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E. Karampini et al.
Retention of von Willebrand factor in the endoplasmic reticulum results in reduced von Willebrand factor secretion in Sec22b-depleted cells
The lack of mature VWF, as well as the shorter WPB, prompted us to investigate how much VWF is stored and secreted in the absence of Sec22b. We observed that in the Sec22b-depleted cells, intracellular VWF levels were slightly increased when compared to the levels in control cells (Figure 5A), potentially because of VWF entrapment in the ER. On the other hand, basal secretion was signif- icantly decreased in Sec22b-silenced cells (Figure 5B). Basal secretion primarily originates from unstimulated WPB release,39,40 suggesting that this compartment is smaller upon Sec22b depletion. In line with this, VWF
release through histamine-induced WPB exocytosis was also significantly reduced (Figure 5C). WPB acquire secre- tion competence during maturation by recruiting Rab GTPases and Rab-effectors,7 so this decrease could poten- tially be attributed to defects in WPB maturation in Sec22b-depleted cells. We assessed whether two matura- tion-dependent components of the exocytotic machinery, Rab27A and Slp4-a,41,42 were recruited to WPB but found no difference between shSec22b and shCTRL cells (Online Supplementary Figure S5). Thus, the simplest explanation for reduction in (stimulated) VWF secretion is failure of sufficient VWF to progress to a stimulus-sen- sitive compartment, i.e. the WPB, because it is retained in the ER.
D
ABC
Figure 5. Sec22b silencing results in reduced basal and stimulated secretion. (A) Intracellular von Willebrand factor (VWF) content in control and Sec22b-silenced endothelial cells measured by enzyme-linked immunosorbent assay (n=5, paired t-test, *P<0.05). (B) Basal VWF release presented as percentage of intracellular VWF content (n=5, paired t-test, *P<0.05) (C) Histamine-stimulated VWF release presented as percentage of intracellular VWF content (n=3 independent experi- ments, paired t-test, **P<0.01). (D) Proposed model of Sec22b-dependent VWF trafficking and Weibel-Palade body size control. SE: sorting endosome; WPB: Weibel- Palade body; iWPB: immature WPB; mWPB: mature WPB; ER: endoplasmic reticulum; rER: rough ER; TGN: trans-Golgi network.
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haematologica | 2021; 106(4)