Endothelial cell function is disrupted by viral inactivation of p53
Session type: Poster / e-Poster / Silent Theatre session
Inactivation of the tumour suppressor protein, p53, is a crucial step in the oncogenic transformation of most cells. Many human oncoviruses also target this pathway by diverse mechanisms, including direct binding of viral proteins and inhibition of p53 activity. Simian Virus 40 (SV40) is also capable of inactivating p53 through expression of Large T Antigen (LTA). Here, we describe a novel gain-of-function activity for virally inactivated p53, principally inhibition of VEGFR2 expression, suppression of endothelial cell (EC) identity, and decreased angiogenesis.
We examined the human brain EC line, hCMEC/D3, for expression of endothelial markers by flow cytometry and Western blotting. Angiogenic capabilities of ECs were assessed in overlay assays, tubulogenesis assays, co-culture models, and in vivo Matrigel plugs. TP53 knockdown cells were generated using p53 shRNA and assessed for restoration of EC marker expression by qRT-PCR and immunofluorescence microscopy. HUVECs directly transduced with LTA were also investigated. Finally, the binding of p53 to gene promoters was examined by chromatin immunoprecipitation.
In comparison to HUVECs, hCMEC/D3 have reduced VEGFR2, CD31, VE-cadherin and CD146 protein levels, correlating with decreased capacity to undergo angiogenesis in response to VEGF-A. hCMEC/D3 were derived by EC immortalisation with LTA and hTERT, with the former known to interact with and disrupt p53. Knockdown of p53 by shRNA restored expression of EC markers, including the blood-brain-barrier component CLDN5, at both the mRNA and protein level. Importantly, angiogenesis was also restored, as shTP53 cells switched from proliferation to differentiation upon VEGFR2 stimulation. Furthermore, HUVECs transduced with SV40 LTA and hTERT largely recapitulated the hCMEC/D3 phenotype, with expression of EC markers decreasing in a p53-dependent manner. Finally, virally inactivated p53 associated directly with KDR and CD34 promoter regions to potentially suppress VEGFR2 expression and EC identity.
The inactivation of p53 in endothelial cells following viral infection not only reduces normal p53 tumour suppressor activities but also inhibits the expression of the critical EC marker, VEGFR2. We have identified a novel gain-of-function activity of virally inactivated p53 in binding to and inhibiting the KDR promoter. Removing inactive p53 from these cells restores VEGFR2 expression, endothelial cell identity and angiogenic potential.