PB0815 - Involvement of the nuclear protein, TC2N, in regulating VWF levels in endothelial cells

Background: Plasma von Willebrand Factor (VWF) levels are influenced by both inherited and acquired factors. Large genome-wide association meta-analyses have been used to identify potential genetic modifiers of this phenotype. Among the loci that have been consistently associated with VWF levels in these studies is the TC2N locus (Tandem C2 domains nuclear protein). The functional explanation for this association remains unclear.

Aims: To investigate the association between TC2N and VWF in human endothelial cells and explore its influence on VWF expression.

Methods: The UCSC Genome Browser (GRCh38/hg38) was used to investigate the TC2N genetic association. QRT-PCR was performed to determine TC2N gene expression in endothelial cells. TC2N protein expression and cellular localization were assessed using western blotting and immunocytochemistry (ICC), respectively. TC2N mRNA was knocked down in Human Lymphatic Endothelial cells (HLEC).

Results: All 7 endothelial cell types investigated expressed low levels of TC2N, with HLEC producing the highest levels, around 200 copies. TC2N expression in HLEC and HUVEC increased 2-fold with 4hr LPS exposure and 1.5 and 2-fold respectively with histamine. TC2N RNA expression was reduced ~75% at 48hr following siRNA knockdown in HLEC, and VWF mRNA expression was reduced by ~30%. However, TC2N protein levels remained stable during this time. ICC showed TC2N localized in 2-5 nuclear bodies in ~75% of HLECs and ~25% of HUVEC. TC2N did not colocalize with VWF in these cells.

Conclusion(s): These results support TC2N’s genetic association with VWF levels and demonstrate that the protein is located in endothelial cell nuclear bodies. TC2N does not colocalize with VWF in endothelial cells. Reduction of TC2N mRNA is associated with reduced VWF expression. While TC2N appears most likely associated with regulating VWF biosynthesis, the details of this association require further study.