LRP-1 ectodomain shedding as a post-transcriptional mechanism to regulate its endocytic activity
(2010)
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- (en) The low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) is a type I integral membrane receptor that mediates endocytosis of a wide array of ligands. LRP-1-dependent endocytosis emerges as a main mechanism controlling the extracellular amounts of various matrix metalloproteinase (MMP) family members. The LRP-1-mediated internalization of MMP-2 was identified as a pivotal mechanism for controlling the extracellular activity of this proteinase. In particular, we have documented that LRP-1 mediates the endocytic clearance of proMMP-2 complexed to tissue inhibitor of metalloproteinases-2 (TIMP-2) in human fibrosarcoma HT1080 cells. However, the endocytic function of LRP-1 in the direct regulation of the extracellular matrix (ECM) remodeling remains hypothetical. Moreover, the mechanistic regulation of LRP-1 is still unclear. Membrane-type MMPs (MT-MMPs) degrade LRP-1 into low-molecular weight fragments, but the intact LRP-1 ectodomain can be selectively shed from the cell surface upon cleavage by a variety of proteolytic enzymes. The general aim of the study was to analyze the relevance of LRP-1-mediated clearance in the regulation of ECM remodeling, and to identify the mechanism by which this process was regulated. We turned to human endometrium, which offers a unique physiological example of efficient regulation of tissue breakdown and reconstruction around the menstrual phase. We further elucidated the mechanism by which LRP-1 is itself regulated in HT1080 cells. First, we addressed the role of LRP-1 in the endocytic clearance of endometrial gelatinases, the expression of which is poorly regulated during the menstrual cycle. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting and immunolabeling. Post-translational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased accumulation of (pro)gelatinases 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 ECM breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by 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 non-bleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP. In the second part of the study, we identified two membrane-associated metalloproteinases, ADAM-12 and MT1-MMP that shed LRP-1 from human fibrosarcoma HT1080 cells. We compared the shedding potential of classical fibroblastoid HT1080 cells with a variant featuring an epithelioid phenotype. Although both fibroblastoid and epithelioid HT1080 cells expressed similar levels of ADAM-12 and MT1-MMP and of their specific inhibitor, TIMP-2, epithelioid cells shed 4-fold less LRP-1 ectodomain. Epithelioid HT1080 cells contained twice more cholesterol than fibroblastoid cells. We demonstrate that cholesterol depletion from epithelioid cells increased LRP-1 ectodomain shedding and conversely, that cholesterol enrichment of fibroblastoid cells impaired their shedding capacity. These results establish cholesterol as a regulator of LRP-1 levels at the plasma membrane.
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Selvais, C. (2010). LRP-1 ectodomain shedding as a post-transcriptional mechanism to regulate its endocytic activity. https://hdl.handle.net/2078.5/130603