Introduction
Interferons (IFNs) are pleiotropic cytokines that mediate anti‐viral responses, inhibit proliferation and participate in immune surveillance and tumor suppression (
Farrar and Schreiber, 1993;
Stark et al., 1998). Transcriptional regulation in response to IFNs is mediated by the Jak–Stat pathway (for recent reviews, see
Leaman et al., 1996;
Darnell, 1997;
Stark et al., 1998). Negative as well as positive regulation of gene expression in response to IFN‐γ has been reported (
Der et al., 1998;
Sharma and Iozzo, 1998). IFN‐α and ‐β activate Stat1 and Stat2, which, with p48, form the transcription factor ISGF3, which binds to IFN‐stimulated response elements. Activated Stat1 dimers translocate into the nucleus and bind to gamma‐activated sequence (GAS) elements. All IFNs cause phosphorylation of Stat1 on Tyr701 and Ser727 (
Wen et al., 1995) and both phosphorylations are required for maximal transactivation (
Wen et al., 1995;
Wen and Darnell, 1997). Stat1 dimers function through interaction with transcriptional co‐activators such as CBP/p300, Nmi and MCM‐5, as well as with other transcription factors, including p48 and SP1 (
Look et al., 1995;
Horvath et al., 1996;
Zhang et al., 1996,
1998;
Zhu et al., 1999). In addition to the IFNs, many growth factors and cytokines also activate Stat1 (
Schindler and Darnell, 1995).
Targeted disruption in mice has confirmed that Stat1 is obligatory for signaling in response to all IFNs and has revealed that Stat1 is also involved in immune surveillance and tumor suppression (
Durbin et al., 1996;
Meraz et al., 1996;
Kaplan et al., 1998). Some tumor cells and tumor‐derived cell lines express little or no Stat1 mRNA or protein (
L.H.Wong et al., 1997;
Abril et al., 1998;
Sun et al., 1998) or fail to activate Stat1 following treatment with IFNs (
Lucas et al., 1998). IFNs inhibit the growth of many cell types (
Balkwill and Taylor‐Papadimitriou, 1978;
Lin et al., 1986;
Kimchi, 1992), and Stat1 that is fully active transcriptionally is required for this effect (
Bromberg et al., 1996). The inhibition of cell growth correlates with the regulation of several cell cycle regulatory genes by IFNs. mRNAs encoding cyclin D and cdc25A decrease in response to IFN‐α and ‐β, and expression of the cyclin‐dependent kinase (CDK) inhibitor p21
waf1 is up‐regulated by IFN‐γ in epidermal carcinoma and glioblastoma cell lines (
Chin et al., 1996;
Tiefenbrun et al., 1996;
Kominsky et al., 1998). In contrast, the inhibition by IFN‐γ of the growth of the colon carcinoma cell line HCT116 is independent of p21 (
Sharma and Iozzo, 1998). IFN‐γ can stimulate rather than suppress the growth of certain cells (
Caux et al., 1992;
Shiohara et al., 1993), but the basis of this paradoxical activity is unclear. Stat1, and indeed other Stats, can also mediate negative regulation of gene expression in response to effectors other than the IFNs. For example, EGF‐induced proliferation correlates with the transient activation of Stat1, whereas EGF‐mediated growth suppression correlates with its sustained activation (
Bromberg et al., 1998).
c‐
myc, a transcription factor that helps to regulate proliferation, is induced rapidly and transiently by many growth factors and cytokines (
Spencer and Groudine, 1991;
Bouchard et al., 1998;
Dang, 1999). The expression of c‐
myc is aberrant in a variety of human tumors (
Marcu et al., 1992). Its ectopic expression overrides both the G
1 and S check points, promoting genomic instability and tumorigenesis (
Chernova et al., 1998;
Felsher and Bishop, 1999). c‐
myc regulates the G
1–S transition by activating cyclin–CDK complexes and, together with its dimerization partner
max, transactivates genes required for entry into S‐phase (
Blackwood and Eisenman, 1991;
Grandori and Eisenman, 1997;
Obaya et al., 1999). Treatment with IFN‐α and ‐β abolishes the formation of transcription factor complexes on the E2F site of the c‐
myc promoter and suppresses c‐
myc expression in both Daudi and M1 cells (
Resnitzsky and Kimchi, 1991;
Melamed et al., 1993).
The constitutive expression of ectopic c‐
myc overcomes IFN‐γ‐mediated arrest of macrophages and vascular smooth muscle cells, indicating that c‐
myc is likely to be involved in the inhibition of proliferation mediated by IFN‐γ (
Bennett et al., 1994;
Vairo et al., 1995). We now find that IFN‐γ inhibits the expression of c‐
myc in wild‐type cells, an effect that is mediated by consensus GAS elements in the c‐
myc promoter to which Stat1 homodimers bind. Furthermore, in Stat1‐null cells, both c‐
myc and c‐
jun are induced transiently and rapidly by IFNs, revealing a novel signaling pathway. In IFN‐γ‐treated PKR‐null mouse cells, serine phosphorylation of Stat1 is defective, transactivation is impaired and c‐
myc mRNA is induced, not suppressed. Furthermore, inhibitors of Raf‐1 activation abrogate the IFN‐dependent induction of c‐
myc in Stat1‐null cells, indicating that Raf‐1 is important in Stat1‐independent signaling.
Discussion
c‐
myc and c‐
jun, required for cell cycle progression (
Grandori and Eisenman, 1997;
Obaya et al., 1999;
Wisdom et al., 1999), are important targets of Stat1‐independent responses to IFN‐γ. c‐
jun is required for progression through G
1 and for trans‐activation of the cyclin D1 gene in fibroblasts (
Wisdom et al., 1999), thus helping to provide a link between the response to growth factors and cell cycle regulation. Deregulated expression of c‐
myc and c‐
jun is likely to be important in the abnormal proliferation of Stat1‐null cells in response to IFN‐γ, which can serve as a growth factor for some cells (
Caux et al., 1992;
Shiohara et al., 1993). IFN‐γ suppressed the expression of c‐
myc in wild‐type cells and is likely to be important in regulating the switch between growth arrest and proliferation. IFN‐γ or ‐β abrogated the induction by PDGF of c‐
myc expression in wild‐type cells but not in Stat1‐null cells, suggesting that Stat1 is required for this response. IFN‐α also abrogated the induction of c‐
myc by PDGF in NIH 3T3 fibroblasts and in Kaposi's sarcoma cells (
Einat et al., 1985;
Koster et al., 1996), although the direct involvement of Stat1 in these responses was not demonstrated. Our results also indicate that c‐
myc, but not the CDK inhibitor p21
waf1, is a target of regulation by IFN‐γ in human fibrosarcoma cells. p21 has been implicated as a mediator of IFN‐γ‐dependent growth arrest in epidermal carcinoma and glioblastoma but not colon carcinoma (HCT116) cell lines (
Chin et al., 1996;
Kominsky et al., 1998;
Sharma and Iozzo, 1998). p21 is induced by IFN‐γ in tumor cell lines harboring mutated p53 but not in cell lines expressing wild‐type p53 such as HT1080 (from which 2fTGH and U3A cells are derived), where its basal expression is high, indicating that the regulation of p21 expression by p53 is dominant over IFN‐γ‐mediated regulation of this gene. Studies in U3A cell variants indicate that tyrosine and serine phosphorylation sites in the C‐terminal transactivation domain of Stat1 are required to suppress c‐
myc expression. These results are consistent with previous data suggesting that transcriptionally competent Stat1 is required for the anti‐proliferative effect of IFNs (
Bromberg et al., 1996).
Transient transfection of a c‐
myc promoter fragment linked to luciferase revealed that a consensus GAS element −1107 to −1099 (relative to the P1 promoter) is required for c‐
myc suppression. This element differs from the previously identified GAS element, which binds to Stat3 preferentially, overlaps the E2F element and functions in the IL‐6‐ and gp130‐mediated transactivation of c‐
myc (
Kiuchi et al., 1999). Stat1 binds to the upstream GAS as a homodimer in extracts of IFN‐γ‐treated wild‐type cells. The upstream element is necessary but not sufficient for suppression of c‐
myc since it lacks intrinsic repressor activity. Therefore, Stat1 is likely to interact with a co‐repressor bound to another site in the c‐
myc promoter to inhibit expression. A likely candidate is Blimp‐1, a member of the Groucho family of co‐repressors that binds to the PRF site of the c‐
myc promoter and mediates repression (
Lin et al., 1997;
Ren et al., 1999). Since Blimp‐1 is expressed exclusively in B‐lymphocytes, other Groucho family members may participate in repressing c‐
myc expression in other cell types. Another candidate is MBP‐1, which represses c‐
myc expression when bound to the E2F site (
Ray and Miller, 1991). IFN‐γ inhibits the transcription of several genes, including those encoding perlecan, bullous pemphigoid antigen 1 and cyclin A (
Tamai et al., 1995;
Sharma and Iozzo, 1998;
Sibinga et al., 1999). Transcriptional repression of the perlecan gene by IFN‐γ requires functional Stat1 and a promoter region containing multiple GAS elements. However, the binding of Stat1 to these GAS elements has not been reported and thus the mechanism of repression is not known (
Sharma and Iozzo, 1998).
In NIH 3T3 cells stably expressing cd2 under the control of the 1.7 kb c‐
myc promoter, treatment with PDGF induced cd2 expression, and simultaneous treatment with IFN‐γ abrogated this induction. The induction of gene expression by PDGF depends on several signal transduction pathways, among which is the
ras/MAPK pathway, and involves the E2F site of the c‐
myc promoter (
Sacca and Cochran, 1990;
Claesson‐Welsh, 1994).
ras/MAPK‐activated Ets factors have been proposed to mediate c‐
myc expression in response to growth factor stimulation (
Roussel et al., 1994;
Aziz et al., 1999;
Cheng et al., 1999). The abrogation by IFN‐γ of c‐
myc induction in response to PDGF might involve a competition between promoter‐bound Ets factors and Stat1 dimers for co‐activators such as CBP/p300 (
Horvai et al., 1997). PDGF activates the formation of complexes involving Stat1, Stats1 and 3 and Stat3 on the SIE element of the c‐
fos gene (
Vignais et al., 1996). However, PDGF did not induce the formation of Stat complexes on the c‐
myc GAS element, indicating that the abrogation by IFN‐γ of c‐
myc induction in response to PDGF does not involve a competition between different Stat dimers for the GAS site.
PKR is involved in regulating anti‐viral, anti‐proliferative and tumor suppressor functions (
Clemens and Elia, 1997), and PKR‐null cells are defective in activating IRF‐1 and NF‐κB in response to double‐stranded RNA (
Kumar et al., 1997a). The defective activation of the GBP and IRF‐1 promoters by IFN‐γ in PKR‐null cells can be rescued by expressing wild‐type but not mutant PKR, indicating that PKR is also required in IFN‐γ‐dependent signaling (
Kumar et al., 1997a). In extracts of IFN‐γ treated cells, a decrease in the mobility of PKR in SDS–PAGE gels was observed, consistent with its phosphorylation (
Kumar et al., 1997a). A dominant‐negative derivative of PKR abrogated both the IFN‐α‐mediated downregulation of c‐
myc expression and the inhibition of cell growth (
Raveh et al., 1996). Our results indicate that PKR‐null cells are defective in phosphorylating Stat1 on Ser727, and that Stat1‐dependent transactivation is 4‐fold lower in PKR‐null than in wild‐type cells, an effect comparable to the reduction observed for the S727A mutant of Stat1 (
Zhang et al., 1998). Furthermore, in PKR‐null cells, c‐
myc mRNA is induced transiently and rapidly in response to IFN‐γ, just as it is in Stat1‐null cells. Therefore, both Stat1 and PKR are required to suppress the expression of c‐
myc in wild‐type cells. PKR associates with Stat1 both
in vitro and
in vivo, although it does not phosphorylate Stat1 directly (
A.H.Wong et al., 1997). Therefore, PKR may be part of a kinase cascade involved in phosphorylating Stat1 on Ser727 in response to IFN‐γ. The phosphorylation of Stat1 on Ser727 is required for maximal transactivation and recruitment of the transcription co‐factor MCM‐5 (
Wen et al., 1995;
Zhang et al., 1998), also a component of the DNA replication licensing factor. The recruitment of MCM‐5 from origins of DNA replication to the transcriptional machinery, mediated by Stat1 in response to IFN‐γ, has been suggested as a mechanism for suppression of proliferation (
Zhang et al., 1998). Nmi, originally identified because it interacts with N‐
myc, was later shown to be inducible by IFN‐α and also to enhance Stat1‐dependent transactivation (
Bao and Zervos, 1996;
Lebrun et al., 1998;
Zhu et al., 1999). Whether PKR is involved in regulating Nmi or MCM‐5 is not known at present.
The kinases directly responsible for phosphorylating Stat1 on Ser727 are not known. Stat1 serine phosphorylation is enhanced by treatment with IFN‐γ or LPS, or by serum stimulation (
Wen et al., 1995;
Kovarik et al., 1998;
Takaoka et al., 1999). The Ser727 phosphorylation site lies within an MAPK consensus sequence and the MAPK Erk2 has been proposed to phosphorylate this residue (
David et al., 1995). The IFN‐γ‐induced activation of Erk2, serine phosphorylation of Stat1 and Stat1‐dependent transactivation are strongly inhibited by overexpression of a dominant‐negative form of the protein tyrosine kinase Pyk2 in a Jak2‐dependent manner (
Takaoka et al., 1999). Evidence for a Stat1 serine kinase that depends on Jak2 and is distinct from MAPK has also been presented (
Zhu et al., 1997). These results indicate that the phosphorylation of Stat1 on serine is likely to be regulated by kinase cascades rather than by a single kinase.
Genetic analyses in yeast and
Drosophila have shown that p50
cdc37 functions both in the cell cycle and in the ras/raf/MAPK pathway in close cooperation with its partner HSP90 (
Reed, 1980;
Cutforth and Rubin, 1994). Recent studies have shown that p50
cdc37 is the primary determinant of HSP90 recruitment to Raf‐1 and of the activation of Raf‐1 by serum and growth factors in mammalian cells (
Grammatikakis et al., 1999). Co‐expression of p50
cdc37 strongly potentiated the v‐
src‐mediated activation of Raf‐1 and, conversely, dominant‐negative p50
cdc37, unable to recruit HSP90 into the Raf‐1 complex, abrogated the activation of Raf‐1 (
Grammatikakis et al., 1999). Inhibition of Raf‐1 activation by pre‐treatment with geldanamycin or expression of dominant‐negative p50
cdc37 abrogated the induction of c‐
myc by IFN‐γ in Stat1‐null cells, suggesting that Raf‐1 activation is critical for this pathway. Jak1 and Jak2 are also implicated in the regulation of c‐
myc expression in response to IFN‐γ. Conditional dimerization of Jak1 or Jak2, leading to their activation, can stimulate the c‐
myc promoter (
Mizuguchi and Hatakeyama, 1998;
Mohi et al., 1998). It is likely that, in the absence of Stat1, IFN‐γ‐activated Jak1 and Jak2 phosphorylate as yet unknown signaling molecules that can activate c‐
myc expression through Raf‐1. Both IFN‐γ and IFN‐β mediate the activation of c‐
myc in the absence of Stat1 and it remains to be determined whether the signals emanating from the two different receptors are the same or different.
A global expression study has shown that growth factor‐dependent stimulation of a mutant PDGF receptor with a restored ras‐GAP binding site promotes the induction of IFN‐γ‐responsive genes rather than of immediate‐early genes (
Fambrough et al., 1999), revealing a link between PDGF‐ and IFN‐γ‐dependent pathways. We have identified several additional genes repressed in wild‐type cells or induced in U3A cells in response to IFN‐γ (
Der et al., 1998; C.V.Ramana and G.R.Stark, unpublished data). Characterization of the promoters of these genes and further analysis of the c‐
myc and c‐
jun promoters should also help to identify the components of this novel pathway.