Cadherin-6B proteolytic N-terminal fragments promote chick cranial neural crest cell delamination by regulating extracellular matrix degradation

Abstract

During epithelial-to-mesenchymal transitions (EMTs), chick cranial neural crest cells simultaneously delaminate from the basement membrane and segregate from the epithelia, in part, via multiple protease-mediated mechanisms. Proteolytic processing of Cadherin-6B (Cad6B) in premigratory cranial neural crest cells by metalloproteinases not only disassembles cadherin-based junctions but also generates shed Cad6B ectodomains or N-terminal fragments (NTFs) that may possess additional roles. Here we report that Cad6B NTFs promote delamination by enhancing local extracellular proteolytic activity around neural crest cells undergoing EMT en masse. During EMT, Cad6B NTFs of varying molecular weights are observed, indicating that Cad6B may be cleaved at different sites by A Disintegrin and Metalloproteinases (ADAMs) 10 and 19 as well as by other matrix metalloproteinases (MMPs). To investigate Cad6B NTF function, we first generated NTF constructs that express recombinant NTFs with similar relative mobilities to those NTFs shed in vivo. Overexpression of either long or short Cad6B NTFs in premigratory neural crest cells reduces laminin and fibronectin levels within the basement membrane, which then facilitates precocious neural crest cell delamination. Zymography assays performed with supernatants of neural crest cell explants overexpressing Cad6B long NTFs demonstrate increased MMP2 activity versus controls, suggesting that Cad6B NTFs promote delamination through a mechanism involving MMP2. Interestingly, this increase in MMP2 does not involve up-regulation of MMP2 or its regulators at the transcriptional level but instead may be attributed to a physical interaction between shed Cad6B NTFs and MMP2. Taken together, these results highlight a new function for Cad6B NTFs and provide insight into how cadherins regulate cellular delamination during normal developmental EMTs as well as aberrant EMTs that underlie human disease.

Draxin alters laminin organization during basement membrane remodeling to control cranial neural crest EMT

“…We hypothesize this abrogation of basement membrane remodeling may be in part due to reduced Cadherin6B (Cad6B) expression. Cad6B is a target of proteolytic cleavage, and it was recently shown that its cleaved fragments promote laminin degradation and basement membrane remodeling during EMT (Schiffmacher et al, 2018;Schiffmacher et al, 2014). Thus, it is possible the premature reduction of Cad6B found with loss of Draxin (Hutchins and Bronner, 2018) subsequently reduces the availability of Cad6B cleavage fragments to modulate laminin.…”

Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

“…In addition, our results indicate that MMP14 acts independently of its catalytic activity strongly suggesting that MMP14 and MMP2 may not belong to the same pathway in the chick embryo. Other MMPs would have to activate MMP2 instead such as MMP15/MT2-MMP (Morrison et al, 2001), MMP16/MT3 (Nakada et al, 1999) which are expressed by cephalic NC cells at the time of EMT (Schiffmacher et al, 2018). Intriguingly, Cadherin-6B can modulate MMP2 activity (Schiffmacher et al, 2018).…”

“…Other MMPs would have to activate MMP2 instead such as MMP15/MT2-MMP (Morrison et al, 2001), MMP16/MT3 (Nakada et al, 1999) which are expressed by cephalic NC cells at the time of EMT (Schiffmacher et al, 2018). Intriguingly, Cadherin-6B can modulate MMP2 activity (Schiffmacher et al, 2018). Cadherin-6B is processed by ADAM proteases and a soluble N-terminal fragment (NTF) is released.…”

Cellular organization and boundary formation in craniofacial development

“…In addition to dismantling adherens junctions to promote delamination and migration, the cleavage of cadherin-6B results in a proteolytic product, CTF2, that functions as a transcriptional regulator to feedback and reinforce the EMT gene regulatory program (Schiffmacher et al, 2016). It was also recently shown that cleavage of cadherin-6B generates shed N-terminal fragments that promote delamination through an increase in extracellular proteolytic activity resulting in the degradation of ECM surrounding NCCs (Schiffmacher, Adomako-Ankomah, Xie, & Taneyhill, 2018). In addition to cadherin-6B, it has been demonstrated in trunk NCCs that levels of N-cadherin must be regulated for chick NCC emigration as its overexpression prevents NCC delamination (Nakagawa & Takeichi, 1998;Shoval, Ludwig, & Kalcheim, 2007).…”