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PubMed2003

Changes in gene expression induced by H(2)O(2) in cardiac myocytes.

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.

Animals↗

PubMed2003

Activation of terminal B cell differentiation by inhibition of histone deacetylation.

A role for histone acetylation, which can alter the accessibility of DNA to transcriptional regulatory proteins and contribute to gene expression, in regulating terminal B cell differentiation was investigated in the mature B lymphoma L10A and mouse splenic B cells. Incubation of the L10A cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and butyrate increased expression of Blimp-1, J chain, and mad genes, decreased expression of c-myc and BSAP/Pax-5 genes, increased the expression of surface CD43 and Syndecan-1, and decreased surface IgM. Incubation of splenic B cells with TSA and dextran conjugated anti-IgD Ab increased Blimp-1 gene and Syndecan-1 surface expression. The alteration in gene expression and cell surface markers was consistent with induction of the onset of terminal B cell differentiation. Co-incubation of L10A cells with TSA and cycloheximide (CHX) abrogated the up-regulation of Blimp-1 expression, indicating that TSA-activated Blimp-1 expression required synthesis of a transcriptional activator. In contrast, mad expression was increased in L10A cells cultured with TSA and cycloheximide or cycloheximide alone, suggesting mad expression may occur independent of Blimp-1 expression and is regulated by a labile, HDAC associated transcriptional repressor. The results demonstrate that histone acetylation regulates transcription of genes controlling terminal B cell differentiation.

Animals↗

PubMed1995

Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans.

We showed previously that the synthesis of heparan sulfate on betaglycan occurs at a Ser-Gly dipeptide flanked by a cluster of acidic residues and an adjacent tryptophan (Zhang, L., and Esko, J.D. (1994) J. Biol. Chem. 269, 19295-19299). A survey of the protein data base revealed that most heparan sulfate proteoglycans contain repetitive (Ser-Gly)n segments (n = 2) and a nearby cluster of acidic residues. To study the role of these amino acid sequences in controlling heparan sulfate synthesis, we have examined the assembly of glycosaminoglycans on Chinese hamster ovary (CHO) cell syndecan-1. The glycosylation sites were mapped by making chimeric proteoglycans containing segments of CHO syndecan-1 cDNA fused to Protein A. Two sites near the transmembrane domain (-EGS205GEQ- and -ETS215GEN-) were used solely for chondroitin sulfate synthesis, whereas three sites near the N terminus (-DGS35GDDSDNFS45GS47GTG-) supported both heparan sulfate and chondroitin sulfate synthesis. The strongest sites for heparan sulfate synthesis consisted of the repeat unit, -S45GS47G-. An unusual coupling phenomenon occurred across the adjacent SG dipeptides, leading to a greater proportion of heparan sulfate than predicted by the behavior of each site acting independently. The clusters of acidic residues adjacent to the heparan sulfate sites play important roles as well. These sequence motifs suggest a set of rules for predicting whether heparan sulfate assembles at glycosylation sites in proteoglycan core proteins.

Amino Acid Sequence↗

PubMed2003

Biological activities of homologous loop regions in the laminin alpha chain G domains.

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.

Actins↗

PubMed2004

Heparan sulfate structure in mice with genetically modified heparan sulfate production.

Using a high throughput heparan sulfate (HS) isolation and characterization protocol, we have analyzed HS structure in several tissues from mice/mouse embryos deficient in HS biosynthesis enzymes (N-deacetylase/N-sulfotransferase (NDST)-1, NDST-2, and C5-epimerase, respectively) and in mice lacking syndecan-1. The results have given us new information regarding HS biosynthesis with implications on the role of HS in embryonic development. Our main conclusions are as follows. 1) The HS content, disaccharide composition, and the overall degree of N- and O-sulfation as well as domain organization are characteristic for each individual mouse tissue. 2) Removal of a key biosynthesis enzyme (NDST-1 or C5-epimerase) results in similar structural alterations in all of the tissues analyzed. 3) Essentially no variation in HS tissue structure is detected when individuals of the same genotype are compared. 4) NDST-2, although generally expressed, does not contribute significantly to tissue-specific HS structures. 5) No change in HS structure could be detected in syndecan-1-deficient mice.

Amidohydrolases↗

PubMed2004

Dermatan sulfate proteoglycan and glycosaminoglycan synthesis is induced in fibroblasts by transfer to a three-dimensional extracellular environment.

Composition and architecture of the extracellular matrix dictate cell behavior. Proteoglycans bind multiple components of the extracellular matrix by serving as important regulators of cell behavior. Given the influence of culture architecture on cell function, we investigated whether switching NIH3T3 fibroblasts from growth on type 1 collagen in monolayer to a collagen gel might influence dermatan sulfate expression. Immunofluorescent staining, immunoblot, and Western blot demonstrated an induction in decorin expression in cells switched to collagen gels. This induction was associated with a 40-fold increase in decorin transcript expression determined by quantitative real time PCR. Disaccharide analysis of extracted glycosaminoglycans from collagen gels showed an increase in total glycosaminoglycan and in the ratio of chondroitin sulfate to heparan sulfate compared with monolayer culture. The ratio of chondroitin sulfate to heparan sulfate likewise increased on syndecan-1 from gel culture. Digestion with chondroitinase B showed that this induced chondroitin sulfate was dermatan sulfate. Syndecan-1 extracted from wounded mouse skin also displayed an increase in dermatan sulfate synthesis compared with unwounded skin. Furthermore, glycosaminoglycans from collagen gel culture activated keratinocyte growth factor, whereas glycosaminoglycans from monolayer culture lacked this ability. These findings suggest that regulation of dermatan sulfate and dermatan sulfate proteoglycan is dependent on extracellular matrix architecture. The ability of collagen gel culture to mimic better the in vivo dermal environment may be due in part to this influence on dermatan sulfate and dermatan sulfate proteoglycan synthesis.

Animals↗

PubMed2002

Proteolysis-inducing factor differentially influences transcriptional regulation in endothelial subtypes.

Proteolysis-inducing factor (PIF) is a novel sulfated glycoprotein initially identified as a protein capable of triggering muscle proteolysis during the process of cancer cachexia. Only skeletal muscle and liver exhibit substantial binding of PIF in adult tissue. Here, we demonstrate that PIF induces transcriptional regulation in both the liver endothelial cell line SK-HEP-1 and in human umbilical vein endothelial cells (HUVECs) but not in pulmonary artery endothelial cells. PIF differentially induces activation of nuclear factor-kappaB, resulting in the induction of proinflammatory cytokines [interleukin (IL)-8 and IL-6] and increased expression of the cell surface proteins intercellular adhesion molecule-1 and vascular cell adhesion molecule in SK-HEP-1 and HUVECs only. In addition, PIF induces the shedding of syndecans from the cell surface. Syndecans are involved in wound repair, metastasis of cancers, and embryonic development. These results suggest that PIF may play additional roles in the proinflammatory response observed in cancer cachexia but may also have a role without the cachectic process.

Blood Proteins↗

PubMed2001

Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis.

In this study, flow cytometry was used to evaluate interleukin-6 (IL-6) production by bone marrow mononuclear cells from 47 patients with multiple myeloma (MM) in different clinical stages and 15 patients with monoclonal gammopathy of undetermined significance. In patients with MM, autocrine IL-6 production paralleled the clinical disease stage. The largest proportion of syndecan-1(+)/IL-6(+) cells was detected in patients with resistant relapse or primary refractory disease, suggesting that tumor progression involves expansion of myeloma cells producing IL-6. The authors assessed autocrine IL-6 production and in vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970, which showed different sensitivities to the addition of exogenous IL-6. Autocrine IL-6 production was observed in IL-6-independent MCC-2, MCC-3, and MCC-5 cloned from patients with aggressive disease and in the IM-9 cell line. In contrast, IL-6-dependent MCC-1, MCC-4, and MCC-6 were syndecan-1(+) and IL-6(-). Blocking experiments with anti-IL-6 monoclonal antibody from clone AH65, which binds IL-6-IL-6Ralpha complexes, prevented cell proliferation of IL-6(+) MCCs. Flow cytometry evaluations after propidium iodide staining revealed different susceptibilities of MCCs to cell death. IL-6-producing MCCs showed minimal spontaneous and dexamethasone-induced apoptosis, whereas a regular amplitude of apoptosis occurred in the IL-6(-) MCCs. These data provide evidence that autocrine IL-6 reflects a highly malignant phenotype of myeloma cells. In fact, autocrine IL-6 production and deregulated apoptosis may induce expansion of selective IL-6(+) myeloma cells resistant to spontaneous and drug-induced cell death.

Apoptosis↗

PubMed1995

Androgen and fibroblast growth factor (FGF) regulation of FGF receptors in S115 mouse mammary tumor cells.

We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.

Animals↗

PubMed1996

Msx1 controls inductive signaling in mammalian tooth morphogenesis.

Members of the Msx homeobox family are thought to play important roles in inductive tissue interactions during vertebrate organogenesis, but their precise developmental function has been unclear. Mice deficient for Msx1 exhibit defects in craniofacial development and a failure of tooth morphogenesis, with an arrest in molar tooth development at the E13.5 bud stage. Because of its potential for experimental manipulation, the murine molar tooth germ provides a powerful system for studying the role of Msx genes in inductive signaling during organogenesis. To further analyze the role of Msx1 in regulating epithelial-mesenchymal interactions during tooth morphogenesis, we have examined the expression of several potential Msx1 downstream genes in Msx1 mutant tooth germs and we have performed functional experiments designed to order these genes into a pathway. Our results show that expression of Bone Morphogenetic Protein 4 (BMP4), the HMG box gene Lef1 and the heparan sulfate proteoglycan syndecan-1 is specifically reduced in Msx1 mutant dental mesenchyme, while expression of the extracellular matrix protein tenascin is unaffected. BMP4 soaked beads can induce Bmp4 and Lef1 expression in explanted wild-type dental mesenchymes, but only Lef1 expression in Msx1 mutant dental mesenchyme. We thus conclude that epithelial BMP4 induces its own expression in dental mesenchyme in a manner that requires Msx1. In turn, we show that addition of BMP4 to Msx1 deficient tooth germs bypasses the requirement for Msx1 and rescues epithelial development from the bud stage to the E14.5 cap stage. Lastly, we show that FGFs induce syndecan-1 expression in dental mesenchyme in a manner that also requires Msx-1. These results integrate Msx1 into a regulatory hierarchy in early tooth morphogenesis and demonstrate that Msx1 is not only expressed in dental mesenchyme in response to epithelial signals, but also in turn regulates the reciprocal expression of inductive signals in the mesenchyme which then act back upon the dental epithelium. We propose that Msx genes function repetitively during vertebrate organogenesis to permit inductive signaling to occur back and forth between tissue layers.

Animals↗

PubMed2003

Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes.

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.

Antibodies, Monoclonal↗

PubMedDate not supplied

Distinct B-cell populations are present in hepatic and intestinal Schistosoma mansoni granulomas.

Although it is generally accepted that schistosomal granuloma formation results from a T-cell dependent host response towards the parasite egg, attention has recently been focused on the involvement of B-cells in the induction of schistosome-induced pathology. In this study we investigated the involvement of two functionally different B-cell populations in the formation of the Schistosoma mansoni granuloma: naive and antigen-stimulated B-cells. In liver granulomas two distinct B-cell populations were found, namely unstimulated B-cells at the periphery of the granuloma and antigen-stimulated, syndecan-1 positive B-cells in the inner part near the deposited egg. Intestinal granulomas differed by their relative lack of unstimulated B-cells at the granuloma periphery, but like hepatic granulomas intestinal granulomas also have syndecan-1 positive B-cells. From our results, we conclude that B-cells are important constituents of the S. mansoni granuloma.

Animals↗

PubMed2002

Neoglycans, carbodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis.

The soluble form of the syndecan-1 heparan sulfate proteoglycan acts as a tumor suppressor molecule that inhibits growth and induces apoptosis of some cancer cell lines in vitro. Analogs of syndecan-1 were produced by carbodiimide (EDAC) conjugation of glycosaminoglycan (GAG) chains to a protein scaffold, thereby generating synthetic proteoglycans that were evaluated for anticancer properties. Surprisingly, when analyzing activities of the controls, we discovered that EDAC modified GAG chains inhibit myeloma cell viability even in the absence of protein. Here, we describe the production and the activities of these novel molecules called neoglycans. The GAG chains heparin and chondroitin sulfate (CS) were exposed to EDAC to generate the neoglycans neoheparin and neoCS, respectively. Heparin and CS in the absence of EDAC modification have no effect or a slight growth promoting effect on cancer and normal cell lines. However, neoheparin and neoCS substantially reduce cell viability by induction of apoptosis of myeloma and breast cancer cells in vitro. NeoCS when injected directly into breast tumors growing in nude mice reduces or abolishes their growth without causing apparent toxicity to the adjacent normal tissue. The neoglycans need not be continuously present in cell cultures because a short pulse exposure is sufficient to reduce cell viability. NeoCS fractions purified by size exclusion chromatography reduce myeloma cell viability, confirming the specificity of neoglycan activity. Collectively, the results of this study demonstrate the anticancer activities of this new class of GAG chain-based molecules and provide the foundation for future development of neoglycans as novel therapeutic agents.

3T3 Cells↗

PubMed1997

Overexpression of B cell-specific activator protein (BSAP/Pax-5) in a late B cell is sufficient to suppress differentiation to an Ig high producer cell with plasma cell phenotype.

The B cell-specific activator protein (BSAP) is a DNA-binding transcription factor expressed in pro-B, pre-B, and mature B cells but not in plasma cells. We explored the role of BSAP in B cell function by creating clones in a late B cell and a plasma cell line transfected with a BSAP expression plasmid. We found that the plasma cell line MPC11, which does not produce BSAP, is still permissive to BSAP production driven by heterologous promoter. Overexpression of BSAP in a late B cell line (CH12.LX.A2) and a plasma cell line augmented cell proliferation and led to greater suppression of Ig synthesis in a late B cell line than in the plasma cell line. The reduction was seen mostly in synthesis of a secretory form of Ig. Overexpression of BSAP reduced Blimp-1 expression in CH12.LX.A2 clones but not in MPC11 clones. In addition, overexpression of BSAP in CH12.LX.A2 cells suppressed spontaneous appearance of cells with high Syndecan-1 expression and high amounts of intracytosolic as well as secreted Ig synthesis. To corroborate the above findings, we cloned nontransfected CH12.LX.A2 cells and found reduced BSAP mRNA expression in the high Ig-secreting clones, which produced more Blimp-1 mRNA with greater Syndecan-1 expression than the low Ig-secreting clones. Taken together, these results indicate that BSAP expression is sufficient to reduce Ig production in late B cells; this effect is mediated in part by suppression of differentiation to cells of plasma-cell phenotype.

Animals↗

PubMed2002

Augmented synthesis and differential localization of heparan sulfate proteoglycans in Duchenne muscular dystrophy.

Muscular dystrophies are characterized by continuous cycles of degeneration and regeneration that result in extensive fibrosis and a progressive diminution of muscle mass. Cell surface heparan sulfate proteoglycans are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with a great variety of ligands, including ECM constituents, adhesion molecules, and growth factors. In this study, we evaluated the expression and localization of three heparan sulfate proteoglycans in the biopsies of Duchenne muscular dystrophy (DMD) patients. Through SDS-PAGE analyses followed by specific identification of heparitinase-digested proteins with an anti-Delta-heparan sulfate specific monoclonal antibodies, we observed an increase of three forms of heparan sulfate proteoglycans, corresponding to perlecan, syndecan-3, and glypican-1. Immunohistochemistry analyses indicated a differential localization for these proteoglycans: glypican-1 and perlecan were found mainly associated to ECM structures, while syndecan-3 was associated to muscle fibers. These results suggest that the amount of specific heparan sulfate proteoglycans is augmented in skeletal muscle in DMD patients presenting a differential localization.

Child↗

PubMed2005

A proteomic approach to characterize protein shedding.

Shedding (i.e. proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fractions, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 45 membrane-associated proteins were identified including 22 that contain at least one transmembrane domain and 23 that indirectly associate with the extracellular surface of the plasma membrane. Of the 22 transmembrane proteins, 18 were identified by extracellular peptides providing strong evidence they originate from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified two proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78 kDa glucose-regulated protein and lactate dehydrogenase A. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78 kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that tandem mass spectrometry can be used to identify shed proteins and to estimate changes in protein abundance.

Biotinylation↗

PubMed1993

Purification of a basic fibroblast growth factor-binding proteoglycan from bovine cardiac plasma membrane.

A heparan sulfate proteoglycan (HSPG) from bovine cardiac plasma membrane was purified to homogeneity using either isoelectric focusing or anion-exchange chromatography, followed by affinity chromatography on immobilized basic fibroblast growth factor (bFGF). Fractions were assayed for bFGF-binding activity using 125I-bFGF as a probe. Purified proteoglycan ran as a broad band on SDS-PAGE, spanning an apparent molecular mass range of 100-200 kDa, and could be incorporated into liposomes. Digestion of radioiodinated proteoglycan with heparitinase yielded a product of 73 kDa, while digestion with chondroitinase ABC did not change the apparent molecular mass. Monoclonal antibody directed against the ectodomain of another plasma membrane HSPG, syndecan, failed to recognize the purified cardiac proteoglycan on immunoblots. We conclude that adult bovine myocardium contains a membrane-associated bFGF-binding heparan sulfate proteoglycan containing little or no chondroitin sulfate and that this HSPG may be distinct from those of the syndecan family of heparan sulfate proteoglycans.

Animals↗

PubMed2006

OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation.

The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.

Acetylcysteine↗