Heparin interaction with a receptor on hyperglycemic dividing cells prevents intracellular hyaluronan synthesis and autophagy responses in models of type 1 diabetes.

Previous studies and ongoing research indicate the importance of an interaction between a putative receptor on dividing cells in hyperglycemia and the non-reducing end motifs of heparin stored in mast cell secretory granules and how this interaction prevents activation of hyaluronan synthesis in intracellular compartments and subsequent autophagy. This suggests a new role for endosomal heparanase in exposing this cryptic motif present in the initial large heparin chains on serglycin and in the highly sulfated (NS) domains of heparan sulfate.

Preferential recognition of isocitrate dehydrogenase by a rabbit monoclonal antibody (ab124797) against the C-terminal peptide of RANKL.

A rabbit monoclonal antibody (Abcam ab124797), with high affinity for a synthetic peptide corresponding to the C-terminal region of the receptor activator of nuclear factor (NF)-κB ligand (RANKL), specifically recognizes a 37 kDa protein by immunoblotting, in good agreement with the molecular mass of RANKL. However, our mass spectroscopy analysis revealed that the protein recognized by the antibody is the α-subunit of NAD(+)-dependent isocitrate dehydrogenase (ICDH), a key Krebs cycle enzyme in mitochondria. Consistently, immunocytochemical staining with the antibody revealed a network organization characteristic of mitochondria, which overlapped with staining by MitoTracker and was lost after the siRNA-mediated downregulation of ICDH. The C-terminal peptide of ICDH contains similar chemical characteristics to that of the RANKL peptide and interacts with the antibody, although the affinity is a hundred times weaker. The present study provides an example of the preferential recognition of a surrogate protein by a rabbit monoclonal antibody.

Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed.

Expression of glucagon-like peptide-1 receptor and glucose­dependent insulinotropic polypeptide receptor is regulated by the glucose concentration in mouse osteoblastic MC3T3-E1 cells.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP­1R) are incretin receptors that play important roles in regulating insulin secretion from pancreatic ß cells. Incretin receptors are also thought to play a potential role in bone metabolism. Osteoblasts in animals and humans express GIPR; however, the presence of GLP-1R in these cells has not been reported to date. Thus, the aim of this study was to determine whether GLP-1R and GIPR are expressed in osteoblastic cells, and whether their expression levels are regulated by the extracellular glucose concentration. Mouse osteoblastic MC3T3-E1 cells were cultured in medium containing normal (5.6 mM) or high (10, 20 or 30 mM) glucose concentrations, with or without bone morphogenetic protein-2 (BMP-2). RT-PCR, western blot analysis and immunofluorescence were carried out to determine GIPR and GLP-1R mRNA and protein expression levels. Cell proliferation was also assessed. The GLP-1R and GIPR mRNA expression levels were higher in the MC3T3-E1 cells cultured in medium containing high glucose concentrations with BMP-2 compared with the cells cultured in medium containing normal glucose concentrations with or without BMP-2. GLP-1R protein expression increased following culture in high-glucose medium with BMP-2 compared with culture under normal glucose conditions. However, the cellular localization of GLP-1R was not affected by either glucose or BMP-2. In conclusion, our data demonstrate that the expression of GLP-1R and GIPR is regulated by glucose concentrations in MC3T3-E1 cells undergoing differentiation induced by BMP-2. Our results reveal the potential role of incretins in bone metabolism.

Degradation of MUC7 and MUC5B in human saliva.

BACKGROUND: Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis. METHODOLOGY/PRINCIPAL FINDINGS: Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours. CONCLUSIONS/SIGNIFICANCE: MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.

Sphingosine kinase 2 inhibitor SG-12 induces apoptosis via phosphorylation by sphingosine kinase 2.

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.

Tetrameric interaction of the ectoenzyme CD38 on the cell surface enables its catalytic and raft-association activities.

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

The proteoglycan repertoire of lymphoid cells.

Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans.

Syndecans reside in sphingomyelin-enriched low-density fractions of the plasma membrane isolated from a parathyroid cell line.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms.

Relationship between oral malodor and glycosylated salivary proteins.

Volatile sulfur compounds (VSCs), which are major sources of oral malodor, are produced by putrefactive activities of bacteria. Saliva provides easily degradable protein substrates, and most proteins are glycosylated. We hypothesized that oral malodor would be associated with enhanced proteolysis or deglycosylation in saliva. The purpose of this study was to evaluate properties of glycoproteins in saliva and assess their association with VSC levels. Subjects were 88 patients who visited "the Fresh Breath Clinic", Dental Hospital, Tokyo Medical and Dental University. They were classified into malodor (n = 67) and non-malodor (n = 21) groups. After collecting saliva, the amounts of the total proteins and carbohydrate were determined. Molecular size distributions of salivary proteins/glycoproteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of the total salivary proteins was significantly higher in the malodor group. Major proteins/glycoproteins observed in SDS-PAGE analyses showed similar distributions between the two groups. In the malodor group, the salivary protein concentrations were positively correlated with the CH3SH levels (p < 0.05), and the carbohydrate contents were negatively correlated with the H2S levels (p < 0.05). These results indicated the possibility that salivary proteins/glycoproteins might be related to the malodor generation.

Extraction and separation of proteoglycans.

Proteoglycans contain a unique carbohydrate component, glycosaminoglycan, which consists of repeating, typically sulfated disaccharides, and is capable of interacting with diverse molecules. Specific, clustered arrangements of sulfate on the glycosaminoglycan backbone form binding sites for many biologically important ligands such as extracellular matrix molecules and growth factors. Core proteins of proteoglycans also show molecular interactions necessary for organizing scaffolds in the extracellular matrix or for anchoring proteoglycans to the plasma membrane. Experimental protocols aiming at extracting maximal amounts of proteoglycans from tissues or cells require disruption of molecular interactions involving proteoglycans by denaturing solvents. Among many of the proteoglycan separation procedures, anion exchange chromatography, which takes advantage of the presence of highly negatively charged glycosaminoglycans in all proteoglycans, serves one of the most convenient general separation techniques.

Alteration of enzymatic properties of cell-surface antigen CD38 by agonistic anti-CD38 antibodies that prolong B cell survival and induce activation.

Leukocyte cell-surface antigen CD38 is a single-transmembrane protein. CD38 ligation by anti-CD38 antibodies triggers the growth or apoptosis of immune cells. Although the extracellular domain of CD38 has multifunctional catalytic activities including NAD(+) glycohydrolase and cyclase, the CD38-mediated cell survival or death appears to be independent of its catalytic activity. It is proposed that a conformational change of CD38 triggers the signalling. The conformational change of CD38 could influence its catalytic activity. However, the agonistic anti-CD38 antibody that alters the catalytic activity of CD38 has not been reported so far. In the present study, we demonstrated that two agonistic anti-mouse CD38 mAbs (CS/2 and clone 90) change the catalytic activities of CD38. CS/2 was clearly more potent than clone 90 in prolonging B cell survival and activation. CS/2 inhibited the NAD(+) glycohydrolase activity of both the isolated extracellular domain of CD38 (FLAG-CD38) and cell-surface CD38. Kinetic analysis suggested a non-competitive inhibition. On the other hand, clone 90 stimulated the NAD(+) glycohydrolase activity of FLAG-CD38 and had little effect on the NAD(+) glycohydrolase activity of cell-surface CD38. CS/2 and clone 90 had no effect on the cyclase activity of FLAG-CD38 and inhibited the cyclase activity of cell-surface CD38. Accordingly, these agonistic antibodies probably induce the conformational changes of CD38 that are evident in the distinct alterations of the catalytic site. The antibodies will be useful tools to analyze the conformational change of CD38 in the process of triggering B cell survival and the activation signal.

Hypoxia/Notch signaling in primary culture of rat lymphatic endothelial cells.

We have developed an improved intralumenal digestion method to get a long-term primary culture of rat lymphatic endothelial cells (rLECs) that maintained their original phenotypes. rLECs in vitro under hypoxia retained their original lymphatic properties observed in the thoracic duct. Blocking Notch signal with a gamma-secretase inhibitor and transfection of a cDNA expressing a dominant negative form of Delta1 indicated that Notch signal downregulated VEGFR-2 under hypoxia and inhibited cell migration. These findings indicated that Notch signal was still operative in mature lymphatic endothelial cells in response to the oxygen concentration.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Immunohistochemical localization of syndecan-1 in the dental follicle of postnatal mouse teeth.

BACKGROUND: Syndecans are cell surface heparan sulfate proteoglycans that modulate the action of growth factors and extracellular matrix components. Syndecan-1 plays important roles during early tooth development, and it is expressed in the dental follicle of fetal tooth germ. However, no studies have followed its expression in the dental follicle during the postnatal period. We hypothesized that syndecan-1 protein expression in the dental follicle may be important for postnatal tooth development, and, thus, examined its expression patterns. METHODS: Syndecan-1 protein expression in the dental follicle of the lower first molar was investigated by immunohistochemistry using embryonic day (E) 18.5 to 21-day-old (d 21) mice. Immunoelectron microscopy was applied to the dental follicle and pulp cells to confirm its membrane localization in mesenchymal cells. RESULTS: Strong syndecan-1 immunostaining was maintained in the dental follicle and the adjacent dental pulp surrounded by the Hertwig's epithelial root sheath (HERS) from d 4 to d 14, but reduced staining was noted at d 21 with the near-completion of tooth eruption. Three dimensionally, syndecan-1-positive areas plugged the apical foramina surrounded by HERS. However, immunostaining was detected constantly in the dental follicle and the dental pulp of the lower incisor at d 21. In addition, membrane localization of syndecan-1 protein was confirmed for the first time in mesenchymal cells, including dental follicle and pulp cells, by immunoelectron microscopy. CONCLUSION: The spatial and temporal expression of syndecan-1 in the dental follicle suggests that this proteoglycan is important for the maintenance of proliferation and/or movement of cells in this region during tooth eruption.

Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes.

We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237-11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468-475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a "versican-like" molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100-94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.

Syntaxin6 separates from GM1a-rich membrane microdomain during granule maturation.

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2. Because syntaxin6 and VAMP4 are known to be related to granule formation, we attempted to separate DRMs from ISGs. To enrich for ISGs, glands were removed from rats 5h after intraperitoneal injection of isoproterenol and used to purify the newly synthesized granules. Compared to mature granules prepared without injection, these newly formed granules were lower in density and contained higher concentrations of syntaxin6, VAMP4, and gamma-adaptin. This composition is consistent with the characterizations of ISGs. DRMs isolated from the newly formed granules were GM1a-rich and contained syntaxin6, VAMP4, and VAMP2 together. Thus, our findings suggest that syntaxin6 and VAMP4 associate with a GM1a-rich membrane microdomain during granule formation but enter a separate membrane microdomain before transport from granules during maturation.

Relationship between total salivary protein content and volatile sulfur compounds levels in malodor patients.

OBJECTIVE: The purpose of this study was to evaluate the relationship between total salivary protein and VSC levels. STUDY DESIGN: A patient group comprised 67 patients from the Fresh Breath Clinic of the Dental Hospital, Tokyo Medical and Dental University, with 18 healthy subjects as a control group. Gas chromatography was used to measure concentrations of VSCs in mouth air. After collecting resting saliva, flow rate, pH, and the amount of total protein in saliva were measured. Salivary protein patterns were analyzed by polyacrylamide gel electrophoresis (PAGE). RESULTS: In the patient group, the amounts of total salivary protein were significantly correlated with CH3SH levels measured by GC, CH3SH/H2S ratio, and organoleptic score. In addition, a significant correlation between flow rate of salivary protein and CH3SH/H2S ratio and the higher levels of salivary proteins from PAGE analyses were found. CONCLUSIONS: Total salivary protein was considered to be involved in the formation of oral malodor as one of the nutrient sources. The total salivary protein could be changed both quantitatively and qualitatively in subjects with malodor.

[Effect of p-nitrophenyl-xyloside on the biosynthesis of proteoglycan in rat ovarian granulosa cells--analyses of glycosaminoglycan synthesis in the Golgi apparatus].

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [³5S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [³5S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [³5S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.

Basement membrane matrix modifies cytokine interactions between lung cancer cells and fibroblasts.

OBJECTIVE: Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated. METHODS: The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel. RESULTS: All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel. CONCLUSION: These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.