Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation
Introduction
Interferon-β (IFNβ) and interferon γ (IFNγ) belong to a group of cytokines that were first discovered as antiviral agents, but are now known to have other properties including immunomodulation, inhibition of proliferation, regulation of differentiation, and anti-tumor activity (Kalvakolanu, 2000, Pestka et al., 1987, Sen, 2001). In brain injury associated with most neurological disorders, interferons are produced at or near injured sites during inflammation. Stem cell therapy has been proposed as a tool to repair and regenerate the CNS but the success of such therapy is hampered by the limited ability of stem cells to survive, proliferate, migrate or differentiate into neurons in the injured adult CNS.
IFNγ (Type II interferon) is one of the inflammatory cytokines produced following brain injury and it is known to exacerbate the symptoms of Multiple Sclerosis. However, it has been shown to exhibit neurotrophic effects (Chang et al., 1990, Turnley et al., 2001) and promote embryonic neural differentiation, neuronal survival, and neurite outgrowth (Barish et al., 1991). In contrast to IFNγ, IFNβ (Type I interferon) is known as an anti-inflammatory cytokine and immunoregulator and is produced by immune and non-immune cells following microbial infection or inflammatory activation. IFNβ has pleiotropic activities in the CNS, some of which may account for its therapeutic effects in Multiple Sclerosis. These include the inhibition of expression of class II major histocompatibility complex induced by IFNγ, modulation of matrix metalloproteinase activities and interleukin-1 and interleukin-1 receptor antagonist expression (Liu et al., 1998, Ransohoff et al., 1991, Yong et al., 1998). It is also reported to reduce blood-brain barrier permeability (Stone et al., 1995) and T cell infiltration to the CNS (Stuve et al., 1996) and to antagonise IFNγ to downregulate expression of cytokines and iNOS in astrocytes (Hua et al., 1998). IFNβ has potent anti-proliferative effects on tumor cells and modulates the activity of immune cells such as microglia, T cells, macrophages and dendritic cells, but its effects on neural stem cells remain to be elucidated.
Both types of IFNs are known to exert their biological effects via the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling pathway and the mechanisms of their signal transduction, including in the nervous system, have been extensively reviewed elsewhere (Campbell, 2005, Platanias, 2005). Various STAT members and signaling pathways have been shown to play an important role in the anti-proliferative effect of interferons. Cells treated with IFNβ or IFNγ typically exhibit inhibition of cell cycle progression, either in the form of S phase accumulation in carcinoma cells (Garrison et al., 1996, Kaynor et al., 2002, Murata et al., 2006, Qin et al., 1997, van Koetsveld et al., 2006, Vannucchi et al., 2000), G0/G1 arrest (Verma et al., 2002) or G2 arrest (Vivo et al., 2001). Such effects have been demonstrated mainly in carcinoma cells and the molecular mechanism is due to one or more of the following: a) induction of cyclin-dependent kinase inhibitor (cdk) p21 or p27 (Giandomenico et al., 1998, Kuniyasu et al., 1997), b) activation of p38 MAPK signalling (Verma et al., 2002), c) downregulation of cyclin E and cyclin A expression and activity mediated by STAT1 (Kortylewski et al., 2004). A link between STAT signalling and cell cycle control has been established, with reports that the production of p21 is increased by IFNγ-induced STAT1 (Chen et al., 2000, Chin et al., 1996, Kominsky et al., 1998) to induce growth arrest. STAT3 also appears to be another important player in cell cycle control, since it upregulates cyclins D and A, and downregulates p21 and p27 (Fukada et al., 1998) to promote cell cycle progression. However, in myeloid derived cells stimulated with IL-6, the activated STAT3 induced G1 arrest (Minami et al., 1996, Nakajima et al., 1996). In glioblastoma cells, activation of STAT3 blocked cell cycle progression and abnormal cell proliferation by activating p21 expression (Barre et al., 2003). Therefore, the effects of IFNs on cell cycle progression appear to be cell-type specific.
Given that IFNγ has inflammatory properties and we found previously that it inhibits the proliferation of neurospheres, promotes neuronal differentiation and neurite outgrowth (Wong et al., 2004), here we investigated whether anti-inflammatory IFNβ plays a similar role in these cells and compared the downstream signaling events activated by IFNβ and IFNγ to block proliferation. IFNβ inhibited neurosphere proliferation but had no effect on neuronal differentiation and neurite outgrowth. The anti-proliferative effect of both IFNs was associated with activation of STAT proteins and cell cycle arrest with modest upregulation of p27.
Section snippets
Cell culture and reagents
Neurospheres were isolated from the subventricular zone of adult C57BL/6 mice and cultured as previously described (Turnley et al., 2002) in neurosphere proliferation media consisting of Dulbecco's modified Eagle's medium:F12 (Gibco) containing 0.6% glucose, 13.4 mM NaHCO3, 5 mM HEPES, 100 U/ml penicillin/streptomycin (Gibco BRL) supplemented with 20 ng/ml epidermal growth factor (EGF; BD Bioscience), 10 ng/ml fibroblast growth factor 2 (FGF2; Roche), 0.1 mg/ml apo-transferrin, 25 µg/ml
IFNβ inhibits proliferation of neurospheres similarly to IFNγ and both IFNs induce G1 cell cycle accumulation
To determine whether IFNβ had an effect on neurosphere proliferation as we showed previously for IFNγ (Wong et al., 2004), we treated cells with various doses of IFNβ for up to 7 days and scored the number of viable cells daily from day 4 onwards. A significant reduction in live cell numbers induced by all doses of IFNβ (10 U/ml-5000 U/ml) was observed from day 6 onwards compared to the untreated control cells (1 way ANOVA, p < 0.001, F(5,12) = 126.3)(Fig. 1A). The decrease in cell numbers due to
Discussion
We have shown here that both IFNβ and IFNγ are anti-proliferative for neural progenitor cells. Whilst the decreased proliferation by IFNγ was linked to increased neuronal differentiation and neurite outgrowth, this was not the case for IFNβ.
IFNβ promoted cell cycle arrest of proliferating progenitors, as well as some longer term toxicity. Its anti-proliferative effect is consistent with that reported in carcinoma cells (Johns et al., 1992, Murata et al., 2006, Vannucchi et al., 2000, Vitale et
Acknowledgements
This work was supported by Bayer Healthcare Pharmaceuticals, (project # NSV-015) and the National Health and Medical Research Council of Australia (project 454384). AMT is a NH&MRC Senior Research Fellow.
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