Stabilin-1, a homeostatic scavenger receptor with multiple functions

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.     

Identification of a sequence in the matricellular protein SPARC that interacts with the scavenger receptor stabilin-1

SPARC (osteonectin/BM-40), a secreted matricellular protein that promotes cellular deadhesion and motility in wound healing, carcinogenesis, and inflammation, binds to the scavenger receptor stabilin-1 in alternatively activated macrophages and undergoes endocytosis and clearance from the extracellular space. Both SPARC and stabilin-1 are expressed by endothelial cells during inflammation, but their interaction in this context is unknown. We have identified a binding site on SPARC for stabilin-1 by a solid-state peptide array coupled with a modified enzyme-linked immunosorbent assay. A monoclonal antibody that recognizes the identified binding site was also characterized that could be an inhibitor for the SPARC-stabilin-1 interaction in macrophages or endothelial cells.     

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.     

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.     

The conserved histidine in epidermal growth factor-like domains of stabilin-2 modulates pH-dependent recognition of phosphatidylserine in apoptotic cells

Clearance of apoptotic cells is involved in the resolution of inflammation, and this mechanism is controlled by the regulation of pro- and anti-inflammatory cytokine production during the ingestion of apoptotic cells. Inflamed areas show extracellular acidity, and low pH stimulates cellular functions of immune cells. However, little is known about the influence of extracellular acidic pH on the function of phagocytic cells. In this study, we showed that stabilin-2-mediated phagocytosis is activated in low pH media (pH 6.8) and examined the molecular mechanisms underlying this pH-dependent enhancement of phagocytic activity. Stabilin-2, which is expressed in human monocyte derived macrophages (HMDM), is a phosphatidylserine (PS) receptor that mediates phagocytosis of apoptotic cells, and releases the anti-inflammatory cytokine, TGF-β. The PS binding activity of stabilin-2 is enhanced in low pH, and a conserved histidine¹⁴⁰³ in close proximity to the PS binding loop is critical for pH-dependent activity. We propose that protonation of His¹⁴⁰³ may rearrange the PS binding loop to enhance binding affinity in low pH, indicating that acidic pH might act as a danger signal to stimulate stabilin-2-mediated phagocytosis to resolve inflammation. Considering that phosphatidylserine is an important target molecule for apoptotic cells in the acidic microenvironment of inflammation and tumors, our results also have implications for pH sensitive targeting of apoptotic cells.     

Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration

Migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. Here, we show that efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. Our results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).     

Progression or resolution of coxsackievirus B4-induced pancreatitis: a genomic analysis

Group B coxsackieviruses are associated with chronic inflammatory diseases of the pancreas, heart, and central nervous system. Chronic pancreatitis, which can develop from acute pancreatitis, is considered a premalignant disorder because it is a major risk factor for pancreatic cancer. To explore the genetic events underlying the progression of acute to chronic disease, a comparative analysis of global gene expression during coxsackievirus B4-induced acute and chronic pancreatitis was undertaken. A key feature of acute pancreatitis that resolved was tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, and embryogenesis and by increased division of acinar cells. Acute pancreatitis that progressed to chronic pancreatitis was characterized by lack of tissue repair, and the expression map highlighted genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis. Furthermore, immune responses appeared skewed toward development of alternatively activated (M2) macrophages and T helper 2 (Th2) cells during disease that resolved and toward classically activated (M1) macrophages and Th1 cells during disease that progressed. Our hypothesis is that growth and differentiation signals coupled with the M2/Th2 milieu favor acinar cell proliferation, while diminished growth signals and the M1/Th1 milieu favor apoptosis of acinar cells and remodeling/proliferation of the extracellular matrix, resulting in fibrosis.     

SPARC (osteonectin/BM-40)

SPARC (Secreted ProteinAcidic and Rich in Cysteine) is a prototype of a family of biologically active glycoproteins that bind to cells and to extracellular matrix (ECM) components. It is expressed spatially and temporally during embryogenesis, tissue remodeling and repair. SPARC is a modular protein (34 kDa) comprised of three structural domains, one or more of which are implicated in the regulation of cell adhesion, proliferation, matrix synthesis/turnover. Rapid proteolysis of SPARC by extracellular proteases accounts for its transient detection in the extracellular environment. The proposed roles of SPARC in the development of cataracts and the regulation of angiogenesis during wound healing and tumor growth account for the recent attention it has received from the biomedical community.     

Adaptor protein GULP is involved in stabilin-1-mediated phagocytosis

The clearance of apoptotic cells is critical during cellular homeostasis as well as inflammation resolution. Recently, we found that stabilin-1 is a phagocytic receptor that is involved in the clearance of apoptotic cells. However, the downstream signaling pathway of stabilin-1-mediated phagocytosis remains to be investigated. Here we identify that GULP is able to specifically interact with the NPxF/Y motif of stabilin-1 cytoplasmic region. The PTB domain of GULP is necessary for interaction with stabilin-1. GULP is enriched around PS-coated beads for phagocytosis and co-localized with stabilin-1. Downregulation of endogenous GULP expression decreased stabilin-1-mediated phagocytosis. Thus, these results indicate that GULP functions as an adaptor protein for stabilin-1-mediated phagocytosis.     

LRP-1--CD44, a new cell surface complex regulating tumor cell adhesion

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.     

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.     

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.     

Induction of endothelial cell activation by a triple helical alpha2beta integrin ligand, derived from type I collagen alpha1(I)496-507

Endothelial cell activation involves the elevated expression of cell adhesion molecules, chemoattractants, chemokines, and cytokines. These expression profiles may be regulated by integrin-mediated cell signaling pathways. In the current study, an alpha2beta1 integrin triple helical peptide ligand derived from type I collagen residues alpha1(I)496-507 was examined for induction of human aortic endothelial cell (HAEC) activation. In addition, a "miniextracellular matrix" composed of a mixture of the alpha1(I)496-507 ligand and a second, alpha-helical ligand incorporating the endothelial cell proliferating region of SPARC (secreted protein acidic and rich in cysteine) was studied for induction of HAEC activation. Following HAEC adhesion to alpha1(I)496-507, mRNA expression of E-selectin-1, vascular and intercellular cell adhesion molecules-1, and monocytic chemoattractant protein-1 was stimulated, whereas that of endothelin-1 was inhibited. Enzyme-linked immunosorbent assay analysis demonstrated that E-selectin-1 and monocytic chemoattractant protein-1 expression was also stimulated, whereas endothelin-1 protein expression diminished. Engagement of the alpha2beta1 integrin initiated a HAEC response similar to that of tumor necrosis factor-alpha-induced HAECs but was not sufficient to induce an inflammatory response. Addition of the SPARC119-122 region had only a slight effect on HAEC activation. Other cell-extracellular matrix interactions appear to be required to elicit an inflammatory response. The alpha2beta1 integrin specific triple helical peptide ligand described herein represents a more general in vitro model system by which gene expression and protein production profiles induced by binding to a single cellular receptor type can be quantified.     

Cross talk between engulfment receptors stabilin-2 and integrin αvβ5 orchestrates engulfment of phosphatidylserine-exposed erythrocytes

Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.     

Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix-associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet-derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C-terminal Ca2+-binding region of the protein. We also report that SPARC arrests monomeric collagen-supported smooth muscle cell proliferation in the late G1-phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin-dependent kinase inhibitors. Cyclin-dependent kinase-2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet-derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin-dependent kinase-2 activity.     

A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response

Lumican is an extracellular matrix protein modified as a proteoglycan in some tissues. The core protein with leucine-rich repeats, characteristic of the leucine-rich-repeat superfamily, binds collagen fibrils and regulates its structure. In addition, we believe that lumican sequestered in the pericellular matrix interacts with cell surface proteins for specific cellular functions. Here we show that bacterial lipopolysaccharide sensing by the Toll-like receptor 4 signaling pathway and innate immune response is regulated by lumican. Primary cultures of lumican-deficient (Lum(-/-)) macrophages show impaired innate immune response to lipopolysaccharides with lower induction of tumor necrosis factor alpha (TNFalpha) and interleukin-6. Macrophage response to other pathogen-associated molecular patterns is not adversely affected by lumican deficiency, suggesting a specific role for the lumican core protein in the Toll-like receptor 4 pathway. An exogenous recombinant lumican core protein increases lipopolysaccharide-mediated TNFalpha induction and partially rescues innate immune response in Lum(-/-) macrophages. We further show that the core protein binds lipopolysaccharide. Immunoprecipitation of lumican from peritoneal lavage co-precipitates CD14, a cell surface lipopolysaccharide-binding protein that is involved in its presentation to Toll-like receptor 4. The Lum(-/-) mice are hypo-responsive to lipopolysaccharide-induced septic shock, with poor induction of pro-inflammatory cytokines, TNFalpha, and interleukins 1beta and 6 in the serum. Taken together, the data indicates a novel role for lumican in the presentation of bacterial lipopolysaccharide to CD14 and host response to this bacterial endotoxin.     

Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape

Using synthetic peptides, we have identified two distinct regions of the glycoprotein SPARC (Secreted Protein Acidic and Rich in Cysteine) (osteonectin/BM-40) that inhibit cell spreading. One of these sites also contributes to the affinity of SPARC for extracellular matrix components. Peptides representing subregions of SPARC were synthesized and antipeptide antibodies were produced. Immunoglobulin fractions of sera recognizing an NH2-terminal peptide (designated 1.1) blocked SPARC- mediated anti-spreading activity. Furthermore, when peptides were added to newly plated endothelial cells or fibroblasts, peptide 1.1 and a peptide corresponding to the COOH terminal EF-hand domain (designated 4.2) inhibited cell spreading in a dose-dependent manner. These peptides exhibited anti-spreading activity at concentrations from 0.1 to 1 mM. The ability of peptides 1.1 and 4.2 to modulate cell shape was augmented by an inhibitor of protein synthesis and was blocked by specific antipeptide immunoglobulins. In addition to blocking cell spreading, peptide 4.2 competed for binding of [125I]SPARC and exhibited differential affinity for extracellular matrix molecules in solid-phase binding assays. The binding of peptide 4.2 to matrix components was Ca+(+)-dependent and displayed specificities similar to those of native SPARC. These studies demonstrate that both anti- spreading activity and affinity for collagens are functions of unique regions within the SPARC amino acid sequence. The finding that two separate regions of the SPARC protein contribute to its anti-spreading activity lead us to propose that multiple regions of the protein act in concert to regulate the interactions of cells with their extracellular matrix.     

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes

Ecto-F₁-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F₁-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y₁₃-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F₁-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F₁-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F₁-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes.This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.     

Suppression of IL-12 production by soluble CD40 ligand: evidence for involvement of the p44/42 mitogen-activated protein kinase pathway

IL-12 is a key cytokine in skewing immune responses toward Th1-like reactions. Human monocytes/macrophages produce high amounts of bioactive IL-12 when a priming signal (IFN-gamma or GM-CSF) precedes a second signal (e.g., LPS). We and others have previously shown that preincubation with LPS before this stimulation procedure can efficiently and selectively suppress the production of IL-12 by human monocytes. In this study, we show that an almost complete suppression of IL-12 production can also be observed after preincubation of monocytes with costimulatory cell surface molecules that bind to members of the TNFR superfamily (CD40 ligand, TNF-related activation-induced cytokine (TRANCE)). The suppression of IL-12 was observable on the mRNA and protein levels and was not due to endogenous production of known IL-12 antagonists (i.e., IL-10, IL-4, and PGE(2)), to an increased number of cells undergoing apoptosis, nor to down-regulation of the IFN-gamma or CD40 receptor. Cell surface expression of the costimulatory molecules CD80 and CD86 was not reduced by the preincubation procedure, and only a moderate reduction of IL-6 production was observed. Several studies have identified signal transduction pathways that are activated by CD40 signaling, including activation of mitogen-activated protein kinases. The presence of the extracellular signal-related kinase-specific mitogen-activated protein kinase kinase 1/2-specific inhibitors PD98059 and U0126 abrogated suppression induced by sCD40 ligand or other second signals. This indicates that activation of extracellular signal-regulated kinase 1/2 contributes to the underlying mechanism of IL-12 suppression. This mechanism may be relevant in other inflammatory responses and may help to develop therapeutic strategies in Th1-mediated diseases.