PB0344 - Does the Tandem C2 domains nuclear protein, TC2N, interfere with Weibel Palade Body biogenesis?

Background: In several large genome-wide association meta-analyses, the TC2N locus has consistently been strongly associated with VWF (von Willebrand Factor) plasma levels. These analyses show the strongest associations with TC2N intronic variants and demonstrate no more plausible locus within 1Mb of TC2N. Although there is robust evidence that TC2N is a genetic modifier of VWF, the functional explanation for this association remains unclear.

Aims: To explore the effects of overexpression of TC2N on VWF biosynthesis in HUVEC.

Methods: Antibiotic resistant HUVECs, expressing TC2N or Lenti-backbone (BB) were selected following transduction of a lentiviral vector containing human TC2N cDNA. QrtPCR was performed to ascertain TC2N and VWF gene expression. TC2N protein and cellular localization were assessed using western blotting and immunofluorescence, respectively. VWF ELISAs were utilized to quantify protein, and multimer profiles were analyzed under resting and exocytosis stimulating conditions. Machine learning analysis was performed in deconvolved confocal 3D images of large areas (0.33mm2) and single cells, focusing on the segmentation of rod-shape ( < 0.4) vs. spheric-shape (>0.4) VWF+ Weibel Palade Bodies (WPB). The average volume of WPBs was compared.

Results: TC2N mRNA increased substantially in HUVEC-TC2N stable cell lines (~243664 copies) compared to HUVEC or HUVEC-BB. VWF mRNA copy number and protein levels in media and lysate did not significantly differ between the groups. No significant difference was observed in the amount of VWF released following stimulated secretion, and multimer analysis showed no difference in the distribution of VWF multimers. Comparing the single cell images, we observed more spheric like WPB structures in TC2N expressing cells (Figure 1), and a reduction in the average structure volume (Figure 2).

Conclusion(s): In this study, overexpression of TC2N did not alter the expression of VWF mRNA or protein, however, it may have an influence on the organization of WPBs within cell. Details of this association require further study.