Cyclooxygenase-2 activates EGFR-ERK1/2 pathway via PGE2-mediated ADAM-17 signaling in testosterone-induced benign prostatic hyperplasia

Abstract

Objective and design: Prostatic inflammation is the driving force in benign prostatic hyperplasia (BPH). This work investigated the potential modulatory effect of COX-2 inhibition on ADAM-17/EGFR/ERK1/2 axis.

Materials or subjects: Adult male Wistar rats were used.

Treatment: Celecoxib (10 and 20 mg/kg; i.p.) was injected i.p. daily for three weeks. Testosterone (TST) (3 mg/kg; s.c.) was used to induce BPH.

Methods: Prostatic inflammation and hyperplasia were assessed by organ weight and histopathology. Inflammatory mediators were measured using ELISA technique. Protein analysis was performed using western blotting and immunohistochemistry. Gene expression analysis was performed using qRT-PCR. Statistical analyses included one-way ANOVA and Tukey's multiple comparison test.

Results: Testosterone-treated rats had a marked increase in COX-2, prostate weight, and index. Moreover, TST-induced COX-2 was inferred from cytoskeletal changes and was attributable to the overexpression of PGE2, NF-κB (p65), and IL-6. COX-2-derived PGE2 increased the activity of ADAM-17, TGF-α, and TNF-α. Consequently, EGFR-ERK1/2 pathway was over-activated, disrupting anti-apoptotic Bcl-2, cyclin D1, and pro-apoptotic Bax. Celecoxib reversed these effects.

Conclusion: COX-2 stimulates the ERK1/2 pathway via PGE2-ADAM-17-catalyzed shedding of TGF-α in testosterone-induced BPH. The results indicate a functional correlation between inflammation and hyperplasia in BPH.

Keywords: ADAM-17 (TACE); Benign prostatic hyperplasia; COX-2; Celecoxib; EGFR–ERK1/2; PGE2.

Fig. 1

TST-induced COX-2 clearly deteriorates the histological architecture of prostate in BPH. Histological examination of rat’s prostate: a Sections of control rats show normal histo-architecture of the ventral prostates. b Treatment with 20 mg of CXB alone showing unremarkable changes to control group. c Induction of BPH with TST exhibiting hypertrophy with increased epithelial thickness and intra-luminar projections and inflammatory infiltrate composed mainly of lymphocytes. d Treatment with 10 mg of CXB showing almost the same records as TST-induced group allover most of glandular acini with few scattered apparent intact acini in between. e Testosterone co-treated with 20 mg/kg CXB showing marked reduction in hypertrophy and hyperplasia. f Thickness of the basal epithelium cells showing significant reduction of cell membrane thickness with CXB-20–treated compared to TST-treated rats. The data are provided as mean ± SEM (n = 6). ^asignificant versus Control; ^bsignificant versus TST; ^csignificant versus CXB-10 + TST at P < 0.05. CXB Celecoxib; TST Testosterone. (Scale bar = 50 μm)

Fig. 2

Summary of inflammatory markers induced by TST in BPH and the subsequent effect of celecoxib (CXB) on these markers: Evaluation of inflammatory markers using quantitative ELISA technique: Prostate content of COX-2 (ng/mg) a, NF-κB (p65) (ng/mg) b, PGE2 (Pg/mg) c and IL-6 (Pg/mg) d are significantly improved with co-administration of CXB. All inflammatory markers are further normalized with higher dose. The data are provided as mean ± SEM (n = 6). ^asignificant versus Control; ^bsignificant versus TST; ^csignificant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone; NF-κB (p65) Nuclear factor kappa-light-chain-enhancer of activated B cells, IL-6 Interleukin 6, COX-2 Cyclooxygenase enzyme isoform 2, PGE2 Prostaglandin E2

Fig. 3

TST-induced COX-2 is critical for phosphorylation of ERK1/2 in BPH. Assessment of phosphorylation of ERK1/2 using western blotting technique: Prostate content of p-ERK1/2/T-ERK1/2 is significantly improved with co-administration of CXB. It is apparent that inhibition of COX-2 is critical for decreasing phosphorylation of ERK1/2. The data are provided as mean ± SEM (n = 6). ^a significant versus Control; ^b significant versus TST; ^c significant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone, ERK1/2 Extracellular signal-regulated kinase 1 and 2

Fig. 4

TST-induced COX-2 triggers metalloproteinase activity of ADAM-17 (TACE) and its substrates in BPH. Evaluation of metalloproteinase activity of ADAM-17 and its substrates using quantitative ELISA technique: Prostate content of ADAM-17 or TACE (ng/mg) a, TNF-α (ng/mg) b and TGF-α (Pg/mg) c are significantly improved with co-administration of CXB. The data are provided as mean ± SEM (n = 6). ^asignificant versus Control; ^bsignificant versus TST; ^csignificant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone; ADAM-17 A disintegrin and metalloproteinase domain-17, TACE Tumor necrosis factor-alpha converting enzyme, TNF-α Tumor necrosis factor alpha, TGF-α Transforming growth factor alpha

Fig. 5

TST-induced COX-2 activates ERK1/2 pathway via phosphorylation of EGFR in BPH. Assessment of phosphorylation of EGFR using western blotting technique: Prostate content of p-EGFR/T-EGFR is significantly improved with co-administration of CXB. It is apparent that inhibition of COX-2 leads to reduce phosphorylation of EGFR. The data are provided as mean ± SEM (n = 6). ^a significant versus Control; ^b significant versus TST; ^c significant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone, EGFR Epidermal growth factor receptor

Fig. 6

TST-induced cyclin D1 is abrogated with inhibition of COX-2. Immunohistochemical analysis of rat’s prostate (Scale bar = 50 μm): a Control sections demonstrate normal expression of cyclin D1. b Treatment with 10 mg of CXB alone showing almost the same normal expression of cyclin D1. c Induction of BPH with TST showing marked high expression of cyclin D1 (brown color). d Treatment with 10 mg of CXB showing almost the same expression of cyclin D1 (brown color) as TST-induced group. e Treatment with 20 mg of CXB showing significant improvement of cyclin D1 expression. f Quantitative analysis of cyclin D1 expression showing normalization of cyclin D1 expression with CXB-20–treated. The data are provided as mean ± SEM (n = 6). ^asignificant versus Control; ^bsignificant versus TST; ^csignificant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone

Fig. 7

TST-induced COX-2 disrupts the normal hemodynamic balance between apoptosis (pro-apoptotic Bax) and cell proliferation (anti-apoptotic Bcl-2). Assessment of proliferative versus apoptotic markers using quantitative PCR technique: Nuclear prostatic content of pro-apoptotic Bax a, and anti-apoptotic Bcl-2 b are significantly modulated after administration of CXB. All values are assigned as mRNA copies/ml (× 10⁴). Together, pro-apoptotic Bax/anti-apoptotic Bcl-2 ratio c is significantly restored to its normal balance with CXB-20 treated rats. The data are provided as mean ± SEM (n = 6). ^asignificant versus Control; ^bsignificant versus TST; ^csignificant versus CXB-10 + TST at P < 0.05. CXB Celecoxib, TST Testosterone Bax Bcl-2 Associated X, Bcl-2 B-cell lymphoma 2

Fig. 8

Graphical diagram of functional correlation between inflammation and hyperplasia in TST-induced BPH. Cycloxygenase-2 (COX-2) upregulates EGFR–ERK1/2 signaling cascade via stimulation of PGE2-induced ADAM-17 and consequent shedding of TGF-α: The left panel (in red color) shows that TST-induced BPH stimulates the nuclear translocation of NF-κB [1–2] resulting in increasing gene transcription of COX-2 [3] and subsequent PGE2 [4]. PGE2 trans-activates EGFR via intracellular activation of ADAM-17 (TACE) [5] leading to shedding of TGF-α [6]. Consequently, active TGF-α phosphorylates EGFR and induce ERK1/2 phosphorylation [7–8]. Together, the nuclear translocation of p-ERK1/2 [9] induced prostatic hyperplasia via enhancing gene expression of cyclin D1 and anti-apoptotic Bcl-2 in consistent with downregulation of pro-apoptotic Bax expression. In the contrast, the panel in the right corner (in blue color) shows the potential effect of COX-2 inhibition in TST-induced BPH