The transcription of the alarmin cytokine interleukin-1 alpha is controlled by hypoxia inducible factors 1 and 2 alpha in hypoxic cells

Abstract

During hypoxia, cells undergo transcriptional changes to adjust to metabolic stress, to promote cell survival, and to induce pro-angiogenic factors. Hypoxia-induced factors (HIFs) regulate these transcriptional alterations. Failure to restore oxygen levels results in cell death by necrosis. IL-1α is one of the most important mediators of sterile inflammation following hypoxia-mediated necrosis. During hypoxia, IL-1α is up-regulated and released from necrotic cells, promoting the initiation of sterile inflammation. This study examined the role of IL-1α transcription in initiation of hypoxic stress and the correlation between IL-1α transcription and HIFα factors. In an epithelial cell line cultured under hypoxic conditions, IL-1α transcription was up-regulated in a process mediated and promoted by HIFα factors. IL-1α transcription was also up-regulated in hypoxia in a fibroblast cell line, however, in these cells, HIFα factors inhibited the elevation of transcription. These data suggest that HIFα factors play a significant role in initiating sterile inflammation by controlling IL-1α transcription during hypoxia in a differential manner, depending on the cell type.

Keywords: DAMPs; HIF-1α; HIF-2; IL-1; alarmin; cytokines and inflammation; sterile inflammation.

FIGURE 1

IL-1α transcription is up-regulated during hypoxia in A549 epithelial cells.(A) A549 cells were cultured in either normoxic or hypoxic conditions for 12 h. Cells lysates were analyzed by Western blot for IL-1α detection. Bands are marked as precursor or mature. (B) A549 cells were cultured in either normoxic or hypoxic conditions for 6 h, and were analyzed for relative quantification (RQ) of IL-1α transcription. Graph represents mean ± SD of three biological repeats.

FIGURE 2

Silencing HIFα proteins down-regulates IL-1α transcription in hypoxic A549 epithelial cells. A549 cells were transfected with HIF-1α siRNA (A) or HIF-2α siRNA (B) 24 h prior to hypoxia (6 h). Each graph represents mean ± SEM of three independent experiments. (C) Cells were transfected with both HIF-1α and HIF-2α mixed siRNA 24 h prior to hypoxia (6 h). Graph represents mean ± SEM of five independent experiments. P values of HIF-2α or HIF-1/2α siRNA-transfected cells vs. control (Ct) siRNA-transfected cells are annotated in the graphs.

FIGURE 3

Silencing HIFα proteins up-regulates IL-1α transcription in hypoxic WI-38 fibroblasts.(A) WI-38 fibroblasts were cultured in either normoxic or hypoxic conditions for 2, 4, or 6 h, and were analyzed by qRT-PCR for RQ of IL-1α transcription. Graph represents the average ± SEM of two different experiments. RQ of IL-1α transcription in WI-38 cells transfected with HIF-1α (B), HIF-2α (C) siRNA 24 h prior to 6 h culture in hypoxic conditions. Each graph represents mean ± SEM of three independent experiments. (D) Cells were transfected with both HIF-1α and HIF-2α mixed siRNA 24 h prior to hypoxia (6 h). Graph represents mean ± SEM of four independent experiments. P values of HIF-1/2α siRNA transfected cells vs. control (Ct) siRNA-transfected cells are annotated in the graphs.

FIGURE 4

Overexpression of HIFα proteins increases IL-1α transcription in A549 and HeLa cells but not in WI-38 fibroblasts.(A) Western blot using anti-HIF-1α or anti-HIF-2α antibodies of nuclear fractions obtained from HEK-T293 cells transfected with the annotated vectors. WI-38 (B), A549 (C), or HeLa (D) cells were transfected with either control vector or with HIF-1α (P402A/P564A) and HIF-2α (P405A/P531A), and 24 h later were analyzed for IL-1α and VEGF RQ of mRNA levels by real-time PCR. Graphs represent mean ± SEM of four independent experiments for each cell type. P values of HIFα-transfected cells vs. control (Ct) transfected are annotated in the graphs.