Surface Of Several Cell Types Research Articles

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Blocking GARP-mediated activation of TGF-β1 did not alter innate or adaptive immune responses to bacterial infection or protein immunization in mice

Transmembrane protein GARP binds latent TGF-β1 to form GARP:(latent)TGF-β1 complexes on the surface of several cell types including Tregs, B-cells, and platelets. Upon stimulation, these cells release active TGF-β1. Blocking TGF-β1 activation by Tregs with anti-GARP:TGF-β1 mAbs overcomes resistance to PD1/PD-L1 blockade and induces immune-mediated regressions of murine tumors, indicating that Treg-derived TGF-β1 inhibits anti-tumor immunity. TGF-β1 exerts a vast array of effects on immune responses. For example, it favors differentiation of TH17 cells and B-cell switch to IgA production, two important processes for mucosal immunity. Here, we sought to determine whether treatment with anti-GARP:TGF-β1 mAbs would perturb immune responses to intestinal bacterial infection. We observed no aggravation of intestinal disease, no systemic dissemination, and no alteration of innate or adaptative immune responses upon oral gavage of C. rodentium in highly susceptible Il22r−/− mice treated with anti-GARP:TGF-β1 mAbs. To examine the effects of GARP:TGF-β1 blockade on Ig production, we compared B cell- and TH cell- responses to OVA or CTB protein immunization in mice carrying deletions of Garp in Tregs, B cells, or platelets. No alteration of adaptive immune responses to protein immunization was observed in the absence of GARP on any of these cells. Altogether, we show that antibody-mediated blockade of GARP:TGF-β1 or genetic deletion of Garp in Tregs, B cells or platelets, do not alter innate or adaptive immune responses to intestinal bacterial infection or protein immunization in mice. Anti-GARP:TGF-β1 mAbs, currently tested for cancer immunotherapy, may thus restore anti-tumor immunity without severely impairing other immune defenses.PrécisImmunotherapy with GARP:TGF-β1 mAbs may restore anti-tumor immunity without impairing immune or inflammatory responses required to maintain homeostasis or host defense against infection, notably at mucosal barriers.

Junctional adhesion molecule-A on dendritic cells regulates Th1 differentiation

The junctional adhesion molecule-A (JAM-A) is an adhesion molecule present in the surface of several cell types, such as endothelial cells and leukocytes as well as Dendritic Cells (DC). Given the potential relevance of JAM-A in diverse pathological conditions such as inflammatory diseases and cancer, we investigated the role of JAM-A in CD4+ T cell priming. We demonstrate that JAM-A is present in the immunological synapse formed between T cells and DC during priming. Furthermore, an antagonistic anti-JAM-A mAb could disrupt the interaction between CD4+ T cell and DC. Antagonism of JAM-A also attenuated T cell activation and proliferation with a decrease in T-bet expression and increased IL-6 and IL-17 secretion. These findings demonstrate a functional role for JAM-A in interactions between CD4+ T cells and DCs during T cell priming as a positive regulator of Th1 differentiation.

Plasminogen‐induced foam cell formation by macrophages occurs through a histone 2B (H2B)‐PAR1 pathway and requires integrity of clathrin‐coated pits

ObjectivePlasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease‐activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen‐mediated FCF by macrophages and if PARs are involved in this process. Approach and ResultsTreating macrophages with plasminogen increases their oxidized low‐density lipoprotein uptake and plasminogen‐mediated foam cell formation (Plg‐FCF) significantly. The magnitude of Plg‐FCF correlates with cell‐surface expression of the H2B level. H2B blockade or downregulation reduces Plg‐FCF, whereas its overexpression or high endogenous levels increases Plg‐FCF. Modulating PAR1 level in mouse macrophages affects Plg‐FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin‐coated pits integrity reduces Plg‐FCF. ConclusionOur data indicate that the magnitude of Plg‐FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin‐coated pits is required for the full effect of Plg‐induced FCF.

Macrophage ICAM-1 functions as a regulator of phagocytosis in LPS induced endotoxemia.

ObjectiveIntracellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein belonging to the immunoglobulin superfamily, plays a critical role in mediating cell–cell interaction and outside-in cell signaling during the immune response. ICAM-1 is expressed on the cell surface of several cell types including endothelial cells, epithelial cells, leucocytes, fibroblasts, and neutrophils. Despite ICAM-1 has been detected on macrophage, little is known about the function and mechanism of macrophage ICAM-1.MethodsTo investigate the role of lipopolysaccharide (LPS) in ICAM-1 regulation, both the protein and cell surface expression of ICAM-1 were measured. The phagocytosis of macrophage was evaluated by flow cytometry and Confocal microscopy. Small interfering RNA and neutralizing antibody of ICAM-1 were used to assess the effect of ICAM-1 on macrophage phagocytosis. TLR4 gene knockout mouse and cytoplasmic and mitochondrial ROS scavenger were used for the regulation of ICAM-1 expression. ROS was determined using flow cytometry.ResultsIn this study, we reported that macrophage can be stimulated to increase both the protein and cell surface expression of ICAM-1 by LPS. Macrophage ICAM-1 expression was correlated with enhanced macrophage phagocytosis. We found that using ICAM-1 neutralizing antibody or ICAM-1 silencing to attenuate the function or expression of ICAM-1 could decrease LPS-induced macrophage phagocytosis. Furthermore, we found that knocking out of TLR4 led to inhibited cytoplasmic and mitochondrial ROS production, which in turn, attenuated ICAM-1 expression at both the protein and cell surface levels.ConclusionThis study demonstrates that the mechanism of ICAM-1-mediated macrophage phagocytosis is depending on TLR4-mediated ROS production and provides significant light on macrophage ICAM-1 in endotoxemia.

Targeting the PD-1 Pathway in MSI-Stable Metastatic Colorectal Cancer

In patients with microsatellite instability-high (MSI-H) metastatic colorectal cancer (CRC), the inhibition of programmed death-1 (PD-1) pathway has achieved promising response [1]. PD-1 is an immune inhibitory receptor, expressed in many cells, including T cells. Its ligand, PD-L1, is expressed on surface of several cell types, especially tumor cells. When PD-L1 binds to PD-1, an inhibitory signal is transmitted into the T cell, which suppresses T-cell proliferation. MSIH metastatic CRC gives rise to high percentage of mutations which is proportional to mutational load. High mutational load of MSI-H CRC correlates with increased PD-L1 expression which indicates a higher likelihood of response to PD-1 inhibitors, compared to microsatellite instability-stable (MSI-S) CRC [2-4]. Нus, MSI-H CRC could respond to single agent PD-1 pathway inhibition.

α-Enolase, a Multifunctional Protein: Its Role on Pathophysiological Situations

α-Enolase is a key glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells and is considered a multifunctional protein. α-enolase is expressed on the surface of several cell types, where it acts as a plasminogen receptor, concentrating proteolytic plasmin activity on the cell surface. In addition to glycolytic enzyme and plasminogen receptor functions, α-Enolase appears to have other cellular functions and subcellular localizations that are distinct from its well-established function in glycolysis. Furthermore, differential expression of α-enolase has been related to several pathologies, such as cancer, Alzheimer's disease, and rheumatoid arthritis, among others. We have identified α-enolase as a plasminogen receptor in several cell types. In particular, we have analyzed its role in myogenesis, as an example of extracellular remodelling process. We have shown that α-enolase is expressed on the cell surface of differentiating myocytes, and that inhibitors of α-enolase/plasminogen binding block myogenic fusion in vitro and skeletal muscle regeneration in mice. α-Enolase could be considered as a marker of pathological stress in a high number of diseases, performing several of its multiple functions, mainly as plasminogen receptor. This paper is focused on the multiple roles of the α-enolase/plasminogen axis, related to several pathologies.

Caveolin-1: A New Locus for Human Lipodystrophy

Caveolae are specialized plasma membrane microdomains appearing as 50- to 100-nm vesicular invaginations (1,2). These organelles were initially identified by Yamada (3) more than 50 yr ago and can be found individually or in clusters at the surface of several cell types, including adipocytes, endothelial cells, fibroblasts, and myocytes. These plasma membrane domains, often referred to as lipid rafts, are rich in cholesterol, glycosphingolipids, and signaling proteins, especially caveolins. Caveolins are major components of the caveolae, and three members of this family (caveolins 1, 2, and 3) have been identified.

Antigen-presenting properties of gingival fibroblasts in chronic adult periodontitis.

Chronic periodontitis is characterized by dense infiltrations of T lymphocytes in the connective tissue, which consists mainly of gingival fibroblasts. It is becoming increasingly clear that T lymphocytes and gingival fibroblasts are capable of influencing each other. For example, the T cell cytokine interferon-gamma (IFN-gamma) is able to induce MHC class II molecules on the surface of several cell types, including gingival fibroblasts. Histological sections of chronically inflamed gingival tissue showed a great number of CD4+ and CD8+ T cells that produced IFN-gamma, and in addition showed abundant expression of MHC class II molecules on gingival fibroblasts. Therefore, we investigated whether these gingival fibroblasts acquire the capacity to carry out MHC class II-restricted functions such as antigen presentation to local T cells. In this study, we show that IFN-gamma-treated gingival fibroblasts were able to function as antigen-presenting cells (APC) for superantigen-mediated T cell proliferation. However, these fibroblasts failed to present whole-cell antigens of periodontitis-associated bacteria. Moreover, gingival fibroblasts inhibited the presentation of the whole-cell antigens of these bacteria by professional APC. This inhibition could be overcome by the addition of IL-2. These results suggest that gingival fibroblasts play an important role in the local specific immune response in chronic inflammatory periodontal lesions by regulating the response of infiltrating T cells.

The F 1F 0 ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study

F 1F 0 ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F 0 subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.

Display of Fas ligand protein on cardiac vasculature as a novel means of regulating allograft rejection.

Fas ligand (FasL) is a potent death-inducing molecule with important functions in immune homeostasis and tolerance to self-antigens. The complex biological activities of FasL and its inefficient expression using conventional gene transfer approaches limit its use for immunomodulation to prevent allograft rejection. We have recently generated a chimeric FasL with core streptavidin (SA-FasL) with potent apoptotic activity and designed a novel approach to display it on the surface of several cell types via biotinylation. We herein tested whether SA-FasL can also be displayed on vascular endothelial cells in the heart and examined its effect on graft survival after transplantation into syngeneic and allogeneic hosts. SA-FasL was efficiently displayed on the vasculature of BALB/c hearts with a half-life of 9 days in vivo. Transplantation of hearts displaying SA-FasL into syngeneic hosts resulted in indefinite graft survival without detectable toxicity to the grafts and hosts. In contrast, transplantation of allogeneic C57BL/10 hearts displaying SA-FasL into BALB/c recipients resulted in graft rejection, but in a delayed fashion as compared with control hearts (mean survival time=17.4+/-5 versus 9.6+/-1 days). Allograft survival was further extended to 21+/-2.6 and 24+/-3 days (P<0.05) by intravenous treatment of graft recipients with 1 dose of SA-FasL-decorated donor splenocytes on days 2 and 6 after transplantation, respectively. This study shows for the first time that exogenous proteins can be displayed on the endothelium of solid organs for therapeutic purposes. This approach provides a convenient and rapid means of displaying exogenous proteins on the surface of cells, tissues, and solid organs, with broad research and therapeutic implications.

The Cleavage of the Urokinase Receptor Regulates Its Multiple Functions

The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88-92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88-92 (P88-92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88-92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88-92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88-92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions.

Cell Surface Targeting of Pregnancy-associated Plasma Protein A Proteolytic Activity: REVERSIBLE ADHESION IS MEDIATED BY TWO NEIGHBORING SHORT CONSENSUS REPEATS

The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

Initial Characterization of the Vitamin D Binding Protein (Gc-Globulin) Binding Site on the Neutrophil Plasma Membrane: Evidence for a Chondroitin Sulfate Proteoglycan

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.

Tetraspanin CD9 Is Associated With Very Late–Acting Integrins in Human Vascular Smooth Muscle Cells and Modulates Collagen Matrix Reorganization

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

Lex glycosphingolipids-mediated cell aggregation.

Glycoconjugates bearing oligosaccharide Lex, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->3R, are found on the surface of several cell types. Although recent studies have indicated that Lexon both glycosphingolipids (GSL) and polylactosaminoglycans can mediate under certain experimental conditions Lex-Lexinteractions, cell-cell interactions based exclusively on LexGSLs have not been demonstrated. In this study we show that preincubation of nonaggregating rat basophilic leukemia (RBL) cells with purified LexGSLs resulted in incorporation of the GSLs into plasma membrane, as determined by immunostaining, and formation of aggregates in the presence of Ca2+; no aggregates were formed after preincubation of the cells with globoside or sphingomyelin. Lex-mediated aggregation was inhibited by removal of Ca2+or by addition of lactofucopentaose III but not by lactose or lacto-N-fucopentaose II. In a mixture of Lex-positive and Lex-negative RBL cells most of the aggregates were composed exclusively of Lex-positive cells. The combined data suggest that interactions between LexGSL on opposite cell surfaces are strong enough to allow formation of stable cell-cell contacts.

Antigen-presenting properties of gingival fibroblasts in chronic adult periodontitis

Chronic periodontitis is characterized by dense infiltrations of T lymphocytes in the connective tissue, which consists mainly of gingival fibroblasts. It is becoming increasingly clear that T lymphocytes and gingival fibroblasts are capable of influencing each other. For example, the T cell cytokine interferon-gamma (IFN-gamma) is able to induce MHC class II molecules on the surface of several cell types, including gingival fibroblasts. Histological sections of chronically inflamed gingival tissue showed a great number of CD4+ and CD8+ T cells that produced IFN-gamma, and in addition showed abundant expression of MHC class II molecules on gingival fibroblasts. Therefore, we investigated whether these gingival fibroblasts acquire the capacity to carry out MHC class II-restricted functions such as antigen presentation to local T cells. In this study, we show that IFN-gamma-treated gingival fibroblasts were able to function as antigen-presenting cells (APC) for superantigen-mediated T cell proliferation. However, these fibroblasts failed to present whole-cell antigens of periodontitis-associated bacteria. Moreover, gingival fibroblasts inhibited the presentation of the whole-cell antigens of these bacteria by professional APC. This inhibition could be overcome by the addition of IL-2. These results suggest that gingival fibroblasts play an important role in the local specific immune response in chronic inflammatory periodontal lesions by regulating the response of infiltrating T cells.

SYNDECANS, CELL SURFACE HEPARAN SULFATE PROTEOGLYCANS, ARE PRESENT IN AND CAN MODIFY THE PROTEOLYTIC BALANCE OF TRACHEAL ASPIRATES. † 1996

An imbalance between proteinases and antiproteinases is thought to mediate, in part, the inflammatory injury that leads to chronic lung disease in mechanically ventilated pre-term infants. The activities of some proteinases and proteinase inhibitors found in inflammatory fluids can be modifiedin vitro by heparin, a mast cell-derived glycosaminoglycan (GAG). The major cellular source of heparin-like GAG is the syndecan family of transmembrane heparan sulfate proteoglycans, found on the surface of several cell types in the lung.

Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia

The development and regeneration of the peripheral nervous system (PNS) is highly dependent on the migration of Schwann cells and the extension of axons toward their distant targets. Plasminogen activators (PAs) are associated with the surface of several cell types of neural origin where they are believed to mediate localized degradation of extracellular matrix, thus facilitating cell motility. In this study, we characterize the expression of tissue-type (tPA) and urokinase (uPA) PAs, as well as the urokinase cell surface receptor (uPAR) during differentiation of cultured cells from mouse dorsal root ganglia. During the first day in culture, the mRNA levels of all three components increase from 75- to 163-fold, as shown using a quantitative PCR method. By 72 hr, the mRNA levels decrease and approach basal levels. This transient increase is in direct correlation with the differentiation of neurons and Schwann cells and the formation of a neuritic network in these regenerating cultures. Densitometric analysis of gel zymographs demonstrates that the elevation in mRNA levels is accompanied by similar increases in the activity levels of tPA and uPA. Interestingly, in situ hybridization studies of the cultures show that tPA mRNA is restricted to small sensory neurons, whereas uPA mRNA is localized predominantly in large sensory neurons. uPAR mRNA is expressed by both neuronal subpopulations and, to a lesser extent, by Schwann cells and fibroblasts. Taken together, these results further support a role for the PA system in facilitating axon extension and cell migration during development and regeneration of the PNS.

The mouse gene coding for high mobility group 1 protein (HMG1).

We have isolated an active gene encoding the mouse HMG1 protein among a multitude of cross-hybridizing sequences, which most likely are retrotransposed pseudogenes. The hmg1 gene contains five exons, of which the first is not translated, and the last contains a long 3'-untranslated sequence and three alternative polyadenylation sites. We found no evidence for a sequence encoding a membrane localization signal in the hmg1 gene, despite the presence of HMG1 protein on the surface of several cell types. The hmg1 promoter coincides with a CpG island, contains no TATA sequence, and derives the expression of reporter genes placed under its control. The hmg1 gene may be a member of a family of closely related genes but appears to be the major or the only active gene coding for HMG1 protein.