A novel GGA-binding site is required for intracellular sorting mediated by stabilin-1.

Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

Cross-talk between endocytic clearance and secretion in macrophages.

Macrophages are essential elements of innate immunity that control inflammatory reactions, healing processes, tissue homeostasis and immune tolerance by the co-ordinated release and clearance of soluble mediators. Scavenger receptors (SRs) constitute a major class of receptors, which direct endocytosed material for degradation. SR-mediated uptake can result in both pro-inflammatory and tolerogenic programming of macrophages. While effects of SRs on the level of signal transduction are well documented, the effect of endocytosis on the regulated secretion, in particular lysosomal secretion, in macrophages remains to be elusive. Stabilin-1, SR identified and functionally characterized by us, mediates clearance of acLDL and universal regulator of tissue turn-over - SPARC. Unexpectedly for SRs, stabilin-1 shuttles between endosomal compartment and biosynthetic compartment and transports newly synthesized stabilin-interacting chitinase-like protein (SI-CLP) to the lysosomal secretory pathway. This sorting function of stabilin-1 is mediated by GGAs, clathrin adaptors responsible for the mannose-6-phosphate receptors (MPR)-mediated transport of lysosomal hydrolases. Moreover, stabilin-1 internalizes hormone placental lactogen, transports it to the trans-Golgi network-associated transcytosis. Thus stabilin-1 is the only known SR that links endocytic clearance, intracellular sorting and trancytosis. We propose novel level of regulation for the secretory repertoire of macrophages: cross-talk of uptake and release at the level of vesicular trafficking.

Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 microg/ml in maternal circulation and stays below 0.5 microg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.

Novel stabilin-1 interacting chitinase-like protein (SI-CLP) is up-regulated in alternatively activated macrophages and secreted via lysosomal pathway.

Mammalian Glyco_18-domain-containing proteins include catalytically active chitinases and chitinase-like proteins with cytokine activity involved in host defense and Th2-type inflammatory reactions. Here, we describe a novel human Glyco_18-domain-containing protein, SI-CLP, as an interacting partner of the endocytic/sorting receptor stabilin-1. Similarly to the chitinase-like cytokines YKL-39, YKL-40, and YM1/2, SI-CLP lacks a chitin-binding domain and catalytic amino acids. Using a novel mAb 1C11, we demonstrated that SI-CLP is sorted into late endosomes and secretory lysosomes in human alternatively activated macrophages. The direct interaction of SI-CLP with stabilin-1, their colocalization in the trans-Golgi network, and the reduced sorting of SI-CLP into lysosomes in macrophages treated with stabilin-1 siRNA suggest that stabilin-1 is involved in intracellular sorting of SI-CLP. Expression of SI-CLP in macrophages was strongly up-regulated by the Th2 cytokine IL-4 and by dexamethasone. This effect was suppressed by IFNgamma but not affected by IL-10. In contrast, expression of YKL-40 was induced by IFNgamma and suppressed by dexamethasone. Macrophages treated with IL-4 secreted SI-CLP, while costimulation with dexamethasone blocked secretion and resulted in intracellular accumulation of SI-CLP. The 1C11 mAb detected SI-CLP in human bronchoalveolar lavage and peripheral-blood leukocytes (PBLs), and can be used to analyze the role of SI-CLP in human disorders.

Phosphatidylinositide 3-kinase activity is required for stabilin-1-mediated endosomal transport of acLDL.

Stabilin-1 is a type 1 transmembrane receptor specifically expressed by tissue macrophages and sinusoidal endothelial cells. We recently demonstrated that stabilin-1 is involved in the endocytic/recycling pathway and shuttles between the endosomal system and the trans-Golgi network (TGN) in human macrophages. In the present study, we designed a model cell system to study the mechanisms of stabilin-1-mediated endocytosis. Using CHO-K1 cells stably transfected with stabilin-1, we demonstrated that acetylated low-density lipoprotein (acLDL) induces recruitment of stabilin-1 into the endocytic pathway. Stabilin-1 mediates internalisation of acLDL and its delivery to early endosomes, and it is translocated together with its ligand to the late endosomal compartment. Treatment with wortmannin, a specific inhibitor of phosphatidylinositide 3-kinase (PI3K), did not block stabilin-1 mediated internalisation of acLDL as well as its trafficking to early endosomes, whereas it induced enlargement of stabilin-1/acLDL positive endosomes as well as partial dissociation of EEA1 from these structures. The major effect of wortmannin was the abrogation of stabilin-1/acLDL trafficking into the late endocytic pathway. In stabilin-1 positive human type 2 macrophages, wortmannin treatment resulted in formation of both enlarged and small stabilin-1 positive endosomes. This effect, however, was significantly weaker in macrophages as compared to CHO-stabilin-1 cells. Our data indicate that PI3K activity is required for the transfer of stabilin-1 and its ligand acLDL from early to late endosomal compartments.