Noncanonical IFN Signaling, Steroids, and STATs: A Probable Role of V-ATPase.

A small group of only seven transcription factors known as STATs (signal transducer and activator of transcription) are considered to be canonical determinants of specific gene activation for a plethora of ligand/receptor systems. The activation of STATs involves a family of four tyrosine kinases called JAK kinases. JAK1 and JAK2 activate STAT1 in the cytoplasm at the heterodimeric gamma interferon (IFNγ) receptor, while JAK1 and TYK2 activate STAT1 and STAT2 at the type I IFN heterodimeric receptor. The same STATs and JAKs are also involved in signaling by functionally different cytokines, growth factors, and hormones. Related to this, IFNγ-activated STAT1 binds to the IFNγ-activated sequence (GAS) element, but so do other STATs that are not involved in IFNγ signaling. Activated JAKs such as JAK2 and TYK2 are also involved in the epigenetics of nucleosome unwrapping for exposure of DNA to transcription. Furthermore, activated JAKs and STATs appear to function coordinately for specific gene activation. These complex events have not been addressed in canonical STAT signaling. Additionally, the function of noncoding enhancer RNAs, including their role in enhancer/promoter interaction is not addressed in the canonical STAT signaling model. In this perspective, we show that JAK/STAT signaling, involving membrane receptors, is essentially a variation of cytoplasmic nuclear receptor signaling. Focusing on IFN signaling, we showed that ligand, IFN receptor, the JAKs, and the STATs all undergo endocytosis and ATP-dependent nuclear translocation to promoters of genes specifically activated by IFNs. We argue here that the vacuolar ATPase (V-ATPase) proton pump probably plays a key role in endosomal membrane crossing by IFNs for receptor cytoplasmic binding. Signaling of nuclear receptors such as those of estrogen and dihydrotestosterone provides templates for making sense of the specificity of gene activation by closely related cytokines, which has implications for lymphocyte phenotypes.

The Significance of Interferon-γ in HIV-1 Pathogenesis, Therapy, and Prophylaxis.

Interferon-γ (IFNγ) plays various roles in the pathogenesis of HIV/AIDS. In an HIV-1 infected individual, the production of IFNγ is detected as early as the acute phase and continually detected throughout the course of infection. Initially produced to clear the primary infection, IFNγ together with other inflammatory cytokines are involved in establishing a chronic immune activation that exacerbates clinical diseases associated with AIDS. Unlike Type 1 IFNs, IFNγ has no direct antiviral activity against HIV-1 in primary cultures, as supported by the in vivo findings of IFNγ therapy in infected subjects. Results from both in vitro and ex vivo studies show that IFNγ can instead enhance HIV-1 replication and its associated diseases, and therapies aimed at decreasing its production are under consideration. On the other hand, IFNγ has been shown to enhance cytotoxic T lymphocytes and NK cell activities against HIV-1 infected cells. These activities are important in controlling HIV-1 replication in an individual and will most likely play a role in the prophylaxis of an effective vaccine against HIV-1. Additionally, IFNγ has been used in combination with HIV-1 vaccine to augment antiviral immunity. Technological advancements have focused on using IFNγ as a biological marker to analyze the type(s) of immunity generated by candidate HIV vaccines and the levels of immunity restored by anti-retroviral drug therapies or novel immunotherapies. Hence, in addition to its valuable ancillary role as a biological marker for the development of effective HIV-1 prophylactic and therapeutic strategies, IFNγ has a vital role in promoting the pathogenesis of HIV.

Feline immunodeficiency virus (FIV) vaccine efficacy and FIV neutralizing antibodies.

A HIV-1 tier system has been developed to categorize the various subtype viruses based on their sensitivity to vaccine-induced neutralizing antibodies (NAbs): tier 1 with greatest sensitivity, tier 2 being moderately sensitive, and tier 3 being the least sensitive to NAbs (Mascola et al., J Virol 2005; 79:10103-7). Here, we define an FIV tier system using two related FIV dual-subtype (A+D) vaccines: the commercially available inactivated infected-cell vaccine (Fel-O-Vax(®) FIV) and its prototype vaccine solely composed of inactivated whole viruses. Both vaccines afforded combined protection rates of 100% against subtype-A tier-1 FIVPet, 89% against subtype-B tier-3 FIVFC1, 61% against recombinant subtype-A/B tier-2 FIVBang, 62% against recombinant subtype-F'/C tier-3 FIVNZ1, and 40% against subtype-A tier-2 FIVUK8 in short-duration (37-41 weeks) studies. In long-duration (76-80 weeks) studies, the commercial vaccine afforded a combined protection rate of at least 46% against the tier-2 and tier-3 viruses. Notably, protection rates observed here are far better than recently reported HIV-1 vaccine trials (Sanou et al., The Open AIDS J 2012; 6:246-60). Prototype vaccine protection against two tier-3 and one tier-2 viruses was more effective than commercial vaccine. Such protection did not correlate with the presence of vaccine-induced NAbs to challenge viruses. This is the first large-scale (228 laboratory cats) study characterizing short- and long-duration efficacies of dual-subtype FIV vaccines against heterologous subtype and recombinant viruses, as well as FIV tiers based on in vitro NAb analysis and in vivo passive-transfer studies. These studies demonstrate that not all vaccine protection is mediated by vaccine-induced NAbs.

IFN signaling: how a non-canonical model led to the development of IFN mimetics.

The classical model of cytokine signaling dominates our view of specific gene activation by cytokines such as the interferons (IFNs). The importance of the model extends beyond cytokines and applies to hormones such as growth hormone (GH) and insulin, and growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). According to this model, ligand activates the cell via interaction with the extracellular domain of the receptor. This results in activation of receptor or receptor-associated tyrosine kinases, primarily of the Janus activated kinase (JAK) family, phosphorylation and dimerization of the signal transducer and activator of transcription (STAT) transcription factors, which dissociate from the receptor cytoplasmic domain and translocate to the nucleus. This view ascribes no further role to the ligand, JAK kinase, or receptor in either specific gene activation or the associated epigenetic events. The presence of dimeric STATs in the nucleus essentially explains it all. Our studies have resulted in the development of a non-canonical, more complex model of IFNγ signaling that is akin to that of steroid hormone (SH)/steroid receptor (SR) signaling. We have shown that ligand, receptor, activated JAKs, and STATs are associated with specific gene activation, where the receptor subunit IFNGR1 functions as a co-transcription factor and the JAKs are involved in associated epigenetic events. We found that the type I IFN system functions similarly. The fact that GH receptor, insulin receptor, EGF receptor, and FGF receptor undergo nuclear translocation upon ligand binding suggests that they may also function similarly. The SH/SR nature of type I and II IFN signaling provides insight into the specificity of signaling by members of cytokine families. The non-canonical model could also provide better understanding to more complex cytokine families such as those of IL-2 and IL-12, whose members often use the same JAKs and STATs, but also have different functions and properties.

Type I IFN receptor controls activated TYK2 in the nucleus: implications for EAE therapy.

Recent studies have suggested that activated wild-type and mutant Janus kinase JAK2 play a role in the epigenetics of histone modification, where it phosphorylates histone H3 on tyrosine 41(H3pY41). We showed that type I IFN signaling involves activated TYK2 in the nucleus. ChIP-PCR demonstrated the presence of receptor subunits IFNAR1 and IFNAR2 along with TYK2, STAT1, and H3pY41 specifically at the promoter of the OAS1 gene in IFN treated cells. A complex of IFNAR1, TYK2, and STAT1α was also shown in the nucleus by immunoprecipitation. IFN treatment was required for TYK2 activation in the nucleus. The presence of IFNAR1, IFNAR2, and activated STAT1 and STAT2, as well as the type I IFN in the nucleus of treated cells was confirmed by the combination of Western blotting and confocal microscopy. Trimethylated histone H3 lysine 9 underwent demethylation and subsequent acetylation specifically in the region of the OAS1 promoter. Resultant N-terminal truncated IFN mimetics functioned intracellularly as antivirals as well as therapeutics against experimental allergic encephalomyelitis without the undesirable side effects that limit the therapeutic efficacy of IFNß in treatment of multiple sclerosis. The findings indicate that IFN signaling is complex like that of steroid signaling.

Steroid-like signalling by interferons: making sense of specific gene activation by cytokines.

Many cytokines, hormones and growth factors use the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway for cell signalling and specific gene activation. In the classical model, ligand is said to interact solely with the receptor extracellular domain, which triggers JAK activation of STATs at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation. Given the limited number of STATs (seven) and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on IFNγ (interferon γ), we have shown that ligand, receptor and activated JAKs are involved in nuclear events that are associated with specific gene activation, where the receptor subunit IFNGR1 (IFNγ receptor 1) functions as a transcription/co-transcription factor and the JAKs are involved in key epigenetic events. RTKs (receptor tyrosine kinases) such as EGFR [EGF (epidermal growth factor) receptor] and FGFR [FGF (fibroblast growth factor) receptor] also undergo nuclear translocation in association with their respective ligands. EGFR and FGFR, like IFNGR1, have been shown to function as transcription/co-transcription factors. The RTKs also regulate other kinases that have epigenetic effects. Our IFNγ model, as well as the RTKs EGFR and FGFR, have similarities to that of steroid receptor signalling. These systems consist of ligand-receptor-co-activator complexes at the genes that they activate. The co-activators consist of transcription factors and kinases, of which the latter play an important role in the associated epigenetics. It is our view that signalling by cytokines such as IFNγ is but a variation of specific gene activation by steroid hormones.

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ.

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The ß-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation.

Controlling Nuclear Jaks and Stats for Specific Gene Activation by Ifn γ and Other Cytokines: A Possible Steroid-like Connection.

The mechanism of specific gene activation by cytokines that use JAK/STAT signalling pathway is unknown. There are four different types of JAKs and seven different types of STATs. In the classical model of signaling, ligand interacts solely with the receptor extracellular domain, which triggers JAK activation at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation, including associated epigenetic changes that cause heterochromatin destabilization. Ligand, receptor, and JAKs play no further role in the classical model. Given the limited number of STATs and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on gamma interferon (IFNγ), we have shown that ligand, receptor, and activated JAKs are involved in nuclear events that are associated with specific gene activation. In this model, receptor subunit IFNGR1 functions as a transcription/cotranscription factor and the JAKs are involved in key epigenetic events that are required for specific gene activation. The model has implications for gene activation in cancer as well as stem cell differentiation.

HSV-1-induced SOCS-1 expression in keratinocytes: use of a SOCS-1 antagonist to block a novel mechanism of viral immune evasion.

Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.

Interferon gamma is recognised by importin alpha/beta: enhanced nuclear localising and transactivation activities of an interferon gamma mimetic.

Interferon (IFN) gamma's ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNgamma and an IFNgamma mimetic peptide by IMPalpha and the IMPalpha/beta heterodimer, where the IFNgamma mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNgamma's anti-viral action, and the potential use of the IFNgamma mimetic in antiviral therapies.