Abstract
MUC1 clearance from the uterine epithelial cell surface is a prerequisite for the creation of an environment conducive to embryo implantation. In some species, reduced mRNA levels along with metabolic turnover account for loss of MUC1 during the receptive phase throughout the uterine epithelium. In other species, MUC1 is rapidly lost solely at the site of blastocyst attachment, suggesting the action of a protease. Correlative studies also indicate the presence of soluble forms of MUC1 in cell culture supernatants in vitro and in bodily fluids in vivo. To characterize the proteolytic activity mediating MUC1 release, shedding of MUC1 was analyzed in a human uterine epithelial cell line (HES) that abundantly expresses and readily sheds MUC1. MUC1 release was stimulated by phorbol 12-myristate 13-acetate and was markedly inhibited by the synthetic peptide hydroxamate metalloprotease inhibitor, tumor necrosis factor-alpha protease inhibitor (TAPI), as well as by an endogenous inhibitor of matrix metalloproteases, tissue inhibitor of metalloproteases (TIMP)-3. These characteristics along with studies conducted with cell lines genetically deficient in various ADAMs (for a disintegrin and metalloprotease) identified tumor necrosis factor-alpha converting enzyme (TACE)/ADAM 17 as a MUC1 sheddase. Furthermore, both TACE and MUC1 were expressed in human uterine epithelia during the receptive phase, and co-immunoprecipitation experiments revealed a physical interaction between TACE and MUC1 in HES cells. These studies establish a proteolytic mechanism for MUC1 clearance from a human uterine epithelial cell line and identify TACE as a MUC1 sheddase.
Highlights
Proteolytic removal of the extracellular domain of numerous transmembrane proteins is responsible for the regulated release of cytokines, growth factors and their receptors, cell adhesion molecules, and ectoenzymes
We provide evidence that MUC1 is cleaved from the surface of a human uterine epithelial cell line, HES, by a protease(s) that is stimulated by phorbol 12-myristate 13-acetate (PMA) and is inhibited by the hydroxamate-based metalloprotease inhibitor, tumor necrosis factor-␣ protease inhibitor (TAPI), and the tissue inhibitor of metalloproteases (TIMP)-3
MUC1 Release Is Rapidly Stimulated by PMA—Shedding of various cell surface proteins has been shown to be stimulated by phorbol esters, such as PMA [20, 41,42,43], presumably through activation of metalloproteolytic sheddases
Summary
Proteolytic removal of the extracellular domain of numerous transmembrane proteins is responsible for the regulated release of cytokines, growth factors and their receptors, cell adhesion molecules, and ectoenzymes (reviewed in Refs. 1–3). Identification of the proteases or “sheddases” involved in the release of transmembrane proteins should provide insight into the regulation of key physiological events under normal and pathological conditions In this regard, members of the ADAM1 (for a disintegrin and metalloprotease) family of metalloproteases appear to mediate the ectodomain release of several proteins Mice that are genetically deficient in catalytically active TACE display an embryonic lethal phenotype [4], which underscores the importance of TACE-mediated shedding in vivo and suggests an essential role for ADAMs in protein ectodomain release In light of these findings and others, numerous in vitro studies attribute proteolytic processing of membrane-anchored proteins to one or more ADAMs. Embryo implantation is a highly regulated process that requires both an attachment competent blastocyst and a receptive uterus. We demonstrate that both TACE and MUC1 are expressed in human uterine epithelia during the receptive phase in vivo and form a stable physical association in HES cells in vitro
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