STAT3 complements defects in an interferon-resistant cell line: Evidence for an essential role for STAT3 in interferon signaling and biological activities

Abstract

STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFN␣͞␤) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFN␣͞␤ signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFN␣͞␤. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-B DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-B DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFN␣͞␤.Interferons (IFNs) are cytokines that block the viral infection of cells, inhibit cell proliferation, and modulate cell differentiation. Type I IFNs (IFN ␣, ␤, and ) compete with each other for binding to a common cell surface receptor, whereas the receptor for type II IFN (IFN␥) is a distinct entity (1). The type I IFN receptor is composed of IFNAR1 and IFNAR2 chains (2-4), which undergo rapid, ligand-dependent tyrosine phosphorylation. Although IFNAR2 is the ligand-binding subunit, IFNAR1 acts as a species-specific transducer for the actions of type I IFN (5-7). IFNs transduce signals from the cell surface resulting in selective gene activation (8-10) through the activation of JAK tyrosine kinases and signal transducers and activators of transcription (STAT) factors (11,12). Upon their tyrosine phosphorylation, IFN-activated STATs (STAT1, STAT2, and STAT3) dimerize and translocate to the nucleus.The crucial role that STAT1 and STAT2 play in the transcriptional response to IFN␣͞␤ and in the induction of antiviral activity has been demonstrated in knockout mice and in mutant human cell lines deficient in these proteins. However, the importance of STAT3 in IFN␣͞␤ action has been unresolved. For example, knockout of the STAT3 gene in mice leads to early embryonic lethality and STAT3-deficient cell lines could not be isolated (13). We recently reported that STAT3 acts as a bridge (adapter) for the IFN-dependent interaction of the IFNAR1 receptor chain and the regulatory 85-kDa (p85) subunit of phosphatidylinositol-3Ј (PI-3) kinase (14). PI-3 kinase is important in the regulation of many cellular events involving protein tyrosine kinases and is an upstream element in a serine kinase transduction cascade (15, 1...

Gene Expression Associated With Interferon Alfa Antiviral Activity in An Hcv Replicon Cell Line

“…Among the STAT proteins, STAT1 appears to play a key role in IFN-␣ signaling, 30 although STAT3 has also been implicated in mediating IFN-␣ activity in other studies. [31][32][33][34] Our study demonstrated that STAT3, not STAT1, was significantly activated by IFN-␣ at the concentrations (50 U or 100 U/mL) that show antiviral activity. The phosphorylated STAT1 did not respond to up to 100 U/mL IFN-␣ treatment, although it did so at 500 U/mL, a relatively higher concentration for antiviral effect in this model.…”

Interferon-resistant Daudi Cell Line with a Stat2 Defect Is Resistant to Apoptosis Induced by Chemotherapeutic Agents

“…As an alternative approach, our laboratory has resorted to long term IFN treatment of cells to isolate naturally arising IFN-resistant mutants, which more closely resemble what occurs in vivo (10,11). Using this strategy, we previously identified a STAT3-defective IFN-resistant cell line (11).…”

“…IFN signals through the activation of the IFN-stimulated gene factor 3 (ISGF3) complex, composed of STAT1, STAT2, and IRF9 (17), as well as STAT1 and STAT3 dimers that bind to ISG promoter elements (11). As shown in Fig.…”

“…DNA Binding Activity Assays-Nuclear extracts (10 g) were incubated with 32 P-labeled probes for the highly conserved ISRE in the promoter region of ISG15 (5Ј-GATCCATGCCTC-GGGAAAGGGAAACCGAAACTGAAGCC-3Ј) or the high affinity c-sis inducible element (SIE) in the c-fos gene (5Ј-AGCT-TCATTTCCCGTAATCCCTAAAGCT-3Ј), respectively, and subjected to electrophoretic mobility shift assay (EMSA) (11). To define the presence of specific STAT proteins in DNA-protein complexes, nuclear extracts were preincubated with a 1:50 dilution of anti-STAT antibodies at 25°C for 20 min before EMSA.…”

“…Moreover, these IFN-resistant cells have undergone multiple mutational events, because complementation of a single defect rescues aspects of IFN signaling but not sensitivity to all of the biological actions of IFN (8, 9). As an alternative approach, our laboratory has resorted to long term IFN treatment of cells to isolate naturally arising IFN-resistant mutants, which more closely resemble what occurs in vivo (10,11). Using this strategy, we previously identified a STAT3-defective IFN-resistant cell line (11).…”