Megalin/LRP2 Expression Is Induced by Peroxisome Proliferator-Activated Receptor -Alpha and -Gamma: Implications for PPARs' Roles in Renal Function

BACKGROUND: Megalin is a large endocytic receptor with relevant functions during development and adult life. It is expressed at the apical surface of several epithelial cell types, including proximal tubule cells (PTCs) in the kidney, where it internalizes apolipoproteins, vitamins and hormones with their corresponding carrier proteins and signaling molecules. Despite the important physiological roles of megalin little is known about the regulation of its expression. By analyzing the human megalin promoter, we found three response elements for the peroxisomal proliferator-activated receptor (PPAR). The objective of this study was to test whether megalin expression is regulated by the PPARs. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of epithelial cell lines with PPARα or PPARγ ligands increased megalin mRNA and protein expression. The stimulation of megalin mRNA expression was blocked by the addition of specific PPARα or PPARγ antagonists. Furthermore, PPAR bound to three PPAR response elements located in the megalin promoter, as shown by EMSA, and PPARα and its agonist activated a luciferase construct containing a portion of the megalin promoter and the first response element. Accordingly, the activation of PPARα and PPARγ enhanced megalin expression in mouse kidney. As previously observed, high concentrations of bovine serum albumin (BSA) decreased megalin in PTCs in vitro; however, PTCs pretreated with PPARα and PPARγ agonists avoided this BSA-mediated reduction of megalin expression. Finally, we found that megalin expression was significantly inhibited in the PTCs of rats that were injected with BSA to induce tubulointerstitial damage and proteinuria. Treatment of these rats with PPARγ agonists counteracted the reduction in megalin expression and the proteinuria induced by BSA. CONCLUSIONS: PPARα/γ and their agonists positively control megalin expression. This regulation could have an important impact on several megalin-mediated physiological processes and on pathophysiologies such as chronic kidney disease associated with diabetes and hypertension, in which megalin expression is impaired

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains

Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts

Vamp-7–dependent secretion at the immune synapse regulates antigen extraction and presentation in B-lymphocytes

Recognition of surface-tethered antigens (Ags) by B-cells leads to the formation of an immune synapse that promotes Ag uptake for presentation onto MHC-II molecules. Extraction of immobilized Ags at the immune synapse of B-cells relies on the local secretion of lysosomes, which are recruited to the Ag contact site by polarization of their microtubule network. Although conserved polarity proteins have been implicated in coordinating cytoskeleton remodeling with lysosome trafficking, the cellular machinery associated with lysosomal vesicles that regulates their docking and secretion at the synaptic interface has not been defined. Here we show that the v-SNARE protein Vamp-7 is associated with Lamp-1(+) lysosomal vesicles, which are recruited and docked at the center of the immune synapse of B-cells. A decrease in Vamp-7 expression does not alter lysosome transport to the synaptic interface but impairs their local secretion, a defect that compromises the ability of B-cells to extract, process, and present immobilized Ag. Thus our results reveal that B-cells rely on the SNARE protein Vamp-7 to promote the local exocytosis of lysosomes at the immune synapse, which is required for efficient Ag extraction and presentation