Abstract
Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is a key mediator of inflammation and stress in the central nervous system (CNS), and is highly expressed in the developing brain. In this study, we investigated the possible role of IL-1β in neuronal differentiation of cortical neural precursor cells (NPCs). We showed that stimulation with IL-1β increased expression levels of neurotrophin-3 (NT3) and neurogenin 1 (Ngn1) and promoted neurite outgrowth. We also found that IL-1β increased mRNA and protein levels of Wnt5a. Knockdown of Wnt5a by transfection with Wnt5a siRNA inhibited IL-1β-induced neuronal differentiation. Moreover, IL-1β-induced Wnt5a expression was regulated by nuclear factor kappa B (NF-κB) activation, which is involved in IL-1β-mediated neuronal differentiation. To examine the role of Wnt5a in neuronal differentiation of NPCs, we exogenously added Wnt5a. We found that exogenous Wnt5a promotes neuronal differentiation, and activates the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway. In addition, Wnt5a-induced neuronal differentiation was blocked by RhoA siRNA, as well as by a specific Rho-kinase inhibitor (Y27632) or a SAPK/JNK inhibitor (SP600125). Furthermore, treatment with RhoA siRNA, Y27632, or SP600125 suppressed the IL-1β-induced neuronal differentiation. Therefore, these results suggest that the sequential Wnt5a/RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation of NPCs.
Highlights
Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine, which is produced as an immune response to both injury and infection [1]
Data are mean ± SD; Student’s t test. * p < 0.05 compared with control siRNA/IL-1β. b and c Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and incubated for 6 h (B) or 2 days (c) with IL-1β (10 ng/ml). b mRNA levels of Nt3 and neurogenin 1 (Ngn1) were analyzed by real-time RT-PCR. n = 3
Western blotting was performed using anti-NT3, anti-Ngn1, anti-RhoA, or anti-calnexin antibodies to detect the respective protein bands. d and e Cells were transiently transfected with control siRNA or RhoA siRNA for 48 h, and incubated for 3 days with IL-1β (10 ng/ml)
Summary
Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine, which is produced as an immune response to both injury and infection [1]. Increased production of IL-1β is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [2]. IL-1β induces an intracellular signaling pathway, leading to altered expression of its target genes and the induction of a pro-inflammatory response [3]. Wnt signaling plays an important role in embryogenesis and in the late stages of development, by regulating cell survival, growth, and, differentiation via various signaling pathways [9, 10]. Wnt5a is involved in IL-1β-mediated cell migration and differentiation. Wnt5a signaling is involved in IL-1β-induced matrix metalloproteinase (MMP)-3-regulated proliferation of ES cell-derived odontoblast-like cells [20]. Wnt5a has been associated with the regulation of proliferation and differentiation in different cell types, little is known about the role of Wnt5a signaling in neuronal differentiation of NPCs
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