Abstract
Brain neural stem cells and transit amplifying cells in the subventricular zone (SVZ) of the lateral ventricles are in direct contact with the microvascular endothelium. The mechanisms/molecules of direct cell contact in the SVZ neurovascular niche are not fully understood. We previously showed that neural stem/progenitor (NS/P) cells induce brain endothelial signaling in direct cell contact through matriptase (MTP) on NS/P cell surface. In the present study, using pull-down and LC-MS/MS, we identified melanoma cell adhesion molecule (MCAM) the brain endothelial molecule that interacts with MTP. MCAM physically binds to the CUB domains of MTP and induces a chain of brain endothelial signaling including p38MAPK activation, GSK3β inactivation and subsequently β-catenin activation; none of these signaling events occurred when either MTP or MCAM is deleted. MTP-MCAM binding and induction of endothelial signaling were all sensitive to cholera toxin. Together, we identified key molecules that may represent a mechanism in neural stem cell vascular niche regulation.
Highlights
Endothelial p38MAPK activation, GSK3β inactivation and subsequent β-catenin activation
We used a Western blot-based screening to search signaling molecules that are activated in brain endothelial cells only after contact co-culture with neural stem/progenitor (NS/P) cells and that their activation depend on the presence of MTP in NS/P cells
We found that only endothelial GSK3β serine residue 9 phosphorylation and β-catenin stability are induced by NS/P-bEnd cell contact and that both depend on neural MTP
Summary
NS/P cell surface MTP induces activation of bEnd cell signaling. To identify brain endothelial surface molecules interacting with neuronal MTP, we first determined the endothelial signaling pathways that are activated depending on interaction with MTP. By examining endothelial p38MAPK phosphorylation, GSK3β serine 9 phosphorylation and IL6 secretion, we asked which of the different MTP constructs failed to restore the knockdown cells’ ability to induce bEnd cell signaling responses Ectopic expression of the protease inactive full-length MTP (Fig. 2A, variant 4) or MTP lacking the four LDLR domains (Fig. 2A, variant 5) restored the MTP-knockdown NS/P cells’ ability to induce endothelial p38 MAPK phosphorylation (Fig 2B, 4 and 5) and GSK3β serine 9 phosphorylation (Fig 2C, 4 and 5), whilst MTP lacking only the two CUB domains failed to do so (Fig. 2A, variant 6; 6 in Fig 2B,C and D) All together, these data demonstrate that CUB motifs of MTP are the key elements for NS/P cell interaction with bEnd cells and induction of endothelial cell signaling. These studies showed that upon binding of MTP and MCAM at cell surface, in addition to p38MAPK, GSK3β bind to MCAM intracellular part and is inactivation phosphorylated by p38MAPK to prevent β-catenin phosphorylation and degradation
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