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Activation of the non-canonical NF-?B/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells.

ABSTRACT: BACKGROUND:The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-?B ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-?B signalling is essential for pro-calcific activation of the underlying SMCs. METHODS:For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25?ng/ml?±?5?ng/ml TRAIL) for 72?h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5?ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS:Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-?B/p52 and canonical NF-?B/p65 pathways in both cell types. In RANKL?±?TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-?B/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-?B2 gene (the molecular forerunner to NF-?B/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY:These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-?B pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.

SUBMITTER: Harper E

PROVIDER: S-EPMC6376867 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells.

Harper Emma E Rochfort Keith D KD Forde Hannah H Davenport Colin C Smith Diarmuid D Cummins Philip M PM

Cellular signalling 20180417

<h4>Background</h4>The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underl ...[more]

PMID: 29678621

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Project description:Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-?B (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-?B (I?B) kinase-? (IKK-?), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-? are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4(+)CD25(+) T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)(+) stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK(+) human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4(+) or FOXP3(+) T cells on human breast cancer prognosis and suggest that the targeting of RANKL-RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

Dataset Information

Activation of the non-canonical NF-?B/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells.

Publications

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells.

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