IFN-beta1b induces transient and variable gene expression in relapsing-remitting multiple sclerosis patients independent of neutralizing antibodies or changes in IFN receptor RNA expression.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Interferon-beta (IFN-beta) therapy for MS is hypothesized to cause short-term and long-term changes in gene expression that shift the inflammation from Th1 to Th2. In vivo gene induction to define kinetics of response to IFN-beta therapy in a large cohort of MS patients is described. Differential gene expression in peripheral blood mononuclear cells (PBMCs) obtained from relapsing-remitting MS patients (RRMS) was assessed using high content microarrays. Rapid onset of gene expression appeared within 4 h of subcutaneous IFN-beta administration, returning to baseline levels at 42 h in clinically stable RRMS. IFN-beta therapy in vivo rapidly but transiently induced strong upregulation of genes mediating immune modulation, IFN signaling, and antiviral responses. RT-PCR showed significant patient-to-patient variation in the magnitude of expression of multiple genes, especially for IFN-beta-inducible genes, such as MxA, IRF7, and CCL8, a Th1 product. Variation among patients in IFN-beta-induced RNA transcription was not explained by neutralizing antibodies or IFN receptor expression. Surprisingly, genes regulated in vivo by IFN-beta therapy do not support a simple Th1 to Th2 shift. A complex interplay between both proinflammatory and anti-inflammatory immune regulatory genes is likely to act in concert in the treatment of RRMS.

IFN-beta-regulated genes show abnormal expression in therapy-naïve relapsing-remitting MS mononuclear cells: gene expression analysis employing all reported protein-protein interactions.

The molecular mechanism by which interferon beta (IFN-beta) is effective in treating multiple sclerosis is not well understood. Mononuclear cells from therapy-naïve MS patients, IFN-beta-1b-treated MS patients, and healthy controls were analyzed to examine mRNA changes that characterize both the disease and its treatment. The scientific literature was comprehensively searched for all protein-protein interactions. In MS patients who had never been treated with IFN-beta, statistical analysis revealed coordinate changes in mRNA expression for proteins reported in the literature as "regulated by IFN-beta." As a positive control for this approach, samples from a separate MS patient cohort showed significant change of these same genes during in vivo treatment with IFN-beta-1b.The strength of effect observed for regulation by IFN-beta was greater than for IFN-alpha, IFN-gamma (Th1), or IL-4 (Th2). Of the sets we investigated, the most strongly affected by disease was the subset defined by regulation by both IFN-beta and IFN-alpha. Changes in cells from therapy-naïve MS patients thus anticipated the importance of IFN-beta in therapy. These findings are a significant step towards marrying MS disease etiology and IFN-beta mechanism of action at a molecular level.

Interferon receptor expression regulates the antiproliferative effects of interferons on cancer cells and solid tumors.

In addition to antiviral effects, Type I interferons (IFN) have potent antiproliferative and immunomodulatory activities. Because of these properties IFNs have been evaluated as therapeutics for the treatment of a number of human diseases, including cancer. Currently, IFNs have been shown to be efficacious for the treatment of only a select number of cancers. The reason for this is unclear. Recent evidence has demonstrated that some cancer cell types seem to be defective in their ability to respond to IFN. It has been suggested that defects in IFN signaling is one mechanism by which cancer cells escape responsiveness to Type I IFNs and growth control in general. We report that transfection and enhanced expression of the Type I IFN receptor chain (IFNAR2c) in 3 different human cancer cell lines markedly increases the sensitivity of these cells to the antiproliferative effects of IFNs. In cancer cells transfected with IFNAR2c, dose response curves demonstrate a significant decrease in the concentrations of IFN required to achieve maximum cell death. Furthermore, in these transfected cells, we observe a significant increase in the number of cells undergoing apoptosis, as measured by DNA fragmentation and Caspase 3 activation. In addition, using an in vivo xenograft tumor model we show an increase in the effectiveness of systemically delivered Betaseron in decreasing tumor burden in animals in which solid tumors were generated from IFNAR2c transfected cells. These data show that specific regulation of IFN receptor expression can play a major role in determining the clinical outcome of IFN-based cancer therapeutics by regulating the relative sensitivity of cancer cells to IFN-dependent growth control.

STAT3 activation by type I interferons is dependent on specific tyrosines located in the cytoplasmic domain of interferon receptor chain 2c. Activation of multiple STATS proceeds through the redundant usage of two tyrosine residues.

Human type I interferons (IFNs) play an important role in the regulation of antiviral defense mechanisms, immunomodulatory activities, and growth control. Recent efforts have demonstrated the importance of IFNs in the activation of signal transducers and activators of transcription (STATs). The role of STAT1 and STAT2 in IFN-dependent JAK-STAT signaling is well established; however, the role of STAT3 and its activation by IFNs remains unclear. Understanding the IFN-dependent regulation of STAT3 is of increasing interest because recent studies have demonstrated that STAT3 may play a role in cancer. Studies have revealed that STAT3 is constitutively active in a number of cancer cell lines and that overexpression of an active form of STAT3 transforms normal fibroblasts. Therefore, STAT3 exhibits properties indicative of known oncogenes. In this report, we define the role of the type I IFN receptor in STAT3 activation and identify for the first time tyrosine residues present in the cytoplasmic domain of IFNAR2c that are critical for STAT3 activation. The regulation of STAT3 activation by IFNs was measured in a human lung fibrosarcoma cell line lacking IFNAR2c but stably expressing various IFNAR2c tyrosine mutants. We show here that in addition to IFN-dependent tyrosine phosphorylation of STAT3, activation using a STAT3-dependent electrophoretic mobility shift assay and a STAT3-specific reporter can also be demonstrated. Furthermore, we demonstrate that type I IFN-dependent activation of STAT3 proceeds through a novel mechanism that is dependent on two tyrosines, Tyr(337) and Tyr(512), present in IFNAR2c and contained within a conserved six-amino acid residue motif, GxGYxM. Surprisingly, both tyrosines were previously shown to be required for type I IFN-dependent STAT1 and STAT2 activation. Our results reveal that type I IFNs activate multiple STATs via the overlapping usage of two tyrosine residues located in the cytoplasmic domain of IFNAR2c.

Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.