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Stat1 is a fascinating and complex protein with multiple, yet contrasting transcriptional functions. Upon activation, it drives the expression of many genes but also suppresses the transcription of others. These opposing characteristics also apply to its role in facilitating crosstalk between signal transduction pathways, as it participates in both synergistic activation and inhibition of gene expression. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes. This review summarizes the well studied involvement of Stat1 in IFN-dependent and growth factor-dependent signaling and then describes the roles of Stat1 in positive, negative and constitutive regulation of gene expression as well as its participation in crosstalk between signal transduction pathways. Oncogene (2000).

Interferon Regulatory Factor (IRF)-1 is a multifunctional transcription factor, involved in cell growth regulation, pathogen response and immune activation. To identify novel gene targets that may contribute to IRF-1 mediated activities, RNA fingerprinting was performed using NIH3T3 cells that inducibly express a hybrid form of IRF-1 under tetracycline regulated control. Secretory leukocyte protease inhibitor (SLPI) - a regulator of inflammation and an inhibitor of the LPS response - was identified as a gene repressed after doxycycline induced IRF-1 expression. Preliminary analysis of the human SLPI promoter identified an ISRE-like site located within the -221 to -200 region to which IRF-1 binds and a second, putative IRF-1 binding site upstream of the TATA box. Furthermore, co-transfection studies demonstrated that SLPI expression was inhibited by IRF-1 co-expression. The identification of SLPI as a target of IRF-1 regulation reveals a unique involvement of IRF-1 in repression of gene transcription and assigns a novel role for IRF-1 in inflammation.

Interferon Regulatory Factor (IRF)-1 has been characterized as an important growth regulatory and immunomodulatory transcription factor. To further characterize the potential targets of IRF-1 antiproliferative activity, IRF-1 was expressed under the control of the tetracycline-inducible system in murine NIH3T3 cells. Due to their ability to mimic IRF-1 transactivator function, the regulatory potential of IRF-1/RelA and IRF-2/RelA fusion proteins consisting of the DNA binding domains of IRF-1 or IRF-2 fused to the transactivation domain of NF-kappaB RelA(p65) was also examined. Cells inducibly expressing IRF-1 or IRF/RelA in response to doxycycline treatment displayed significantly reduced growth rates compared to control cells, and inhibition of cell growth correlated directly with the level of transgene expression. Interestingly, IRF-1 and IRF/RelA expression also induced a low level of apoptosis, as detected by microscopic analyses. Furthermore, expression of the interferon inducible, double stranded RNA dependent kinase PKR was increased following IRF-1 or IRF/RelA induction. Most strikingly, induction of IRF-1 and IRF/RelA expression resulted in a significant increase in STAT1 (p91) protein and increased ISGF3 DNA binding activity, suggesting that IRF-1 tumor suppressor activity may involve a novel mechanism which activates the JAK-STAT pathway through STAT1. WAF1 levels were also constitutively elevated in IRF-1 and IRF-1/RelA cells. These studies demonstrate that inducible expression of the transactivation function of IRF-1 or IRF/RelA mediates tumor suppressor activity by inducing cell growth arrest, apoptosis and the differential activation of growth regulatory genes such as PKR, STAT1 and WAF1.

The Interferon Regulatory Factors-1 and -2 (IRF-1 and IRF-2) play a transcriptional role in the regulation of the IFN-beta gene as well as other immunoregulatory genes. IRF-1 serves as a transcriptional activator whereas IRF-2 acts as an antagonistic transcriptional repressor. IRF-1 and IRF-2 also play opposing functional roles in cell growth regulation, and are implicated as a potential antioncogene and oncogene, respectively. To analyse the relationship between DNA binding/transcriptional repression and oncogenic transformation, NIH3T3 cells expressing C-terminal deletions of IRF-2 were established and assayed for transformation by saturation density analysis, anchorage independent growth in soft agar and tumor formation in nude mice. Cells expressing an IRF-2 protein of at least 160 N-terminal amino acids were transformed in vitro and tumorigenic in vivo, thus mapping IRF-2 oncogenic activity to its DNA binding/transcriptional repression domain. Overexpression of wild-type and truncated IRF-2 proteins resulted in reduced IFN-beta mRNA levels following induction by dsRNA. However, there was no effect of IRF-2 on IFN-beta inducibility by Sendai virus infection, suggesting the involvement of multiple IFN-beta induction pathways. In DNA binding assays, recombinant IRF-2 was found to preferentially bind to the IFN-beta PRDI site compared to IRF-1. These studies indicate that the transformed phenotype resulting from overexpression of IRF-2 may be due to constitutive engagement of the IRF-E recognition site, thus preventing DNA binding and transactivation of putative tumor suppressor genes by the IRF-1 anti-oncogene.

In this study we examine the relationship between p21CIP1/Waf1 (CIP1), a 21 kDa protein that binds to and modulates the activity of several cyclin dependent kinases and expression of wild-type (WT) p53 in human breast epithelial cells. Basal CIP1 protein, but not CIP1 mRNA levels correlated well with expression of WT p53 in human breast epithelial cells. To obtain more direct evidence that WT p53 regulated the level of CIP1 protein, the Human Papilloma Virus (HPV) E6 protein was introduced into immortalized 184B5 breast cells. Residual WT p53 levels correlated well with CIP1 protein but not CIP1 mRNA levels in isolated clones of transfected cells. CIP1 protein was increased at early times after growth factor arrested cells were stimulated to proliferate. The rise in CIP1 protein was due to a concomitant increase in CIP1 mRNA levels in MCF10, but not in normal mammary epithelial cells. DNA damage induced by ionizing radiation resulted in a transient increase in WT p53 levels but a prolonged induction of CIP1 protein. The sustained increase in CIP1 protein 24 h after radiation could not be attributed to a concomitant increase in CIP1 mRNA levels. Although the half-life of the CIP1 protein was not altered following irradiation, a fourfold increase in the amount of radioactivity incorporated into CIP1 protein was detected. When considered together these data suggest that wild-type p53 affects CIP1 protein accumulation at a posttranscriptional level in human breast epithelial cells under different physiologic and stress conditions.