IGF-1 induced HIF-1α-TLR9 cross talk regulates inflammatory responses in glioma Sanchari Sinha, Nitin Koul, Deobrat Dixit, Vivek Sharma, Ellora Sen ⁎ National Brain Research Centre, Manesar, Haryana 122 050, India abstract article info Article history: Received 10 June 2011 Accepted 27 June 2011 Available online 3 July 2011 Keywords: IGF-1 HIF-1α TLR9 Glioblastoma The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes in glioblastoma multiforme (GBM). As HIF-1α links inflammatory and oncogenic pathways in GBM, we investigated whether IGF-1 affects HIF-1α to regulate inflammatory response in glioma cells under normoxia. IGF-1 induced Ras and Calmodulin-dependent kinase II (CaMKII) regulated HIF-1α transcriptional activity in glioma cells. Increase in HIF-1α was concurrent with decreased Toll-like receptor (TLR9) and CXCR4 expression and elevated suppressor of cytokine signaling (SOCS3) levels. Interestingly, while synthetic CpG containing oligodeoxynucleotide TLR9 agonist (CpG DNA) decreased IGF-1 mediated increase in HIF-1α activity, siRNA mediated knockdown of HIF-1α decreased TLR9 levels. This suggested that IGF-1 induced HIF- 1α-TLR9 axis is regulated by both positive and negative feedback loops. Importantly, TLR9 agonist reversed the effect of IGF-1 on CXCR4 and SOCS3 expression. While knockdown of HIF-1α abrogated IGF-1 mediated increase in SOCS3 it elevated IGF-1 induced decrease in CXCR4 levels. Thus HIF-1α positively and negatively regulates SOCS3 and CXCR4 expression respectively, in glioma cells. Though TLR9 agonist had no additive effect on IGF-1 mediated increase in pro-inflammatory cytokines IL-1β, IL-6 and IL-8, treatment with TLR9 agonist alone elevated expression of these pro-inflammatory cytokines. Our studies indicate that a complex HIF-1α-TLR9 cross-talk sustains a self-regulating cycle of inflammatory response through intrinsic negative and positive feedback mechanisms. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Signaling pathways emanating from the insulin-like growth factor-I receptor (IGF-IR) — a transmembrane receptor tyrosine kinase activated by IGF, is associated with the growth and prolifer- ation of several tumor types including glioblastoma [1–3]. Although HIF-1α is a key regulator of cellular response to hypoxia [4] it can also be activated under normoxia in response to IGF [5,6]. HIF-1α influences tumor growth [7] by regulating expression of genes implicated in angiogenesis, metabolism and cell survival [8]. IGF-1 is known to induce HIF-1α accumulation in Kaposi Sarcoma [9] and neuroblastoma [10]. HIF-1α-regulated genes are induced by IGF-1 in glioblastoma [11]. Suppression of IGF-1R inhibits tumorigenic potential of rat glioblas- toma cells [12]. We have demonstrated IL-1β induced Ras/Akt/Erk mediated HIF-1α activation in glioma cells [13]. Activated Ras elevates IGF-I mediated induction of the HIF-1α target VEGF [14]. Oncogenic Ras activation occurs in GBMs [15] and inhibition of Ras down- regulates HIF-1α activity in GBM [16]. CaMKII which translates intracellular changes in calcium is associated with glioma migration [17], and regulates Ras mediated Erk activation [18]. CaMKII regulates HIF-1α transcriptional activity under intermittent hypoxia [19] as well as under inflammatory conditions [20]. By demonstrating that Ras rewires HIF1α-driven IL-1β autocrine loop [13,21], we have emphasized the pivotal role of HIF-1α in linking inflammation and tumorigenesis [22]. HIF-1α is involved in host immune response during bacterial infection [23], and LPS is a potent inducer of HIF-1α [24]. Also, HIF-1α accumulation and target gene expression are impaired upon induction of endotoxin tolerance [25]. Activation of toll like receptors (TLRs) which recognize pathogen- associated molecular patterns triggers signaling events that initiate innate immunity and inflammatory response [26]. Interestingly, HIF-1α regulates hypoxia induced TLR4 expression in macrophages [27]. While TLR9 increases metastatic potential of cancer cells through CXCR4 expression [28], the later modulates TLR9 mediated signaling [29]. TLR activation is modulated by negative regulators such as suppressor of cytokine signaling (SOCS) that feed back upon and inhibit TLR activation [30]. Moreover, SOCS3 which is constitutively expressed in GBM is not only involved in inducing radioresistance [31], but it also regulates CXCR4 function [32]. Given that TLR9 is expressed in GBM [33], we investigated whether IGF-1 affect HIF-1α to modulate TLR9 mediated signaling in glioma. Our study to understand the involvement of IGF-1 in HIF-1α regulated inflammatory responses in GBM indicates that IGF-1 induced Ras/CaMKII enhances HIF-1α activation, under normoxia. Increase in Cellular Signalling 23 (2011) 1869–1875 ⁎ Corresponding author. Tel.: + 91 124 2338921x235; fax: + 91 124 2338910/28. E-mail addresses: sancharisinha79@gmail.com (S. Sinha), nitinkoul11@gmail.com (N. Koul), dev25@nbrc.ac.in (D. Dixit), viveksharmabt@gmail.com (V. Sharma), ellora@nbrc.ac.in (E. Sen). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.06.024 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig