse-chase experiments reveal that, respective consensus promoter element (Ohmori et al., although activated Stat1 is short-lived, the Stat1 1997; Pine, 1997). Stat1-NFkB association is also protein is relatively stable (Haspel et al., 1996). responsible for synergistic activation of inducible nitric Multiple mechanisms are involved in down regulating oxide (iNOS) and IFNg-inducible protein-10 (IP-10)

Oncogene Functions of Stat1 CV Ramana et al 2623

Figure 4 Functional antagonism between the IFNg signaling pathway and the pathways responding to TGFb, CSF1 and IL4. Activated Stat1 abrogates AP1/ets-mediated transactivation of the macrophage-scavenger receptor in CSF1 signaling by competing for a limiting amount of the CBP/p300 coactivator. Stat1 also enhances the expression of SOCS1 and Smad7, which inhibit the IL4 and TGFb signaling pathways, respectively

Figure 3 Synergistic activation of IRF1 transcription in response to IFNg plus TNFa. IFNg-activated Stat1 and TNFa- prevents the interaction of Smad3 with the TGFb activated NFkB bind simultaneously to the IRF1 promoter to receptor. This response requires Jak1 and Stat1 (Ulloa activate transcription (see text for details) et al., 1999). IFNg-mediated inhibition of IL4 signaling occurs through a Stat1-dependent mechanism involving the induction of the silencer of cytokine signaling expression in response to IFNg plus bacterial cell wall (SOCS1) protein, a general inhibitor of cytokine lipopolysaccharide (LPS) (Gao et al., 1997; Ohmori signaling pathways (Venkataraman et al., 1999). and Hamilton, 1994). Whether synergism between Competition of activated Stat1 with the AP1/Ets Stat1 and other transcription factors involves physical transcription factors for the transcriptional coactivator interaction has not been clearly demonstrated. A CBP/p300 represents yet another mechanism for Stat1- synergistic eect of retinoic acid on Stat1 signaling mediated transcriptional repression in response to IFNg, involves induction of the Stat1 and IRF1 genes as well relevant to colony stimulating factor-1 (CSF1)-induced as enhancement of IFN-induced Stat1 activation macrophage scavenger receptor gene expression (Hor- (Chelbi-Alix and Pelicano, 1999). vai et al., 1997) and possibly also to the PDGF- Recent studies reveal an alternative mechanism mediated induction of c-myc (Ramana et al., 2000). through which dierent inducers can synergize through Functional antagonism between the IFNg and TGFb, Stat1, based on enhancement of its transactivation CSF1 or IL4 signaling pathways is summarized potential. Stat1 activation by IFNg involves the schematically in Figure 4. phosphorylation of S727, which increases its transcriptional activity. LPS and TNF induce Stat1 S727 phosphorylation only, thereby increasing IFNg-induced Stat1 as a constitutive transcription factor phosphorylation and enhancing IFNg activation of a GAS-driven reporter (Kovarik et al., 1998, 1999). The ®rst evidence that Stat1-induced transcription can Synergy mediated by IL2 and IL12 is also dependent occur without ligand-mediated tyrosine phosphorylation on increased Stat1 and Stat3 serine phosphorylation, in a came from an analysis of caspase gene expression in the p38 MAP kinase-dependent process (Gollob et al., 1999). mutant cell line U3A, which lacks Stat1. Unlike their Interestingly, while Stat1 participates in the syner- parents, U3A cells are insensitive to apoptosis-inducing gistic activation of some genes in response to IFNg signals and have very low basal expression of caspases 1, plus LPS, it is also required for the repression of other 2 and 3. Surprisingly, caspase expression is rescued in genes in response to the same two factors. LPS- U3A cells reconstituted with Stat1 variants that cannot inducible expression of the Type II TNF receptor form homodimers (Y701F or mutant in the SH2 domain) (TNFRII) and KC (GRO/melanoma growth stimulat- (Kumar et al., 1997). Recent analysis of lymphocytes ing activity) genes is suppressed by IFNg in murine from Stat1-null mice revealed reduced levels of caspase 1 macrophages (Tannenbaum et al., 1993; Ohmori and and caspase 11 (Lee et al., 2000) and a requirement for Hamilton, 1994) and the suppression of both genes is Stat1 for constitutive expression of major histocompat- Stat1-dependent (TA Hamilton, personal communica- ibility class I antigens (Lee et al., 1999). tion). The participation of Stat1 in IFNg-mediated Low molecular mass polypeptide 2 (LMP2) is a down-regulation of gene expression can also involve component of the b subunit of the 20S proteasome. the synthesis of speci®c signaling inhibitors. Inhibition The bi-directional promoter that regulates LMP2 of transforming growth factor-b (TGFb) signaling by expression contains overlapping interferon consensus IFNg involves the rapid synthesis of Smad7, which sequence 2 (ICS2) and GAS sites. The LMP2 gene is

Oncogene Functions of Stat1 CV Ramana et al 2624 IFN-inducible, but the gene is also transcribed at a induces the expression of c-myc and c-jun, thus revealing lower level in the absence of IFN (Min et al., 1996). In a novel signal transduction pathway (Ramana et al., vivo footprinting of the ICS2/GAS revealed protein- 2000). Global expression studies involving the use of DNA contacts at both the ICS2 and GAS subsites in gene-chip technology also indicate that IFNs regulate the unstimulated HeLa cells (White et al., 1996). Stat1 is expression of several genes in Stat1-null cells (CV required for basal expression of the LMP2 gene, and Ramana and GR Stark, unpublished data; R Schreiber, LMP2 transcription requires that Stat1 and IRF1 both personal communication). Treatment with geldanamy- bind to the composite ICS2/GAS site (Chatterjee- cin, an HSP90-speci®c inhibitor, or expression of a Kishore et al., 1998). LMP2 transcription was not dominant negative mutant of cdc37 which is unable to detected in U3A cells but could be reconstituted by recruit HSP90 to the Raf1 complex, both inhibited the introducing the Y701F variant of Stat1 (M Chatterjee- IFNg-induced expression of c-myc in Stat1-null cells, Kishore and GR Stark, unpublished data). indicating that Raf1 activation is critical for the Stat1- Serine phosphorylation does not aect the ability of independent pathway (Ramana et al., 2000). Stat1 to bind to DNA. Since serine phosphorylation of Stat1 can be induced independently of tyrosine phosphorylation (Zhu et al., 1997), Stat1 will be phosphory- Stat1 in antiproliferative responses, immune surveillance lated only on serine in response to some ligands, and tumor suppression probably inducing the Stat1-dependent transcription of a distinct set of genes. However, since tyrosine Interferons inhibit cell growth, and transcriptionally phosphorylation is essential for the tight binding of active Stat1 is required for this antiproliferative activity Stat1 dimers to DNA, it is important to ®nd out how (Bromberg et al., 1996). The negative eect on cell Stat1 binds to DNA in the absence of tyrosine growth correlates with the transcriptional regulation by phosphorylation. The mutual binding of two Stat1 IFN of several cell cycle genes. The mRNAs encoding monomers without the SH2-phosphotyrosine interaction cyclin D and the CDK phosphatase cdc25A are down would appear possible from the structures of DNA- regulated in response to IFNa (Tiefenbrun et al., 1996) bound Stat1 and Stat3b (Chen et al., 1998; Becker et al., and the CDK inhibitor p21(WAF1) is up-regulated by 1998) and the N-terminal portion of Stat4 (Vinkemeier et IFNg in epidermal carcinoma and glioblastoma cell al., 1998). Each monomer of DNA-bound Stat1 dimer lines (Chin et al., 1996; Kominsky et al., 1998). In contacts only half of the palindromic GAS element. The contrast, IFNg-dependent inhibition of cell growth is N-terminal domain of Stat1 is likely to have very few independent of p21 in HCT116 colon carcinoma cells contacts with DNA and thus is essentially free for (Sharma and Iozzo, 1998). Treatment with IFNa protein-protein interactions (Vinkemeier et al., 1998; abolishes the formation of transcriptional complexes Chen et al., 1998), explaining how two dimers of Stat1 on the E2F site of the c-myc promoter and suppresses bound to adjacent GAS elements can contact each other c-myc expression in Daudi and M1 myeloblast cells through their N-terminal domains to form highly stable (Melamed et al., 1993). The ectopic expression of tetramers (Xu et al., 1996). The N-terminal coiled-coil deregulated c-myc overcomes macrophage growth domain of Stat1 is probably also involved in interactions arrest and inhibition of vascular smooth muscle cell with other proteins too, for example with p48 (Horvath proliferation in response to IFNg, indicating that et al., 1996). Therefore, the interaction of Stat1 with IFNg-induced c-myc has a major role in down- other proteins, either transcription factors that bind to regulating proliferation (Bennett et al., 1994; Vairo et adjacent sites or accessory factors and coactivators, may al., 1995). Deregulated expression of c-myc and c-jun is allow unphosphorylated Stat1 to bind to DNA well likely to be responsible for the abnormal proliferation enough to activate transcription. A comparison of the of Stat1-null cells in response to IFNs (Ramana et al., expression of over 6000 genes in U3A cells and U3A cells 2000). Abnormal proliferation and genomic instability expressing Stat1 Y701F suggests that the basal expres- are two hallmarks of tumor development and even a sion of many more genes is regulated by unpho- transient excess of c-myc can promote genomic sphorylated Stat1 (M Chatterjee-Kishore and GR instability (Felsher and Bishop, 1999). Mice lacking Stark, unpublished data). the IFNg receptor or Stat1 develop tumors more Stat1 has been found in multimeric complexes in the rapidly and with greater frequency following challenge cytoplasm of quiescent cells by surface plasmon with chemical carcinogens (Kaplan et al., 1998; Lee et resonance analysis (Lackmann et al., 1998). Pre-formed al., 2000). IFNg functions in enforcing the recognition complexes of Stat1 with Stat2 or Stat3 have been of transformed cells by the immune system and as an observed in unstimulated HeLa cells (Stancato et al., integral part of immune surveillance and tumor 1996). Furthermore, Stat1 has also been found in the suppression mechanisms (Kaplan et al., 1998). A nuclei of unstimulated cells (Schindler et al., 1992). These corollary is that tumor cells with acquired defects in observations are related to how Stat1 can function as a IFN-dependent signaling can evade detection by the constitutive transcription factor. Recent studies indicate immune system. Consistent with this hypothesis, some that other Stats may also be able to function in the same tumor cells and tumor-derived cell lines express little or way (Richer et al., 1998; Johnson et al., 1999). no Stat1 mRNA or protein or fail to activate Stat1 following treatment with IFNs (Abril et al., 1998; Wong et al., 1997; Sun et al., 1998). Stat1-null cells A Stat1-independent pathway in IFN signaling resist apoptosis in response to TNF or IFNg due to low constitutive expression of caspases (Kumar et al., IFNs induce the expression of many genes and it now 1997; Lee et al., 2000), thus conferring a selective seems likely that at least some of these are regulated by a advantage to tumor cells that lack Stat1. Identi®cation Stat1-independent pathway. In Stat1-null cells, IFNg of IFNg-insensitive tumors may therefore have prog-

Oncogene Functions of Stat1 CV Ramana et al 2625 nostic and therapeutic relevance. In contrast, constitu- study, including the identi®cation of serine kinases tive activation of Stat1 has been reported in several involved in phosphorylating Stat1 on S727 and the cancers (Weber-Nordt et al., 1996; Gouilleux-Gruart et additional proteins involved in Stat1-dependent tran- al., 1996), in in¯ammatory conditions involving muscle scriptional regulation. Stat1 expression is absent or (Illa et al., 1997) and in the airway epithelium of defective in a variety of tumor cells and its role in chronic asthma patients (Sampath et al., 1999). Thus, immune surveillance and tumor suppression needs to the association of aberrant Stat1 expression or be clari®ed further. Recent developments in the activation with disease is complex. technology of analysis of gene expression will un- doubtedly reveal the full repertoire of Stat1 transcriptional targets in dierent tissues, under dierent Conclusion physiological conditions. Stat1 interacts with a variety of transcription factors and participates in extensive Although dramatic progress has been made in recent crosstalk with other signal transduction pathways, years in establishing the essential role of Stat1 in IFN- making it a likely target for therapeutic intervention dependent signaling, several aspects require further in several pathophysiological conditions.

References

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Oncogene