Functional characterization of interferon regulatory factor 3a (IRF-3a), an alternative splice isoform of IRF-3.

Virus infection of numerous cell types results in the transcriptional induction of a subset of virus- and interferon (IFN)-stimulated genes. The beta IFN (IFN-beta) gene is one of these rapidly induced genes; it serves as a fundamental component of the cellular defense response in eliciting potent antiviral, immunomodulatory, and antiproliferative effects. One of the transcription factors involved in the stringent regulation of IFN-beta production following virus infection is interferon regulatory factor (IRF) 3 (IRF-3). We have characterized an alternatively spliced isoform of IRF-3 that we have called IRF-3a. IRF-3a can selectively and potently inhibit virus-induced activation of the IFN-beta promoter. IRF-3a lacks half of the DNA binding domain found in IRF-3 and is unable to bind to the classical IRF binding elements, IFN-stimulated response elements. These studies suggest that IRF-3a may act as a modulator of IRF-3.

Dual utilization of an acceptor/donor splice site governs the alternative splicing of the IRF-3 gene.

Interferon regulatory factors constitute a family of transcriptional activators and repressors involved in a large number of vital cellular processes. Interferon regulatory factor-3 (IRF-3) has been implicated in virus and double-stranded RNA mediated induction of IFNbeta and RANTES, in DNA damage signaling, and in virus-induced apoptosis. With its critical role in these pathways, the activity of IRF-3 is tightly regulated in myriad ways. Here we describe novel regulation of IRF-3 at the level of RNA splicing. We show that an unprecedented dual utilization of a splice acceptor/donor site within the IRF-3 mRNA governs the production of two alternative splice isoforms.

Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways.

A hallmark of human papillomavirus (HPV) associated carcinogenesis is the integration of the viral DNA into the cellular genome, usually accompanied by the loss of expression of the viral E2 gene. E2 binds to and represses the viral promoter directing expression of the E6 and E7 oncogenes. The re-introduction and expression of exogenous E2 in HPV-positive cancer cells results in cellular growth arrest, while growth in the context of exogenous E2 can be restored through the expression of exogenous E6 and E7. Here we examine the individual contributions of the viral E6 and E7 genes to this phenotype. E6 alone displays moderate activity, whereas both E7 and adenovirus E1A display high activity in reversing E2-mediated cellular growth suppression. Using defined mutants of E7 and E1A, we show that an intact retinoblastoma interaction domain is required for this function. In addition, we show that the E2-mediated growth arrest of HPV-positive cells results in cellular senescence, and implicate the cyclin/cdk inhibitor p21(CIP) as a downstream E2 effector in this phenotype.

Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity.

Interferon regulatory factor-3 (IRF-3) was found to specifically interact with HPV16 E6 in a yeast two-hybrid screen. IRF-3 is activated by the presence of double-stranded RNA or by virus infection to form a stable complex with other transcriptional regulators that bind to the regulatory elements of the IFNbeta promoter. We show that IRF-3 is a potent transcriptional activator and demonstrate that HPV16 E6 can inhibit its transactivation function. The expression of HPV16 E6 in primary human keratinocytes inhibits the induction of IFNbeta mRNA following Sendai virus infection. The binding of HPV16 E6 to IRF-3 does not result in its ubiquitination or degradation. We propose that the interaction of E6 with IRF-3 and the inhibition of IRF-3's transcriptional activity may provide the virus a means to circumvent the normal antiviral response of an HPV16-infected cell.

Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1.

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.

MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes.

We tested whether activation of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase-1 (MEK1) is required and sufficient for extracellular signal-regulated kinase (ERK) activation in airway smooth muscle cells. First, we transiently cotransfected bovine tracheal myocytes with an epitope-tagged ERK2 and a dominant-negative or a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the dominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)-induced ERK2 activation, whereas expression of the constitutively active MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the effect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 microM) inhibited MEK1 and ERK activation, confirming that MEK1 is required for ERK activation in bovine tracheal myocytes. PD-98059 had no effect on Src or Raf-1 activity, evidence that PD-98059 is a specific inhibitor of MEK in this system. Finally, PD-98059 reduced PDGF-induced [(3)H]thymidine incorporation in a concentration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is required for PDGF-induced ERK activation in bovine tracheal myocytes and that MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.