RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

ESCRT machinery components are required for Orthobunyavirus particle production in Golgi compartments.

Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.

Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells.

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

Mast Cells Interact with Endothelial Cells to Accelerate In Vitro Angiogenesis.

Angiogenesis is a complex process that involves interactions between endothelial cells and various other cell types as well as the tissue microenvironment. Several previous studies have demonstrated that mast cells accumulate at angiogenic sites. In spite of the evidence suggesting a relationship between mast cells and angiogenesis, the association of mast cells and endothelial cells remains poorly understood. The present study aims to investigate the relationship between mast cells and endothelial cells during in vitro angiogenesis. When endothelial cells were co-cultured with mast cells, angiogenesis was stimulated. Furthermore, there was direct intercellular communication via gap junctions between the two cell types. In addition, the presence of mast cells stimulated endothelial cells to release angiogenic factors. Moreover, conditioned medium from the co-cultures also stimulated in vitro angiogenesis. The results from this investigation demonstrate that mast cells have both direct and indirect proangiogenic effects and provide new insights into the role of mast cells in angiogenesis.

Recombinant ArtinM activates mast cells.

BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines.

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators.

Differential effects of chemoattractants on mast cell recruitment in vivo.

Rats were injected with rat recombinant (rr) IL3, rrSCF, rrIL-3+rrSCF, rrRANTES and LTB4. Six hours after subcutaneous injection of rrIL-3 or rrIL-3+rrSCF, there was an increase in mast cell numbers in the skin and spleen. Peritoneal mast cells were recruited following i.p. injection of rrIL-3, but with rrIL-3+rrSCF recruitment was delayed. Immunostaining with a mast cell specific antibody showed that immature orthochromatic mast cells were being recruited. rrIL-3 induced recruitment of mast cells to the peritoneal cavity was blocked by anti-integrin antibodies. Mast cell recruitment depended on the target tissue and the time of exposure to the chemoattractant.

Tyrosines in the carboxyl terminus regulate Syk kinase activity and function.

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcepsilonRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcepsilonRI-induced degranulation, nuclear factor for T cell activation and NFkappaB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.

The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, ß-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

Relative expression of KLK5 to LEKTI is associated with aggressiveness of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a challenging cancer to treat despite all the advances of the last 50 years. Kallikrein 5 (KLK5) is among the serine proteases implicated in OSCC development. However, whether the activity of KLK5 promotes carcinogenesis is still controversial. Moreover, knowledge regarding the role of the KLK5 cognate inhibitor, Lympho-Epithelial Kazal-Type related Inhibitor (LEKTI), in OSCC is scarce. We have, thus, sought to investigate the importance of KLK5 and LEKTI expression in premalignant and malignant lesions of the oral cavity. METHODS: KLK5 and LEKTI protein expression was evaluated in 301 human samples, which were comprised of non-malignant and malignant lesions of the oral cavity. Moreover, a bioinformatic analysis of the overall survival rate from 517 head and neck squamous cell carcinoma (HNSCC) samples was performed. Additionally, to mimic the uncovered KLK5 to serine peptidase inhibitor (SPINK5) imbalance, the KLK5 gene was abrogated in an OSCC cell line using CRISPR-Cas9 technology. The generated cell line was then used for in vivo and in vitro carcinogenesis related experiments. RESULTS: LEKTI was found to be statistically downregulated in OSCCs, with increased KLK5/SPINK5 mRNA ratio being associated with a shorter overall survival (p = 0.091). Indeed, disruption of KLK5 to SPINK5 balance through the generation of KLK5 null OSCC cells led to smaller xenografted tumors and statistically decreased proliferation rates following multiple time points of BrdU treatment in vitro. CONCLUSION: The association of increased enzyme/inhibitor ratio with poor prognosis indicates KLK5 to SPINK5 relative expression as an important prognostic marker in OSCC.

Evidences of a role for eukaryotic translation initiation factor 5A (eIF5A) in mouse embryogenesis and cell differentiation.

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors.

BACKGROUND: Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. RESULTS: Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. CONCLUSIONS: In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

The lectin ArtinM activates RBL-2H3 mast cells without inducing degranulation.

Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells.

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcvarepsilonRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcvarepsilonRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcvarepsilonRI, LAT and alpha-galactosyl derivatives of ganglioside GD(1b) mobilized to lipid raft domains following FcvarepsilonRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of beta-hexosaminidase activity after FcvarepsilonRI activation. The two mutant cell lines have a reduced release of beta-hexosaminidase activity after FcvarepsilonRI stimulation, but not after exposure to calcium ionophore. These results indicate that the alpha-galactosyl derivatives of ganglioside GD(1b) are important in the initial events of FcvarepsilonRI signaling upstream of Ca2+ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcvarepsilonRI.

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach.

BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma. OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides. METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program. RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6. CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

The low affinity IgG receptor Fc gamma RIIB contributes to the binding of the mast cell specific antibody, mAb BGD6.

The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of approximately 110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to Fc gamma RIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab')(2) fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to Fc gamma RIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab')(2) fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have Fc gamma RIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a approximately 110 kDa protein and the Fc tail interacts with Fc gamma RIIB (CD32).

Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits.

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and ß1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis.

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan.

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.