Ras regulates interleukin-1β-induced HIF-1α transcriptional activity in glioblastoma

Abstract

We observed elevated levels of pro-inflammatory cytokine IL-1β in glioblastoma multiforme tumor samples. Since hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in linking inflammatory and oncogenic pathways, we investigated the effect of IL-1β on HIF-1α expression in glioma cells under normoxia. IL-1β-mediated elevation of HIF-1α transcriptional activity was dependent on Ras-induced NF-κB activation, as IL-1β failed to induce NF-κB and HIF-1α activity in cells transfected with dominant negative RasN17. Increased Ras expression was accompanied by increased phosphorylation of Ras effectors AKT, ERK, JNK, and p38MAPK. While inhibition of these effectors individually failed to block the IL-1β-mediated increase in HIF-1α induction, co-inhibition of both AKT and ERK resulted in a significant decrease in IL-1β-induced HIF-1α activation. Interestingly, IL-1β elevated Wnt-1 expression in a Ras-dependent manner, and small interfering RNA (siRNA)-mediated knockdown of Wnt-1 decreased HIF-1α activity. Although Wnt-1-mediated HIF-1α was independent of the canonical Wnt/β-catenin signaling pathway, it regulated HIF-1α through NF-κB. siRNA-mediated HIF-1α knockdown attenuated elevated IL-1β mRNA levels induced upon IL-1β treatment. This was accompanied by increased interaction of HIF-1α with HIF responsive element on the IL-1β promoter upon IL-1β treatment, under normoxia. Our studies highlights for first time that (1) Ras is a key mediator of IL-1β-induced NF-κB and HIF-1α activation, under normoxia; (2) Wnt-1 regulates IL-1β-mediated HIF-1α induction via NF-κB; (3) Ras and Wnt-1 are intermediaries in the canonical IL-1β-NF-κB signaling pathway downstream of MyD88; and (4) IL-1β-induced HIF-1α drives a HIF-1α-IL-1β autocrine loop to maintain persistently elevated IL-1β level.

Tumor Necrosis Factor Alpha-Induced Hypoxia-Inducible Factor 1α–β-Catenin Axis Regulates Major Histocompatibility Complex Class I Gene Activation through Chromatin Remodeling

“…Glioma cells were seeded into the wells of 24-well plates or onto 90-mm tissue culture dishes in DMEM without antibiotics, and transfection with 50 nmol/liter duplex HIF-1␣, ␤-catenin, or a nonspecific siRNA (Thermo Fischer Scientific) was carried out with Lipofectamine RNAiMAX reagent (Life Technologies-Invitrogen) as described previously (18). Cells were stimulated at 18 h posttransfection with 50 ng/ml TNF-␣ (R&D Systems).…”

“…Western blot analysis was performed with protein from whole lysates and nuclear extracts of cells transfected with different constructs or siRNA and treated with different inhibitors in the presence or absence of TNF-␣ as described previously (18), with antibodies against MHC-I, ␤ 2 -microglobulin (␤2m), ␤-catenin, HIF-1␣, CREB, pCREB, CBP, Brg1, Brm, RFX5, and NF-Y subunit beta (NF-YB). Antibodies were purchased from Cell Signaling Inc. unless otherwise mentioned.…”

“…After the addition of a chemiluminescent reagent (Millipore), blots were exposed to the Chemigenius bioimaging system (Syngene) for development and images were captured with GeneSnap software (Syngene). The blots were stripped and reprobed with anti-␤-actin (Sigma) or anti-C-23 (Santa Cruz) antibody to determine equivalent loading as described previously (18). For endogenous IP, nuclear extracts from glioma cells were incubated with 4 g of the indicated antibodies for 16 h. Lysate was then incubated for 4 h with a mixture of protein G-Sepharose (Amersham Pharmacia).…”

Hypoxia-inducible factor–1 and associated upstream and downstream proteins in the pathophysiology and management of glioblastoma

“…14 Hypoxia in these pseudopalisading cells surrounding micronecrotic areas encourages infiltration by inflammatory cells, which can further stimulate the normoxic activation of HIF-1. 100,192,206 Localized inflammation is an important contributing factor in the development of GBM, as demonstrated by the presence of infiltrating immune cells and the expression of inflammatory cytokines. 225 Because HIF-1 is connected with prominent proinflammatory mediators such as interleukin (IL)-1b and nuclear factor (NF)-kB, it is a central player in linking inflammation and tumorigenesis in GBM.…”

“…225 Because HIF-1 is connected with prominent proinflammatory mediators such as interleukin (IL)-1b and nuclear factor (NF)-kB, it is a central player in linking inflammation and tumorigenesis in GBM. 206 The IL-1β gene is a target for HIF-1 activation and is a proinflammatory cytokine with pluripotent activity, including the promotion of angiogenesis, tumor growth, and metastasis. 249 This link suggests a mechanism for inflammation stimulating HIF-1 activity in the normoxic areas of tumors, particularly in the mesenchymal subtypes of GBM, which show activated Ras and immune cell infiltrates.…”