[Mechanisms of EGF regulation of COX-2 through the STAT5 signaling pathway in human lung adenocarcinoma A549 cells]

Abstract

Background: It has been proved that cyclooxygenase-2 (COX-2) is a key factor in lung cancer oncogenesis. COX-2 can be induced by a number of cytokines and growth factors and can be regulated by the JAK/STAT signaling pathway. Inhibiting the expression of COX-2 can prevent the development of lung cancer. The aim fo this study is to investigate whether the epidermal growth factor (EGF) can stimulate the signal transducers and activators of transcription 5 (STAT5) as well as to discover the effects of the STAT5 signaling pathway on the COX-2 in human lung adenocarcinoma A549 cells.

Methods: The phenomenon of STAT5 activation stimulated by the EGF was assayed through immunofluorescence and Western blot. The adenovirus containing the wild-type (WT)-STAT5 (AdWT-STAT5) plasmid, dominant-negative (DN)-STAT5 (Ad-CMV5Stat5aΔ740) plasmid, and STAT5 siRNA were transfected into A549 cells. The latter two groups were stimulated using EGF. Reverse transcriptase polymerase chain reaction was used to detect the mRNA expression of COX-2.

Results: STAT5 was not activated in A549 cells in vitro. EGF stimulation significantly increased the level of the p-STAT5 protein and induces the shuttling of p-STAT5 from the cytoplasm into the nucleus. STAT5 activation was crucial for the COX-2 expression induced by the EGF. STAT5 was required for COX-2 expression, but can mediated the effects of the COX-2 expression through pathways that were independent of transcriptional activation.

Conclusions: COX-2 expression is dependent on STAT5 phosphorylation. A second pathway does not require STAT5 phosphorylation.

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A549细胞和EGF刺激A549细胞后p-STAT5的免疫荧光染色分析。A:p-STAT5在A549细胞中的表达; B:A549细胞的核染色; C:A与B的重叠图像; D:EGF刺激后p-STAT5在A549细胞中的表达; E:A549细胞的核染色; F:D与E的重叠图像。 Immunofluorescence of p-STAT5 in resting and EGF-stimulated human lung adenocarcinoma A549 cells.Upper panels, no EGF stimulation; lower panels, EGF stimulation.(A) and (D), p-STAT5 staining; (B) and (E), Hoechst33258 staining of nuclei; (C) and (F), merged images.

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A549细胞和EGF刺激A549细胞后STAT5的免疫荧光染色分析。A:STAT5在A549细胞中的表达; B:A549细胞的核染色; C:A与B的重叠图像; D:EGF刺激后STAT5在A549细胞中的表达; E:A549细胞的核染色; F:D与E的重叠图像。 Immunofluorescence of STAT5 in resting and EGF-stimulated human lung adenocarcinoma A549 cells.Upper panels, no EGF stimulation; lower panels, EGF stimulation.(A) and (D), STAT5 staining; (B) and (E), Hoechst33258 staining of nuclei; (C) and (F), merged images.

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EGF刺激对A549细胞核p-STAT5(A)和细胞浆STAT5(B)表达影响的免疫印迹分析 P-STAT5 (A) and STAT5(B) expression induced by EGF in human lung adenocarcinoma A549 cells.Western blot analysis of nucleus extracts from EGF-stimulated and resting A549 human lung adenocarcinoma cells showing upregulation of nucleus p-STAT5 (A) and no significant of cytoplasmic STAT5 (B).

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A549细胞转染STAT5 siRNA后以及受到EGF刺激后对STAT5表达影响的免疫印迹分析(A)和COX-2 mRNA表达影响的电泳分析(B)。A549:未转染组; CtrlsiRNA:转染阴性对照siRNA组; EGF:EGF刺激组; EGF+CtrlsiRNA:转染阴性对照siRNA加EGF刺激组; EGF+siRNA:转染STAT5 siRNA加EGF刺激组; STAT5 siRNA:转染STAT5 siRNA组。 Western blot analysis of STAT5 expression (A) and electrophoresis analysis of COX-2 expression (B) in A549 cells transfected with STAT5 siRNA together with or without EGF stimulation.A549:untransfected; CtrlsiRNA:transfected with control siRNA; EGF:stimulation with EGF; EGF+CtrlsiRNA:transfected with control siRNA and stimulation with EGF; EGF+STAT5 siRNA:transfected with STAT5 siRNA and stimulation with EGF; STAT5 siRNA:transfected with STAT5siRNA.

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A549细胞转染野生型STAT5, STAT5显性负突变体和STAT5 siRNA后以及受到EGF刺激后对STAT5(A)、p-STAT5(B)表达影响的免疫印迹分析和COX-2 mRNA表达影响的电泳分析(C)。Ctrl:未转染组; WT-STAT5:转染野生型STAT5组; EGF:EGF刺激组; EGF+DN-STAT5:转染STAT5显性负突变体加EGF刺激组; EGF+STAT5 siRNA:转染STAT5 siRNA组加EGF刺激组。 Western blot analysis of STAT5 (A), p-STAT5 (B) and electrophoresis analysis of COX-2 (C) expression in A549 cells transfected with WT-STAT5, DN-STAT5 and STAT5 siRNA together with or without EGF stimulation.Ctrl, transfected with control adenovirus; WT-STAT5, transfected with wild-type STAT5;EGF, stimulation with EGF; EGF+DN-STAT5, transfected with dominant negative STAT5 and stimulation with EGF; EGF+STAT5 siRNA, transfected with STAT5 siRNA and stimulation with EGF.

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A549细胞转染野生型STAT5, STAT5显性负突变体和STAT5 siRNA后以及受到EGF刺激后对STAT5 DNA结合力表达影响的分析。与其它组相比, ^*P < 0.05, n=3。 STAT5 DNA binding assay in A549 cells nuclei transfected with WT-STAT5, DN-STAT5 and STAT5 siRNA with or without EGF stimulation.Data are means± SEM.^*P < 0.05 when compared with the other four groups.n=3.

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STAT5对COX-2可能存在的两种调控机制。STAT5通过磷酸化及非磷酸化双途径来实现对COX-2的调控。 Schematic depiction of two different potential mechanisms of regulation.STAT5 regulates COX-2 by pathways dependent of phosphorylation and unphosphorylation.