IFNγ signaling—Does it mean JAK–STAT?

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Cytokine & Growth Factor Reviews 19 (2008) 383–394 www.elsevier.com/locate/cytogfr

Survey

IFNg signaling—Does it mean JAK–STAT? Daniel J. Gough a, David E. Levy a, Ricky W. Johnstone b, Christopher J. Clarke b,* a

Department of Pathology, NYU Cancer Institute, New York University Langone School of Medicine, 550 1st Avenue, New York, NY 10016, USA b Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, Australia Available online 16 October 2008

Abstract The molecular pathways involved in the cellular response to interferon (IFN)g have been the focus of much research effort due to their importance in host defense against infection and disease, as well as its potential as a therapeutic agent. The discovery of the JAK–STAT signaling pathway greatly enhanced our understanding of the mechanism of IFNg-mediated gene transcription. However, in recent years it has become apparent that other pathways, including MAP kinase, PI3-K, CaMKII and NF-kB, either co-operate with or act in parallel to JAK–STAT signaling to regulate the many facets of IFNg biology in a gene- and cell type-specific manner. The complex interactions between JAK/STAT and alternate pathways and the impact of these signaling networks on the biological responses to IFNg are beginning to be understood. This review summarizes and appraises current advances in our understanding of these complex interactions, their specificity and proposed biological outcomes. # 2008 Elsevier Ltd. All rights reserved. Keywords: Interferon gamma; STAT1 independent

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological and biochemical evidence for a non-STAT1-signaling mechanism Proteins recruited to the IFNg receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . IFNg signaling downstream of the IFNgR . . . . . . . . . . . . . . . . . . . . . . . . 4.1. MAP kinase pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. PI3-K, Akt and protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . IFNg activates transcription factors other than STAT1 . . . . . . . . . . . . . . . . 5.1. IFNg-activated STAT1-dependent or co-operative transcription factors 5.1.1. IRF family transcription factors . . . . . . . . . . . . . . . . . . . . . 5.1.2. MHC class II trans-activator . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. CCAAT/enhancer binding protein beta . . . . . . . . . . . . . . . . 5.1.4. PU.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. STAT1-independent, IFNg-induced transcription factors . . . . . . . . . . 5.2.1. STAT3 and STAT5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. NF-kB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. AP-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STAT1-independent IFNg biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Cell cycle arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. E-mail address: [email protected] (C.J. Clarke). 1359-6101/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2008.08.004

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1. Introduction IFNs were initially described as a biological activity that was able to interfere with viral infection [1]. The subsequent isolation of the proteins and cloning of the many IFN genes has led to the discovery that they affect a plethora of cellular functions including, anti-viral responses, anti-microbial responses, apoptosis and control of the cell cycle. The IFN molecules are secreted soluble proteins of three classes—type I (a, b, v and t), type II (g) and type III (l) and this review will focus on type II, IFNg. IFNg, the only type II IFN family member is secreted by activated immune cells—primarily Tand NK cells, but also Bcells, NKT cells and professional APCs [2]. The most striking phenotype from mice lacking either IFNg or its receptor is increased susceptibility to bacterial and viral pathogens [2]. Additionally, it has become clear that IFNg is critical for tumor immuno-surveillance as assessed using spontaneous, transplantable and chemical carcinogen-induced experimental tumors [3]. More subtle phenotypes exist including, leukocyte homing, cellular adhesion, immunoglobulin class switching, Th cell polarity, antigen presentation, cell cycle arrest and apoptosis which have been reviewed elsewhere [4]. In humans, complete IFNgR deficiency is associated with frequent infection and ultimately death from the usually poorly virulent mycobacterium—Bacille Calmette-Guerin [5]. Patients with partial IFNgR deficiency have similar but milder clinical presentation [6]. Similarly, the inability to secrete IFNg or the development of auto-antibodies neutralizing endogenous IFNg resulted in the death of a patient by overwhelming mycobacterium infection [7]. More subtle immune defects have also been observed in patients unable to secrete IFNg, these patients have poor neutrophil trafficking and NK cell activation [8]. The biological effects of IFNg are elicited through activation of intracellular molecular signaling networks, the best characterized of which is the JAK–STAT pathway (Fig. 1). Conventional IFNg signaling follows the binding of IFNg to its cell surface receptors IFNgR1 and IFNgR2, resulting in oligomerization of the receptor. The JAK (Janus kinase) family of kinases JAK1 and JAK2 are pre-associated with IFNgR1 and IFNgR2, respectively and receptor oligomerization brings these kinases into close proximity with one another, allowing them to trans-phosphorylate each other and the cytoplasmic domains of the receptors. Tyrosine phosphorylation of IFNgR1 on residue 440 provides a docking site for the SH2 domain of the latent STAT1 transcription factor. Docked STAT1 is phosphorylated in the C-terminus on tyrosine Y701 (most likely by JAK2 [9]), enabling a homodimer of tyrosine phosphorylated STAT1 to form and dissociate from the receptor. A second independent C-terminal phosphorylation on S727 occurs following IFNg stimulation [10]. This phosphorylated STAT1 homodimer translocates to the nucleus and initiates transcription of genes containing a gamma activated sequence (GAS, TTCN(24)GAA [11]) in their promoter region. To a lesser extent IFNg

Fig. 1. Canonical IFNg signaling. IFNg stimulation initiates the oligomerization of receptor subunits, activation of JAK kinases 1 and 2 facilitating trans-phosphorylation of the JAKs and the receptor subunits. STAT1 is then recruited to the receptor where it becomes phosphorylated on tyrosine enabling a homodimer to form which is serine phosphorylated. The phosphorylated homodimer translocates to the nucleus and binds to GAS elements in promoters to initiate gene transcription.

stimulation can activate an ISRE (AGTTTCNNTTTCNC) binding complex (reviewed elsewhere [2,4]). There is some evidence to suggest that STAT1 can form a homodimer and initiate transcription of a subset of genes independent of tyrosine phosphorylation [12], but most STAT1-mediated transcription is entirely dependent on tyrosine phosphorylation. STAT1 S727 phosphorylation has no impact on the formation of the STAT1 homodimer, Y701 phosphorylation [13], nuclear translocation and DNA binding [14]. However, S727 phosphorylation is important for STAT1 function because STAT1-mediated transcription is markedly attenuated [10] as is the IFNg-mediated anti-viral state in its absence [15]. The importance of the JAK/STAT1 pathway in IFNg biology is clearly evident because defects in anti-viral, antimycobacterial and anti-tumor immunity observed in STAT1-

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deficient mice grossly resemble the phenotypes of IFNg/ IFNgR knockout mice [16,17]. Moreover, heightened susceptibility to viral and mycobacterial infections in humans is associated with STAT1 mutations as well as IFNgR mutations [18,19].

2. Biological and biochemical evidence for a nonSTAT1-signaling mechanism Some facets of IFNg biology occur in the absence of JAK/STAT1 signaling. The elucidation of these STAT1independent mechanisms is required to complete our understanding of the underlying biochemistry of multiple immune phenotypes and diseases. For example, STAT1deficient mice are more resistant to some viral challenges (murine cytomegalovirus (MCMV), Dengue virus or Sindbis virus) than mice lacking the IFNa/b/g receptors [20,21]. Interestingly, the strength and duration of IFNg-mediated signaling must be tightly regulated to ameliorate infection because when STAT1-deficient mice are chronically infected with Lymphocytic Choriomeningitis (LCMV) they die from an IFNg-dependent wasting disease [22] rather than viral load. This suggests that some aspects of the STAT1independent response are pathogenic in absence of STAT1dependent negative feedback signals (such as SOCS). Finally, at a molecular level, microarray analysis revealed that approximately one-third of IFN-stimulated genes (ISGs) are still regulated by IFNg in the absence of functional STAT1 [20,23]. Such responses would not be expected if STAT1-mediated signaling was sufficient for a complete IFNg response and implies that STAT1-independent pathways can play an important role in IFNg biology. It has been proposed that, in the absence of STAT1, IFNg signaling is at least partially maintained through compensation by other STAT family members. IFNg can activate both STAT3 [24] and STAT5 [25], and STAT3 expression and phosphorylation was reported to be augmented in the absence of STAT1 [24,26]. However, we found no evidence of such compensation by STAT3 [27], and the reason for this discrepancy is unclear. There is some controversy over the role that STAT2 can play in IFNg signaling. STAT2 phosphorylation has been detected in response to IFNg, but at very low levels in comparison to the induction by type I IFN stimulation [28]. However, there is an implied role for STAT2 in IFNg signaling. The MCMV gene product M27 specifically impedes STAT2 function and potently suppresses IFNg-mediated responses in addition to inhibiting type I IFN-mediated anti-viral immunity [29]. Therefore, STAT2 is likely to have some impact on IFNg biology but the nature of this activity and whether it is dependent on phosphorylation remains unknown. It is unlikely that compensation by other STATs fully accounts for the responses to IFNg observed in STAT1deficient mice. There is a subset of IFNg-induced genes that are transcribed to a similar extent in wild-type and STAT1-

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deficient cells, implying that STAT1-independent pathways are not a cellular contingency plan for the rare circumstance of STAT1 deficiency. Indeed, there is strong evidence (discussed in Sections 3–5) of the existence of signaling pathways that act entirely in parallel to JAK/STAT1 signaling following IFNg stimulation. Ironically, a key step in the paradigm shift from a single JAK–STAT1 pathway of IFNg signaling to a more multi-faceted signaling network concept came from studying STAT1 activation. STAT1 requires phosphorylation on S727 and Y701 to achieve full transcriptional and anti-pathogenic activity [10,15], demonstrating that IFNg must, in addition to JAK tyrosine kinases, activate serine kinase(s). A diverse array of kinases have been proposed to phosphorylate STAT1 on S727 including phosphoinositide 3-kinase (PI3-K)/Akt [30], protein kinase C (PKC) isoforms [31,32], calcium/calmodulin kinase II (CaMKII) [33] and mitogen activated protein (MAP) kinases [34]. Identifying these kinases has provided some insight into the non-JAK/STAT pathways that may be activated by IFNg as, in addition to phosphorylating STAT1 on S727, these kinases will likely activate other molecular pathways that in turn elicit their own specific biological responses.

3. Proteins recruited to the IFNg receptor JAK1 and JAK2 are constitutively associated with IFNgR1 and IFNgR2 chains of the IFNg receptor respectively and STAT1 is recruited to phosphorylated Y440 after receptor ligation [2]. However, several studies proposed that the IFNgR can form higher order receptor complexes (Fig. 2A). IFNg stimulation led to the rapid (within 5 min) JAK2-dependent trans-activation of the epidermal growth factor receptor (EGFR) in A431 and HeLa cells, though not in HEK293 cells [35]. This pathway is likely to engage non-STAT1-signaling mechanisms and warrants further study, although the physiological significance remains undefined. Additionally, the type I IFN receptor I (IFNAR1) was shown to interact with the IFNgR in membrane calveolae and this heterodimerization of type I and II IFN receptors was necessary for efficient IFNg signaling and the IFNg-mediated anti-viral state [36]. A potentially simpler explanation of the impact of the loss of IFNAR1 on IFNg anti-viral response is that in its absence cells lack type I IFN priming which is of known importance to IFNg biology [37]. This priming between the interferon subclasses is typically associated with the increase in gene expression necessary for a rapid IFN response, e.g. ISGF3 components such as STAT1 and other inducible signaling components [38]. It should therefore prove informative to assay IFN receptor deficient cells for the expression of IFN signaling intermediaries. However, priming would likely be expected only to enhance the efficiency of existing JAK– STAT signaling, not engage alternate pathways. Less is known about proteins other than JAK and STAT that may be recruited to the IFNgR following IFNg

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stimulation and whether they simply contribute to conventional JAK/STAT signaling or initiate alternate signaling pathways. This is partly due to the technical difficulty in identifying proteins which only interact transiently following stimulation; however the use of sensitive technologies like affinity purification and mass spectrometry or fluorescent microscopy techniques will give insights into the molecular mechanisms of IFNg signaling diversity. Conventional biochemical techniques have been used to identify a number of potential candidates for IFNgR-interacting proteins that could initiate alternative signaling cascades including Src kinases, PI3-K, adaptor molecules, guanidine exchange factor (GEFs) and GTPases (Fig. 2B). The Src family non-receptor tyrosine kinases c-Src, Fyn and Lyn are phosphorylated in response to IFNg treatment in a JAK-dependent manner [24,26,39]. While JAK1 and JAK2 are necessary for IFNg-stimulated c-Src and Lyn phosphorylation, only Fyn is known to directly interact with JAK kinases [40] and none of these Src kinases are known to directly interact with the IFNgR complex. Co-immunoprecipitation studies indicate that phospholipase-C (PLCa or g) forms a complex with JAK1/2 and c-Src/Lyn following IFNg stimulation and IFNg-induced activation of Src kinases is blocked by chemical inhibitors of PLC [39].

However, it is unclear whether this represents an alternative pathway, because the only endpoint of these investigations was the activation of STAT1. PI3-K is an attractive prospect for a signaling molecule recruited to the IFNgR, in part because IFNg and PI3-K are involved in regulating similar biological activities including survival, apoptosis, and proliferation, but primarily because IFNg activates PI3-K equally well in cells devoid of JAK1 or 2 as it does in wild-type cells [30]. This could closely parallel the signaling of type I IFNs during which the p85 subunit of PI3-K has been reported to interact directly with IFNAR1 [41], independent of JAK mediated phosphorylation of the receptor [42], though this has not been formally tested. It is also possible that compensation by other JAKs allows for IFNg-mediated stimulation of PI3-K in JAK1- or 2-deficient cells and this remains to be tested. IFNg stimulation engages molecules with adaptor functions, such as MyD88 [43] and the c-Cbl proto-oncogene [44], and GTPases, such as Ras and Rap1 (Fig. 2B). MyD88 is better known as an adaptor molecule that is critical for signaling through toll-like receptors (TLR) to activate MAP kinase and NF-kB pathways [45]. Recently, MyD88 was found to co-immunoprecipitate with IFNgR1 and as there is no defect in IFNg-induced STAT1 activation in the absence of

Fig. 2. Proteins recruited to the IFNg receptor. (A) IFNg treatment results in higher order receptor complexes between the type I and II IFN receptor or IFNgR and the EGFR. (B) IFNg stimulates the recruitment of kinases like PI3-K, PLC and Src family kinases in addition to GTPases Ras and Raf1 and the adaptor molecule CrkL to initiate multiple downstream signaling cascades.

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MyD88, this may act as a component of an alternate pathway. Consistent with this hypothesis a role has been identified for MyD88 in stabilization of ISG mRNA [43]. IFNg treatment results in the phosphorylation of c-Cbl with similar kinetics to phosphorylation of JAK1/2 [44]. c-Cbl is an E3 ubiquitin ligase that generally acts as a negative regulator of cell signaling. However, the tyrosine residues of c-Cbl that become phosphorylated in response to IFNg can act as docking sites for SH2 domain containing proteins including CrkL which in turn recruits and activates the GEF C3G via its SH3 domain. C3G acts as a GEF to activate the GTPase Rap1 [44]. This C3G-CrkL pathway activated by IFNg contrasts with that activated by type I IFN which recruits CrkL to IFNAR1 via JAK1 and Tyk2 and results in the activation of a transcription factor complex comprised of STAT5 and CrkL, because of the lack of a STAT5-CrkL complex in response to IFNg [44]. In summary, a number of proteins including PI3-K, c-Cbl and MyD88 are recruited to the ligated IFNgR (Fig. 2B). These proteins are either linked to signaling pathways that are independent of STAT1, or can augment STAT1 activity. In most cases JAK kinase activity is required; however it is likely that the activation of the PI3-K pathway is activated by a JAK-independent mechanism.

4. IFNg signaling downstream of the IFNgR The recruitment of proteins Src, PI3-K, c-Cbl and MyD88 to the IFNgR leads to the initiation of signaling cascades that involve proteins such as MAP kinase, AKT, CaMKII, and IkB kinase (IKK) (summarized in Fig. 3). In many cases these signaling events were identified as candidates for phosphorylation of STAT1 on S727. Because so many different kinases are potentially capable of targeting this residue it is unclear which of them is most physiologically relevant, particularly as they all have other well-documented substrates. These substrates (including MAP kinase, IKK/NF-kB and Akt) probably constitute key signal transduction proteins in STAT1-independent IFNg signaling. 4.1. MAP kinase pathways IFNg triggers the activation of the MEK1/ERK1/2 pathway in a range of cell lines [46] and primary cells [27] which could occur through a number of different molecular pathways. First, activation of ERK can occur through trans-activated EGFR, however ERK can be activated by other pathways when this route is unavailable [35]. The Raf1 serine kinase can associate with JAK2 and become phosphorylated and activated by IFNg resulting in the phosphorylation of ERK. While Ras also appears to be required in this pathway its mechanism of activation is unknown [47]. Interestingly, the Ras and Raf1 pathway to ERK activation is independent of MEKK1 [48], which implies that there are multiple mechanisms in place to

Fig. 3. Alternate IFNg signaling cascades. IFNg signaling engages multiple pathways including the MAP kinase and IKK/NF-kB pathways through the activation of PI3-K, GTPases, Pyk2, and PKC.

transmit the IFNg signal to ERK. Not only are there multiple pathways to IFNg-stimulated ERK activation, but there also appears to be two waves of ERK activation. One that occurs within 30 min of stimulation leads to the activation of AP-1 independent of the JAK–STAT1 pathway [27], a second pathway is active following a 2 h exposure to IFNg, which is necessary for STAT1 S727 phosphorylation [46]. The activation of p38 MAP kinase by IFNg is somewhat controversial. IFNg-stimulated recruitment of MyD88 to the receptor was shown to trigger activation of the MKK6/p38 MAP kinase pathway [43]. Furthermore c-Src activation at the IFNgR resulted in the activation of the calcium dependent kinase Pyk2 leading to the activation of the Mekk4/MKK6/p38 MAP kinase pathway [49]. However, others have been unable to demonstrate phosphorylation of p38 MAP kinase in response to IFNg [27,50]. Most groups have reported that IFNg does not activate JNK MAP kinase [27,51]. However, it was recently reported that IFNg can activate JNK in macrophages where it appeared to be required for the expression of genes associated with antigen presentation [52]. Together these data suggest that ERK MAP kinase is the major IFNg activated MAP kinase, however contributions from p38 and JNK cannot be discounted, especially in specific cell lineages.

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4.2. PI3-K, Akt and protein kinase C IFNg-mediated activation of PI3-K has several outcomes that may not necessarily be mutually exclusive (Fig. 3): the serine phosphorylation of STAT1 and the activation of downstream signaling proteins including Akt and PKC. Although phosphorylation of STAT1 on S727 in response to IFNg can be blocked by inhibitors of PI3-K [30], the enzyme from this pathway required to phosphorylate STAT1 is unlikely to be PI3-K itself, because the phosphorylation can be also be prevented by inhibitors of kinases that act downstream of PI3-K, such as mTOR [30], Akt [30] or PKCd [54]. The phosphorylation of Akt [30] or PKCe [31] in response to IFNg is blocked by expression of dominant negative PI3-K or PI3-K inhibitors confirming that these kinases are acting downstream of PI3-K. IFNg-induced activation of PKC isoforms (a, d, e) may be a point of divergence in IFNg signaling. Three PKC isoforms are activated by IFNg but only PKCd directly bound to and phosphorylated STAT1 on S727 [54]. Unlike PKCd, IFNgactivated PKCa and PKCe had no effect on STAT1 phosphorylation. PKCa augments IFNg-induced transcription of MHC class II and ICAM [39,55]. IFNg-induced PKCe activation led to the phosphorylation of ERK1/2 MAP kinase [31]. Because these two PKC isoforms impact on IFNg signaling and biology without affecting STAT1 they may prove to be important mediators of STAT1-independent IFNg signaling. However, it is important to note that different laboratories have reported distinct results and the identity of the IFNg activated S727 kinase remains unclear. In summary, following binding of IFNg to its receptor, many downstream effector molecules are activated in addition to the canonical JAK–STAT pathway (Fig. 3). The best characterized of which are PI3-K and MAP kinase pathways. The activation of these pathways have several outcomes pivotal to IFNg signaling: (i) phosphorylation of STAT1 on S727 thus increasing its transcriptional efficacy; (ii) phosphorylation and activation of additional substrates result in the stimulation of alternate signal transduction cascades leading to STAT1-independent gene transcription and subsequent biological outcomes. Other less well-characterized enzymes include CamKII which co-purifies with STAT1 resulting in S727 phosphorylation and Ca2+ flux [33], but it is unclear whether its activation has consequences other than STAT1 S727 phosphorylation.

5. IFNg activates transcription factors other than STAT1 Whilst the STAT1 homodimer is the primary and best characterized route to IFNg-induced transcription, IFNg additionally activates other transcription factors which will be discussed in the following sections. These factors fall into three categories: (i) those transcription factors that are

activated downstream of STAT1, i.e. they are transcriptional targets of STAT1 and therefore represent secondary responses (‘‘STAT1 dependent’’, e.g. IRF-1 and CIITA); (ii) those transcription factors that form a protein complex with STAT1 on gene promoters to augment IFNg-mediated transcription (‘‘STAT1 co-operative’’, e.g. ISGF3 [28], STAT1/IRF9 [37] and STAT1/c-Jun [56]); and (iii) those transcription factors that can be activated independently of STAT1 and thus can function in parallel with STAT1 (‘‘STAT1-independent’’, e.g. NF-kB and AP-1). 5.1. IFNg-activated STAT1-dependent or co-operative transcription factors 5.1.1. IRF family transcription factors An important family of transcription factors activated by IFNg downstream of STAT1 is the interferon regulatory factor (IRF) family. In the context of IFNg the most important are IRF-1 and IRF-8 which have the capacity to bind to ISRE and other IFN regulated response elements in the promoter of genes and initiate the transcription of both ISGs and the IFNs themselves (reviewed in [57]). Loss of IRF-1 confers a severe defect in anti-microbial and pathogenic immunity and the loss of many IFNg-stimulated genes. IRF-1 is not directly activated by IFNg, rather its expression is induced by IFNg in a STAT1-dependent fashion over a period of hours [58,59]. IRF-1 cooperates with STAT1 to transcribe ISGs such as GBP1/2 [60] and gp91phox [61] which require intact IRF-1 and STAT1-binding sites to be optimally transcribed [60]. Promoters of other ISGs such as LMP2 appear to contain sites that bind to complexes of IRF-1 and STAT1—a novel mode of ISG induction which does not require STAT1 to be phosphorylated on Y701 [62]. IRF-8 is specifically activated by IFNg, not IFNa/b [63,64]; however its expression is restricted to macrophage and lymphoid lineages, where it can co-operate with other transcription factors including PU.1 and IRF-1 to drive the differentiation of these lineages [65]. Importantly, cells expressing phosphorylation site mutants of STAT1 cannot induce IRF-8 or drive differentiation of U937 cells [66]. Thus like IRF-1, IRF-8 appears to be dependent on and co-operate with conventional STAT1 signaling. 5.1.2. MHC class II trans-activator MHC class II trans-activator (CIITA) is a transcription factor whose primary role is to regulate the expression of MHC II [67], although it is also reported to affect the expression of other genes [68]. Regulation of CIITA expression is complex because it can involve four independent promoters (pI–IV). Induction of CIITA by IFNg requires the pIV promoter and this is entirely dependent of the expression of STAT1 and IRF-1 [53,69,70]. The induction of CIITA itself provides a further example of co-operation of STAT1 with other transcription factors. A complex consisting of upstream factor I (USF-1) and STAT1 is required to efficiently induce CIITA in response to IFNg [69].

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5.1.3. CCAAT/enhancer binding protein beta The transcription factor CCAAT/enhancer binding protein beta (C/EBPb) binds to a unique IFNg-responsive enhancer element known as gamma activated transcription element (GATE) found in the promoter of the ISG p48 (IRF9, ISGF3g) [71]. A recent report has shown that a subset of IFNg stimulated genes lose responsiveness to IFNg in CEBP/b deficient macrophages and that these cells don’t growth arrest following IFNg stimulation [72]. IFNg enhances the expression of C/EBPb [46], however, how IFNg activates C/EBPb is unclear. C/EBPb activation was reported to require phosphorylation by ERK1/2 in a STAT1-dependent manner [46]. However our studies found that, at least at early time points, ERK activation in response to IFNg is normal in STAT1/ cells [27]. Thus C/EBPb is likely to be required for the induction of a subset of ISGs, however it is unlikely to be necessary for STAT1-independent IFNg biology. 5.1.4. PU.1 PU.1 is an ETS domain containing transcription factor important for myeloid development. The activity of PU.1 is largely regulated at the level of expression which is unaffected by IFNg; however, stimulation of cells with IFNg does augment PU.1-dependent transcription of genes such as FcgRI [73], by means of three distinct molecular mechanisms. The first activity is direct, as IFNg can induce PKC-dependent phosphorylation of PU.1 [74]: an activity that may prove to be STAT1 independent. Secondly, PU.1 can form complexes with IRF-8 [75]. Finally, PU.1 co-operates with STAT1 to induce genes like gp67phox in myeloid cells [76], although in this case, PU.1, IRF-8 and STAT1 bind to the gp67phox promoter independently of one another. 5.2. STAT1-independent, IFNg-induced transcription factors In addition to STAT1-dependent and co-operative transcription factors, IFNg activates some transcription factors equivalently in wild-type and STAT1/ cells. These factors appear to function in parallel to STAT1 to mediate the biological effects of IFNg. A subset of ISGs is transcribed equivalently in wild-type and STAT1/ cells suggesting that STAT1-independent mechanisms of signaling would operate in all cells, not just the rare instances of STAT1 deficiency. Therefore, it will be important to identify transcription factors activated by IFNg with the same kinetics and to a similar magnitude in wild-type and STAT1-deficient cells. Only four transcription factors may satisfy these criteria, STAT3/5, NFkB and AP-1. 5.2.1. STAT3 and STAT5 STAT3 and STAT5 are activated by IFNg and may compensate for the absence of STAT1 in certain circumstances [25,26,77], but STAT3 cannot compensate for the loss of STAT1 with respect to anti-viral responses [78]. Two reports demonstrated that expression and/or

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phosphorylation of STAT3 was enhanced in cells deficient in STAT1 [26,77], however our work could not confirm this finding [27]. The reason for this discrepancy is unclear, but could relate to the strains of STAT1-deficient animals used in each study. The activation of STAT5 is dependent on cell lineage and maturation state. STAT5 is not phosphorylated in HeLa, cells but is in U937 cells and this phosphorylation is diminished when U937 cells are differentiated along the monocytic lineage [25]. Therefore, whilst STAT3 and STAT5 may represent a STAT1-independent mechanism of IFNg-mediated transcription, they are only likely to be engaged in the rare circumstance of STAT1 deficiency, or in a cell lineage dependent manner. 5.2.2. NF-kB The importance of the NF-kB pathway in IFNg signaling is still emerging. IFNg can induce DNA binding of NF-kB in a STAT1-independent manner [32]. However activation of NF-kB DNA binding is restricted to specific cell lineages—primarily fibroblasts suggesting this may not be a major mechanism of STAT1-independent transcription. Commonly, the inhibitor of kB kinases (IkB) holds the NFkB components in an inactive state. In response to stimuli (including IFNg) it is phosphorylated by the IKK leading to IkB degradation and thus the release of active NF-kB components. As expected IFNg stimulation led to the phosphorylation of IKKa and b subunits, and in the absence of these proteins the induction of subsets of ISGs was attenuated [79,80]. Although this pathway requires expression of the NF-kB subunit RelA, it does not involve NF-kB DNA binding [79,80]. Therefore, there are two discrete NFkB related pathways; (i) activation of the NF-kB transcription factor and (ii) the activation of IKK and RelA independent of NF-kB DNA binding. As yet, it is not known how IKKa/b and RelA affect IFNg-mediated transcription, but it is known that the pathway is functional in STAT1deficient cells and as such could represent an important STAT1-independent mechanism of IFNg-mediated transcription. 5.2.3. AP-1 The AP-1 transcription factors are rapidly activated by IFNg and are required for transcription of several ISGs [27,81]. IFNg stimulation results in a rapid increase in AP-1 DNA binding activity, independent of JAK1/2 or STAT1 [27]. Importantly, the rapid activation of AP-1 is necessary and sufficient to initiate the transcription of a subset of ISGs either by co-operating with STAT1 or acting entirely independently of STAT1 [27]. While activation of AP-1 is commonly associated with phosphorylation of Jun-family proteins by JNK1 and/or JNK2, the JNK pathway is not activated by IFNg [51] and instead the ERK pathway is used by IFNg to phosphorylate and activate c-Jun [27]. Although AP-1 can act entirely independent of STAT1 and a subset of genes depend only on c-Jun expression, AP-1 and STAT1 cooperate to

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activate other subsets of ISGs [27]. In an analysis of the a2macroglobulin gene induction in response to IL-6 it was discovered that STAT3 and c-Jun directly interact with each other and the a2-macroglobulin promoter to enhance its transcription [56]. It is possible that c-Jun and STAT1 can physically interact in response to IFNg treatment to augment IFNg-mediated transcription. Alternatively, the co-operation between c-Jun and STAT1 could simply be a consequence of independent promoter binding of two distinct complexes, these two hypotheses would need to be formally tested. Interestingly, extended IFNg treatment (greater than 12 h) inhibited both AP-1 DNA binding and transcription [82] possibly due to negative feedback mechanisms involving proteins of the SOCS family. These data show that despite the number of transcription factors activated by IFNg only AP-1, IKK/RelA, NF-kB and STAT3/5 are sufficient for gene induction independent of STAT1. Whilst this is a small repertoire of transcription factors, they are commonly activated by the alternate pathways engaged by IFNg and have overlapping biological properties with IFNg.

STAT1-dependent or co-operative transcription factors like STAT5, GATA1, IRF8 and PU.1 could determine the cell specific effect of IFNg on cellular proliferation. IFNg may be able to induce cell cycle arrest through a STAT1-independent mechanism in macrophages. Exposure of macrophages to IFNg causes them to arrest at the G1/S phase transition of the cell cycle [90,91] which is largely controlled by pRb and c-Myc. These two genes are reported to be regulated by IFNg independent of STAT1 [20,23,92] suggesting that IFNg may regulate the cell cycle at least in part via a STAT1-independent pathway. Another STAT1independent IFNg-signaling pathway is implicated in growth arrest. Depletion of CrkL using anti-sense technology impaired IFNg-mediated growth retardation of primary erythroid and granulocyte/macrophage cultures [93]. Therefore, IFNg-activation of CrkL is necessary for antiproliferative effects in cultures of hematopoietic progenitor cells, it is still necessary to determine whether this occurs by engaging the pathways identified to be downstream of IFNg-activated CrkL (Rap1, PI3-K and/or MAP kinase [44]).

6. STAT1-independent IFNg biology

6.2. Adhesion

While many aspects of IFNg biology are STAT1 dependent there is good reason to believe that several facets of IFNg biology that are independent of STAT1; such as cell cycle arrest, leukocyte adhesion and some aspects of the anti-viral response.

An examination of the ontogeny of STAT1-independent ISGs suggests that leukocyte recruitment and adhesion may be STAT1-independent processes. Microarray studies comparing the genes induced by IFNg in bone marrow derived macrophages from wild-type and STAT1-deficient mice identified chemokines and their receptors as a group of genes induced in both genotypes [23]. This is supported by direct experimental evidence because IFNg-induced leukocyte adhesion has a STAT1-independent component. IFNgstimulated monocyte adhesion is dependent on PI3-K, but is unaffected by the loss of STAT1 [94]. In addition, the IFNg-induced expression of a macrophage adhesion molecule, ICAM-1, is dependent on a non-JAK/STAT pathway. Over-expression of a dominant negative c-Src protein, or addition of small molecule c-Src inhibitors attenuated the induction of ICAM-1 by IFNg [39,55]. It is a fascinating possibility that IFNg-stimulated recruitment and extravasation of leukocytes is mediated by two ‘‘alternate’’ IFNg-responsive enzymes (PI3-K and c-Src) and could be independent of STAT1, however this hypothesis requires further investigation. The pathways described in this review remain largely untested in the context of STAT1-independent IFNg biology. It will prove informative to assess the anti-viral, adhesion and cell cycle responses to IFNg in vivo or ex vivo in mice devoid of NF-kB or AP-1 components. However, deletion of IKKb, RelA or c-Jun – components of NF-kB and AP-1 – results in embryos terminating in utero at mid to late gestation from hematopoietic and liver defects [95–97]. Therefore it will be necessary to rely on conditional deletion of these genes from tissues or cell lineages relevant to the biological process being interrogated.

6.1. Cell cycle arrest While IFNg mediates growth arrest in most cell types, it robustly induces proliferation in Th1 lymphocytes [83]. However IFNg can also induce apoptosis in Th1-cells following the engagement of the TCR [84]. These differing responses have been attributed to signal strength as a result of differences in the levels of expression of IFNgR2 [85], however there may be other potential explanations including differences in expression of other components of IFNgactivated signaling pathways. One factor affecting IFNg-responsiveness of cells may be the expression level of STAT1. Although STAT1 is ubiquitously expressed, the absolute expression levels can vary substantially between cell types and this may impact on cellular responses to IFNg [59,86–88]. For example, STAT1 can be induced by priming with type I and type II IFNs and IFN-primed cells respond more efficiently to subsequent IFNg treatment than non-primed cells [89]. Moreover, the levels of STAT1 increase following monocyte differentiation of U937 cells [86]. The concept of signal strength may apply to this phenomenon, i.e. altering the level of STAT1 expression could impinge on the magnitude of the IFNg response; however, the influence of non-STAT pathways may also be more substantial in cells expressing low levels of STAT1. Cell specific expression of other IFNg-activated,

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7. Concluding remarks Initially, IFNg signal transduction was viewed as a linear process in which exogenous IFNg transmitted a signal from an activated cell surface receptor (IFNgR, JAKs) to a transcription factor (STAT1) capable of initiating gene transcription to mediate biological responses. However, in this model a diverse array of biological outcomes is controlled by a single transcription factor (STAT1); conversely, other cytokines utilize JAK–STAT1 signaling to mediate biological responses very different to that of IFNg (e.g. Oncostatin M). Therefore, a complex IFNg signaling network is more plausible than a single discrete linear pathway model of signaling. Whilst STAT1 is of great importance to IFNg biology, there are aspects of IFNg biology that are evident in the absence of STAT1. This is not only relevant in the rare instances of STAT1 deficiency in humans, but also following infection with virus which inactivate the STAT molecules [98]. In these circumstances, the alternate pathways activated independent of STAT still remain to help ameliorate infection. Additionally, the activation of multiple pathways/transcription factors allows a higher degree of control of transcription which is of great importance with a stimulus like IFNg which is capable of eliciting such potent and varied biological processes. However, despite more than 50 years of research we are only just beginning to uncover the complexities of IFNg signaling networks and their biological consequences.

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Prof. David E. Levy holds the Dr. Louis A Schneider Chair in Molecular Pathology at the New York University School of Medicine. He has dedicated his career to understanding of basic mechanisms of gene regulation and the actions of inflammatory cytokines. His contributions to the field have included describing and identifying transcription factor complexes required for type I IFN and IFNstimulated gene regulation and exploring their mechanisms of action and their roles in inflammation, both in vitro and in animal models.

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D.J. Gough et al. / Cytokine & Growth Factor Reviews 19 (2008) 383–394 Ricky W. Johnstone, PhD is a Pfizer fellow and Assistant/Director of the Research Division of the Peter MacCallum Cancer Centre and Assistant/Professor of Pathology at the University of Melbourne. The study of interferon-induced gene expression has been a major focus of his work; in particular focusing on activation of AP-1 as a mode of transducing STAT1-independent signals.

Dr. Christopher J. Clarke is a post-doctoral fellow in the Laboratory of A/Prof Johnstone at Peter MacCallum Cancer Centre. He has had a long-standing interest in the study of cytokine signal transduction and is currently focused on links between type I and type II interferon signaling.