Environmental Factor, July 2008, National Institute of Environmental Health Sciences

Intramural Papers of the Month

By Robin Arnette
August 2008

The Binding of Cell-Surface Nucleolin and P-selectin Promote Metastasis

NIEHS scientists determined that the cell-surface form of nucleolin binds P-selectin — a vascular adhesion molecule — to the surface of Colo-320 human colon carcinoma cells; this binding leads to the activation of signal transduction pathways that promote tumor cell metastasis. Generally, the protein nucleolin exists inside the cell and shuttles between the nucleus and the cytoplasm, but the cell-surface form of nucleolin acts as a P-selectin receptor molecule. This work sheds light on how cancer cells establish new colonies at distant sites in the body.

The team used affinity chromatography, mass spectrometry, immunoblots and RNAi knockdown to identify cell-surface nucleolin as a P-selectin receptor on Colo-320 cells. The data indicated P-selectin binding to the cell initiated the phosphoinositide-3 kinase and p38 mitogen-activated protein kinase signaling pathways, which in turn activated the transmembrane glycoprotein α5β1 integrin. These actions resulted in the increase of cell attachment and cell spreading on fibronectin substrates.

The finding that cell-surface nucleolin is a P-selectin receptor is a novel discovery and provides a potential target for therapeutics that inhibit the progress of metastatic disease.

Citation: Reyes-Reyes EM, Akiyama SK . 2008. Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells. Exp Cells Res 314(11-12):2212-2223.

NOS and Nitroglycerin-mediated Vasodilation

Nitroglycerin helps patients with angina and a past history of heart attacks by relaxing the smooth muscles around blood vessels, allowing more blood to reach cardiac muscles, but the exact mechanism involved in nitric oxide synthase (NOS) activation was unknown. Researchers at NIEHS and the University of Sao Paulo School of Medicine have found evidence that nitroglycerin triggered constitutive NOS activation using cell cultures, isolated vessels and whole animals. The work may offer insight into the molecular mechanisms involved in nitrate resistance.

The team’s studies indicated that endothelial NOS was phosphorylated at Ser1177 on the endothelial isoform and Ser852 on the neuronal isoform in the aortae of mice and rats treated with nitroglycerin, which confirmed that isoforms of NOS were involved in vasorelaxation. Aortic ring studies determined that high doses of nitroglycerin (300 nm) produced vasodilation that was independent of the endothelium and could not be annulled by NOS inhibitors. At higher doses nitroglycerin is known to be bioactivated to nitric oxide.

Citation: Bonini MG, Stadler K, Silva Sde O, Corbett J, Dore M, Petranka J, Fernandes DC, Tanaka LY, Duma D, Laurindo FR, Mason RP . 2008. Constitutive nitric oxide synthase activation is a significant route for nitroglycerin-mediated vasodilation. Proc Natl Acad Sci USA 105(25):8569-8574.

The Involvement of the MEK-MSK1 Pathway in the Immune Response

According to NIEHS scientists, farnesol, an isoprenoid alcohol used as a flavoring ingredient, induces the expression of several inflammatory genes in human lung adenocarcinoma H460 cells including IL-6, CXCL3, IL-1α and COX-2. The response was dependent on the NF-κB pathway and involved the MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA (Ser276).

The researchers utilized microarray analysis, western blots, siRNA knockdowns and electrophoretic mobility shift assay (EMSA). One of the findings of the studies demonstrated that the farnesol treatment reduced the level of IκBα protein and overexpression of several immune response and inflammatory genes. The results also suggest that activation of the NF-κB pathway by farnesol is part of a pro-cell survival response.

Citation: Joo JH, Jetten AM . 2008. NF-kappaB-dependent transcriptional activation in lung carcinoma cells by farnesol involves p65/RelA(Ser276) phosphorylation via the MEK-MSK1 signaling pathway. J Biol Chem 283(24):16391-16399.

Low Doses of UVA Promotes Tumor Growth in Human Keratinocytes

Extended exposure to the sun has long been associated with human skin cancer, but researchers at NIEHS have found that low, nonlethal doses of UVA (315–400nm) — a wavelength of light that makes up 95% of the UV irradiation in natural sunlight — induces dose-dependent cell cycle progression in human HaCaT keratinocytes. The study, performed by NIEHS scientists, provided evidence that even low amounts of UVA could promote skin cancer and that the mechanism involved disintegrin and metalloprotease/epidermal growth factor receptor (EGFR)/AKT/Cyclin D1 pathways.

The research team, who used siRNA knockdowns, western blotting and confocal microscopy, determined that AKT activation was mediated by the EGFR pathway following UVA exposure. In addition, knockdown of cyclin D1 reduced the G1-S transition triggered by UVA exposure, which indicated cyclin D1’s role in the process. The study also discovered that the metalloprotease ADAM proteins were also involved in cell cycle progression because the use of a metalloprotease inhibitor prevented cell cycle progression after UVA exposure.

The authors acknowledged that other signaling pathways may be involved in UVA-induced cell proliferation, but concluded that the ADAM/EGFR/AKT pathway was required. The work may further the development of safe and effective chemopreventative and therapeutic strategies for skin cancer.

Citation: He YY, Council SE, Feng L, Chignell CF . 2008.UVA-induced cell cycle progression is mediated by a disintegrin and metalloprotease/epidermal growth factor receptor/AKT/Cyclin D1 pathways in keratinocytes. Cancer Res 68(10):3752-3758.

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