Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs

Abstract

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues

“…These results were confirmed by immunofluorescence showing that the intracellular distribution of heparin was associated to lysosomes. Our observations go ahead with previous data in the literature about the systemic clearance of heparin from the circulation mediated by a hyaluronan receptor for endocytosis (HARE), derived from stabilin-2 by proteolysis 48,49 . This primary scavenger receptor had an affinity not only for hyaluronic acid and chondroitin sulfate but also for heparin and other ligands, binding and internalizing these macromolecules for further degradation in lysosomes 40 .…”

Physiological and pathogenic role of scavenger receptors in humans

“…Экспрессия SR-H1 в основном выявляется на макрофагах РЭС и M2, а SR-H2 -дополнительно и на эндотелиоцитах. Общими свойствами этих рецепторов является участие в поглощении продуктов апоптоза, oxLDL, продуктов коагуляции крови, обеспечение контакта клеток с экстраклеточным матриксом и, вероятно, кофакторное связывание отдельных бактерий и продуктов их деградации [7,68,122,166]. При этом гепарин плазмы крови связывается с несколькими факторами роста, включая FGF-2, PDGF и HGF и их рецепторами [247], всего 22% белков плазмы связываются с гепарином [193].…”

“…Контакт SR-H, в частности SR-H2, с лигандом запускает сигнальный путь MFPK-ERK1/2 и активацию NF-κB, но это, как правило, не приводит к выраженному клеточному стрессу и даже способствует продукции антивоспалительного цитокина -TGF-β [165,166]. Поэтому эти рецепторы могут активно функционировать в физиологических условиях.…”

“…Поэтому эти рецепторы могут активно функционировать в физиологических условиях. Так, макрофагальные и эндотелиальные SR-H участвуют в ангиогенезе в норме и при воспалении, в заживлении ран, но также и в онкогенезе (васкуляризации опухолевой ткани) [126,166,216]. Подавление у мышей экспрессии SR-H1 на макрофагах приводит к увеличению продукции TNFα, а также опосредованному цитокинами усилению продукции IgG и IgM [46].…”

Ligand Binding and Signaling of HARE/Stabilin-2

“…The significance of HA size may have to do with physical properties of how the polysaccharide interacts with the protein receptor such as the rod-to-coil transition of HA at about 200 kDa. Likewise, low-molecular-weight heparin does not activate NF-kB unless there is a relatively high concentration in contrast to heparin that induces the NF-kB signal at much lower concentrations [88]. Low-molecular-weight heparin has fewer 3- O -sulfated residues to interact with Stabilin-2 than the unfractionated or unprocessed heparin [58,65].…”