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Transcription factor interactions mediate EGF-dependent COX-2 expression.

ABSTRACT: Cyclooxygenase-2 (COX-2) is linked to poor prognosis in patients with malignant gliomas. Amplification/overexpression of epidermal growth factor receptor (EGFR) is commonly seen in these tumors. EGFR signaling, through activation of the p38-MAPK/PKC-?/Sp1 cascade, plays an essential role in the regulation of COX-2 expression in glioma cells. Here, we report that Src kinase contributes upstream to this signaling cascade. In addition, more detailed analysis revealed the involvement of FOXM1, a member of the forkhead box family of transcriptional activators, in EGF-dependent COX-2 induction. FOXM1 protein increased after stimulation with EGF, although its role in modulating COX-2 expression does not depend on this increase. While a conventional FOXM1 responsive element resides in a distal region (-2872/-2539 relative to the transcriptional start site) of the COX-2 promoter, this is not required for EGF-dependent induction of COX-2. Instead, FOXM1 forms a cooperative interaction with Sp1 at the Sp1-binding site (-245/-240 relative to the start site) of the COX-2 promoter to mediate EGF-induced COX-2 expression. Definition of this novel interaction provides a clearer understanding of the mechanistic basis for EGF induction of COX-2.These data provide a guide for the evaluation of potential newer therapeutic targets that have relevance in this disease.

SUBMITTER: Xu K

PROVIDER: S-EPMC3748214 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Transcription factor interactions mediate EGF-dependent COX-2 expression.

Xu Kaiming K Shu Hui-Kuo G HK

Molecular cancer research : MCR 20130501 8

<h4>Unlabelled</h4>Cyclooxygenase-2 (COX-2) is linked to poor prognosis in patients with malignant gliomas. Amplification/overexpression of epidermal growth factor receptor (EGFR) is commonly seen in these tumors. EGFR signaling, through activation of the p38-MAPK/PKC-δ/Sp1 cascade, plays an essential role in the regulation of COX-2 expression in glioma cells. Here, we report that Src kinase contributes upstream to this signaling cascade. In addition, more detailed analysis revealed the involvem ...[more]

PMID: 23635401

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Project description:BACKGROUND: Endothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. OBJECTIVE: The goal of this study was to examine whether ET-1-induced COX-2 expression and prostaglandin E2 (PGE2) release were mediated through a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in brain microvascular endothelial cells (bEnd.3 cells). METHODS: The expression of COX-2 induced by ET-1 was evaluated by Western blotting and RT-PCR analysis. The COX-2 regulatory signaling pathways were investigated by pretreatment with pharmacological inhibitors, short hairpin RNA (shRNA) or small interfering RNA (siRNA) transfection, chromatin immunoprecipitation (ChIP), and promoter activity reporter assays. Finally, we determined the PGE2 level as a marker of functional activity of COX-2 expression. RESULTS: First, the data showed that ET-1-induced COX-2 expression was mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. Next, we demonstrated that ET-1 stimulated activation (phosphorylation) of c-Src/EGFR/Akt/MAPKs (ERK1/2, p38 MAPK, and JNK1/2) and then activated the c-Jun/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. The activated c-Jun/AP-1 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Ultimately, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in bEnd.3 cells. CONCLUSIONS: These results demonstrate that in bEnd.3 cells, c-Src-dependent transactivation of EGFR/PI3K/Akt and MAPKs linking to c-Jun/AP-1 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.

Project description:Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis.NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.

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Transcription factor interactions mediate EGF-dependent COX-2 expression.

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Transcription factor interactions mediate EGF-dependent COX-2 expression.

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