Matriptase Cleaves EpCAM and TROP2 in Keratinocytes, Destabilizing Both Proteins and Associated Claudins

Abstract

The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells

“…In the absence of the protease inhibitor HAI-2, the serine protease matriptase cleaves EpCAM, thereby targeting EpCAM and claudin-7 for lysosomal degradation. We have reported that this pathway exists in keratinocytes, and that in these cells, TROP2 is also a matriptase substrate [30]. Perhaps TROP2 and EpCAM turn over at different rates in crypt versus villous IEC via matriptase-mediated or other mechanisms.…”

Trop2: Jack of All Trades, Master of None

“…Mechanistic studies revealed that Trop2 silencing blocked the phosphorylation of Claudin 1 and Occludin, altered their proper cellular localization and inhibited hepatitis C virus infection at the level of viral entry into hepatocytes [ 49 ]. Recently, membrane-anchored epithelial protease Matriptase was shown to cleave Trop2 and EpCAM proteins [ 50 , 51 , 52 ]. Interestingly, elimination of both proteins was necessary to reduce claudin levels in keratinocytes confirming the functional redundancy of both protein in regulation of epithelial barrier function [ 50 ].…”

“…Recently, membrane-anchored epithelial protease Matriptase was shown to cleave Trop2 and EpCAM proteins [ 50 , 51 , 52 ]. Interestingly, elimination of both proteins was necessary to reduce claudin levels in keratinocytes confirming the functional redundancy of both protein in regulation of epithelial barrier function [ 50 ].…”

EpCAM as Modulator of Tissue Plasticity

“…The lack of reports on similar effects in other epithelial tissues and cell lines is likely due to the frequent coexpression of EpCAM and EpCAM2/Trop2. The redundancy of the two genes was recently demonstrated in keratinocytes [ 52 ].…”

“…Thus, the release of the EpCAM in the cytoplasm by RIP [ 28 , 29 , 30 , 31 ] would be predicted to abolish EpCAM inhibitory function. Another potential protease-dependent regulatory mechanism involves cleavage of the extracellular domain by an extracellular protease called matriptase [ 52 , 59 ]. In this case, the small N-terminal product remains connected to the rest of the protein through disulphide bridges, but the properties of EpCAM are affected, including stability at the plasma membrane [ 52 , 59 ], but also cis-dimerization [ 60 ] and lateral interactions with claudins [ 59 ], all events that could impact on EpCAM function.…”