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Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells.

ABSTRACT: Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.

SUBMITTER: Lee PC

PROVIDER: S-EPMC6855450 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundReceptor signaling is central to vascular endothelial function and is dysregulated in vascular diseases such as atherosclerosis and pulmonary arterial hypertension (PAH). Signaling pathways involved in endothelial function include vascular endothelial growth factor receptors (VEGFRs) and G protein-coupled receptors, which classically activate distinct intracellular signaling pathways and responses. The mechanisms that regulate these signaling pathways have not been fully elucidated and it is unclear what nodes for cross talk exist between these diverse signaling pathways. For example, multifunctional β-arrestin (ARRB) adapter proteins are best known as regulators of G protein-coupled receptor signaling, but their role at other receptors and their physiological importance in the setting of vascular disease are unclear.MethodsWe used a combination of human samples from PAH, human microvascular endothelial cells from lung, and Arrb knockout mice to determine the role of ARRB1 in endothelial VEGFR3 signaling. In addition, a number of biochemical analyses were performed to determine the interaction between ARRB1 and VEGFR3, signaling mediators downstream of VEGFR3, and the internalization of VEGFR3.ResultsExpression of ARRB1 and VEGFR3 was reduced in human PAH, and the deletion of Arrb1 in mice exposed to hypoxia led to worse PAH with a loss of VEGFR3 signaling. Knockdown of ARRB1 inhibited VEGF-C-induced endothelial cell proliferation, migration, and tube formation, along with reduced VEGFR3, Akt, and endothelial nitric oxide synthase phosphorylation. This regulation was mediated by direct ARRB1 binding to the VEGFR3 kinase domain and resulted in decreased VEGFR3 internalization.ConclusionsOur results demonstrate a novel role for ARRB1 in VEGFR regulation and suggest a mechanism for cross talk between G protein-coupled receptors and VEGFRs in PAH. These findings also suggest that strategies to promote ARRB1-mediated VEGFR3 signaling could be useful in the treatment of pulmonary hypertension and other vascular disease.

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Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells.

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