Glucocorticoid-induced reversal of interleukin-1β-stimulated inflammatory gene expression in human oviductal cells

Abstract

Studies indicate that high-grade serous ovarian carcinoma (HGSOC), the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE). Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events. Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1), tumor necrosis factor (TNF), and cortisol. Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect. The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated. To examine the response of FTE cells and the ability of glucocorticoids to oppose this response, an immortalized human FTE cell line, OE-E6/E7, was treated with IL1β, dexamethasone (DEX), IL1β and DEX, or vehicle and genome-wide gene expression profiling was performed. IL1β altered the expression of 47 genes of which 17 were reversed by DEX. DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1β treatment altered the expression of 784 genes. Network and pathway enrichment analysis indicated that many genes altered by DEX are involved in cytokine, chemokine, and cell cycle signaling, including NFκΒ target genes and interacting proteins. Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, PI3 and TACC2 in OE-E6/E7 cells. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1β that was blocked by DEX. A parallel experiment using primary cultured human FTE cells indicated similar effects on PTGS2, IL8, IL23A, PI3 and TACC2 transcripts. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that dysregulation of glucocorticoid signaling could contribute to increased risk for HGSOC.

Figure 1. Increased PTGS2 levels in OE-E6/E7 cells by IL1β and TNFα treatment is blocked by DEX.

A, Cells were treated with 50/ml IL1β for 24 h or 48 h and Western blot analysis was performed for PTSG2 and tubulin. B, Cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with IL1β or vehicle and harvested 24 h later. Western blot analysis was performed for PTGS2 and tubulin. C, Western blot analysis was performed on OE-E6/E7 cells for glucocorticoid receptor and tubulin levels with BT20 breast cancer cells used as positive control. D, Cells were treated with DEX or vehicle 30 h prior to treatment with TNFα or vehicle. Cells treated with TNFα alone were harvested at 24 and 48 h after treatment and cells treated with DEX+TNFα were harvested at the 48 h time point. Western blot analysis was performed for PTGS2 and tubulin. Histograms summarize quantification of PTGS2 levels normalized to tubulin in 3 to 6 immunoblots. Bars represent the mean ± SEM relative to control. Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test (p<0.05).

Figure 2. Number and overlap of genes differentially expressed due to IL1β, DEX, and IL1β+DEX treatment.

Cells were treated with 10/ml IL1β or vehicle and harvested 18 h later. Total RNA was extracted and gene expression profiling was performed on Illumina HT-12 v4.0 BeadChips. The venn diagram shows the overlap of genes differentially expressed by the different treatments relative to control treated cells.

Figure 3. Network analysis of differentially expressed genes in human FTE OE-E6/E7 cells treated with DEX.

Module annotations were performed with false discovery rate (FDR)<0.05. Genes up-regulated in human FTE OE-E6/E7 cells treated with DEX are depicted by red outer circles, whereas genes down-regulated in human FTE OE-E6/E7 cells treated with DEX are depicted by green outer circles. Linker genes are depicted as diamond shaped nodes. Direct activating or inhibitory interactions are indicated with the symbols → and -|, respectively. Indirect interactions involving additional proteins are depicted with dashed lines.

Figure 4. DEX affected NFκB target genes and interacting proteins across four network modules identified by network-based analysis.

Genes up-regulated by DEX are depicted by red outer circles, whereas genes down-regulated by DEX are depicted by green outer circles. NFκB subunits are depicted by yellow diamonds. Direct activating or inhibitory interactions are indicated with the symbols → and -|, respectively. Indirect interactions involving additional proteins are depicted with dashed lines.

Figure 5. Transcript levels of IL8, IL23A, PI3 and TACC2 following IL1β and/or DEX treatment.

OE-E6/E7 cells were treated with 10 nM DEX or vehicle 30 h prior to treatment with 50 ng/ml IL1β or vehicle and harvested 18 h later. Total RNA was extracted and RT-qPCR was performed for IL8 (A), IL23A (B), PI3 (C), TACC2 (D) and were normalized to TBP. Bars represent the mean ± SEM (n = 3 biological replicates performed in triplicate). Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test (p<0.05).

Figure 6. Transcript levels of PTGS2, IL8, IL23A, PI3 and TACC2 following IL1β and/or DEX treatment.

Primary FTE cells were treated with 10/ml IL1β or vehicle and harvested 18 h later. Total RNA was extracted and RT-qPCR was performed for PTGS2 (A), IL8 (B), IL23A (C), PI3 (D), TACC2 (E) and were normalized to the geometric mean of 4 reference genes. Bars represent the mean ± SEM (n = 3). Bars with different letters are statistically different from one another as determined by ANOVA followed by a Student-Newman-Keuls post-hoc multiple comparison test (p<0.05).