Metalloproteinase-Dependent Shedding of Low-Density Lipoprotein Receptor-Related Protein-1 Ectodomain Decreases Endocytic Clearance of Endometrial Matrix Metalloproteinase-2 and -9 at Menstruation

Abstract

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

LRP-1–CD44, a New Cell Surface Complex Regulating Tumor Cell Adhesion

“…Recombinant RAP was purified by gravity-flow chromatography using a nickel-charged resin (Ni-nitrilotriacetic acid [NTA]-agarose from Qiagen, Courtaboeuf, France) and then controlled after SDS-PAGE by Coomassie blue and immunoblotting using both anti-HA tag and anti-RAP antibodies. The LRP-1 binding capacity was confirmed using a BIAcore X system (BIAcore AB, Uppsala, Sweden), as already reported (49).…”

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

“…LRP-1 comprises two chains, a 515-kDa ␣-chain that interacts with ligands via its four clusters of LDL receptor type A repeats and a noncovalently associated 85-kDa ␤-chain that tethers the receptor in the membrane and interacts with intracellular adaptors to direct endocytosis via clathrin-coated pits (20). LRP-1 can be shed from cell membranes, releasing a soluble form of the receptor, sLPR-1, that can act as a competitive inhibitor of ligand endocytosis (21,22).…”

Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis

“…The ectodomain of the LRP1b chain is cleaved by metalloproteinases (Quinn et al, 1999;Liu et al, 2009;Selvais et al, 2009Selvais et al, , 2011 and the membrane-associated b-secretase BACE1 (von Arnim et al, 2005) during RIP to generate the membraneassociated 25-kDa LRP1-CTF fragment. LRP1b-CTF then becomes a substrate of intramembrane g-secretases to produce cytoplasmic LRP1b-ICD.…”

“…A soluble fragment of LRP1, composed of the a-chain and the extracellular part of the b-chain, has been detected in the blood stream (Quinn et al, 1997;Quinn et al, 1999). The shedding of the LRP1b-ectodomain is thought to be catalysed by one or more membrane-associated metalloproteases (Quinn et al, 1999;Rozanov et al, 2004;Liu et al, 2009;Selvais et al, 2009Selvais et al, ., 2011 and the membrane-associated b-secretase BACE1 (von Arnim et al, 2005). LRP1b-CTF is then cleaved by g-secretases to give cytoplasmic LRP1b-ICD (May et al, 2002).…”

“…RIP is a process that involves two cleavages, as described for Notch (De Strooper et al, 1999), APP (amyloid precursor protein) (De Strooper et al, 1998) andLRP6 (Mi andJohnson, 2007). The first cleavage, performed by metalloproteinases (Quinn et al, 1999;Rozanov et al, 2004;Liu et al, 2009;Selvais et al, 2009Selvais et al, , 2011 and by the membrane-associated b-secretase, BACE1 (von Arnim et al, 2005), occurs in the extracellular region, close to the plasma membrane, and leads to shedding of the LRP1b ectodomain. This produces the membrane-associated carboxyl-terminal fragment, LRP1b-CTF, which is then cleaved by g-secretases within its transmembrane domain (Hass et al, 2009).…”