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Cell surface heparan sulfate proteoglycans: target and partners of the basic fibroblast growth factor in rat Sertoli cells.

Basic fibroblast growth factor (bFGF) regulates diversified biological functions in rat Sertoli cells. This report demonstrates that bFGF inhibits steroidogenesis in developing rat Sertoli cells. Follicle stimulating hormone (FSH)-stimulated estradiol production was reduced by bFGF. Moreover, the amount of cytochrome P450 aromatase, responsible for the irreversible transformation of androgens into estrogens, is decreased by bFGF at the transcriptional level. The bFGF inhibitory effect was also observed in the presence of dibutyryl-cAMP, cholera toxin or RO-20-1724, all inducing high levels of cAMP, the second messenger of FSH. Heparan sulfate proteoglycans (HSPGs) were shown to be required as cofactors for bFGF signaling. Indeed, sodium chlorate, described to drastically decrease proteoglycan sulfation, abolishes the bFGF downregulation of FSH-stimulated estradiol synthesis previously observed. Glypican-1, syndecan-1 and -4, potential bFGF coreceptors, are mainly regulated at the transcriptional level. This report shows that the bFGF regulation of their expression specifically depends on the nature of HSPG and of the Sertoli cell developmental stage. In conclusion, HSPG are partners and the target of bFGF in rat Sertoli cells.

Animals↗

[Transcriptional and electron microscopic analysis of extracellular matrix proteoglycans in acute acetaminophen intoxication].

UNLABELLED: Acetaminophen (APAP) OTC regimen is not compatible with kidney and liver toxic effects. In order to further understand the determinism of the repair process, we have studied the expression of some target extracellular matrix proteoglycan components in an animal model. MATERIALS AND METHODS: An acute APAP toxic dose (800 mg/bw) was administered to 45 mice aged 1 month. At 24 hours, tissue samples were processed for light microscopy, electromicroscopy and RT-PCR analysis. RESULTS: Extensive necrotic lesions involving hepatocytes and tubular epithelial cells associate strong positivity for proteoglycans, detected as convoluted filaments with lateral projections, frequently associated with collagen fibers, cell surface and other matrix components. Overexpression for perlecan, syndecan 1 and 4 and low expression for biglycan was assessed by RT-PCR. CONCLUSIONS: Extracellular matrix and cell surface components are early involved in both lesional mechanism and repair reaction post acetaminophen injury.

Acetaminophen↗

Characterization and immunohistochemical localization of the glycoconjugates of the rabbit bladder mucosa.

An impairment of the mucosal glycoconjugates could be an important factor in the development of bladder disorders such as interstitial cystitis. However, very little definitive biochemical information is available on the glycoco-jugate components of the mammalian bladder mucosa. In this-study, the mucosa from metabolically radiolabeled rabbit bladder was separated, delipidated, and digested with protease, and the released glycosaminoglycans and glycopeptides were fractionated. About 80 and 36% of the nondialyzable tritium and 35S activities, respectively, was associated with the sialoglycopeptide fractions. The balance of the total tritium activity in the protease digest was in glycosaminoglycans identified as hyaluronan, chondroitin sulfates, and heparan sulfate. Immunohistochemical examination using anti-heparan sulfate antibodies, including one against mouse syndecan-1, indicated the presence of heparan sulfate proteoglycan in the epithelium. In contrast, there was no significant staining of the bladder epithelium with anti-chondroitin-4- and 6-sulfate antibodies or hyaluronan-binding protein. The lamina propria and muscle layers showed strong staining with anti-chondroitin-4-sulfate antibody and hyaluronan-binding protein and weak staining with anti-chondroitin-6-sulfate antibody. The insignificant levels of glycosaminoglycans in the glycocalyx of bladder mucosa epithelium suggest that glycosaminoglycans may be less important than other glycoconjugates in maintaining normal epithelial function and in bladder disorders such as interstitial cystitis.

Animals↗

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate.

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.

Animals↗

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Acetic Acid↗

Sweet syndrome in multiple myeloma: a series of six cases.

BACKGROUND: Sweet syndrome (SS), a paraneoplastic syndrome characterized by fever, neutrophilia, multiple erythematous painful plaques, and a dense dermal neutrophilic infiltration, has a known association with hematologic malignancies such as acute myelogenous leukemia. However, no clear association with multiple myeloma (MM) has been reported. MATERIALS AND METHODS: Pathology reports of the 2357 patients with multiple myeloma at the University of Arkansas for Medical Sciences were reviewed to retrieve cases who had developed SS. Cytogenetic studies and immunoglobulin secretory status were retrieved. Five cases of SS in MM and 25 cases of SS in patients without MM underwent syndecan-1 immunohistochemistry. OBSERVATIONS: Six cases of SS occurring in the setting of MM showed a predominance in patients secreting IgG paraprotein. Five of the six patients received granulocyte-colony stimulating factor while the sixth received granulocyte-monocyte-colony stimulating factor. Fifty percent showed a non-specific cytogenetic anomaly. CONCLUSIONS: There is no specific cytogenetic anomaly associated with SS in the setting of MM. This paraneoplastic syndrome may be secondary to elevated levels of granulocyte colony stimulating factor (G-CSF), possibly with a component of enhanced sensitivity to endogenous G-CSF. The immunoglobulin secretory status parallels that seen in MM with cutaneous involvement, but IgG secretors may be at an increased risk of developing SS compared with their counterparts who secrete other immunoglobulins.

Adult↗

The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells.

Ectopic activation of the Wnt signaling pathway is highly oncogenic for many human tissues. Here, we show that ectopic Wnt signaling increases the effective stem cell activity in mouse mammary glands in vivo. Furthermore, Wnt effectors induce the accumulation of mouse mammary epithelial progenitors (assayed by Hoechst dye exclusion, a surrogate stem cell marker, side population cells) both in vivo and in vitro. The longevity of stem cells makes them good candidate tumor precursors, and we propose that Wnt-induced progenitor amplification is likely to be key to tumor initiation. In support of this notion, mammary glands from a tumor-resistant strain of mice (carrying a null mutation in syndecan-1) contain fewer side population cells. When this strain is crossed to mice that overexpress effectors of the beta-catenin/T cell factor Wnt pathway, the amplification of progenitors is reduced, together with all subsequent events of tumor development. We propose that the growth dynamic of the stem cell fraction is a major determinant of tumor susceptibility.

Animals↗

Phenotypically distinct B cell development pathways map to the three B cell lineages in the mouse.

A recent article by Montecino-Rodriguez et al. [Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. (2006) Nat. Immunol.7, 293-301] has distinguished the early progenitors for B-1 cells, which principally develop in neonates, from early progenitors for B-2 cells, which principally develop in adult bone marrow. Here we introduce syndecan-1 (CD138) and MHC class II (I-A) as markers of early B cell development [Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. (1991) J. Exp. Med. 173, 1213-1225; Hardy fractions B-D] and show that the expression of these markers distinguishes the predominant B cell development pathway in neonates from the corresponding predominant pathway in adults (both progenitors are present but differently represented in each case). We show that pre-B cells (Hardy fraction D) in the predominant adult pathway express high levels of CD138 and intermediate levels of I-A, whereas the corresponding pre-B cells in the pathway that predominates in neonates do not express either of these markers. As expected, because most of the pre-B cells in adults express CD138, we find that sorted CD138+ adult pre-B cells differentiate to IgM+ B cells in vitro. Sorted CD138- pre-B cells from neonates, the majority subset at this age, also mature to IgM+ cells (without passing through a CD138+ stage). Importantly, our studies here confirm the differential representation of adult and neonatal progenitor populations and further demonstrate that CD138 expression subdivides the adult CD19+, B220-6B2-/low population shown to contain B-1 progenitors in a way consistent with the predominance of B-1b progenitors in adults. Thus, CD138 expression provides a key route to distinguishing early B cell development pathway for what now are clearly three B cell lineages.

Animals↗

Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo.

The factors controlling cationic liposome-DNA complex (CLDC)-based gene transfer in cells and in animals are poorly understood. We found that cell surface heparin/heparan sulfate-bearing proteoglycans mediate CLDC-based gene transfer and expression both in cultured cells and following intravenous gene delivery into animals. CLDC did not transfect Raji cells, which lack proteoglycans, but did efficiently transfect Raji cells stably transfected with the proteoglycan, syndecan-1. Fucoidan, heparin, or dextran sulfate, all of which are highly anionic polysaccharides, each blocked CLDC-mediated transfection both in cultured cells and following intravenous injection into mice, but had no effect on transfection by either recombinant adenovirus infection or electroporation. Intravenous pretreatment of mice with heparinases, which specifically cleave heparan sulfate molecules from cell surface proteoglycans, blocked intravenous, CLDC-mediated transfection in mice, confirming that proteoglycans mediate CLDC gene delivery in vivo. Modulation of proteoglycan expression may prove useful in controlling the efficiency of, as well as targeting the sites of, CLDC-based gene transfer in animals.

Adenoviridae↗

Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content.

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.

Animals↗

Cyclic expression of mRNA transcripts for connective tissue components in the mouse ovary.

In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation.

Animals↗

Shedding light on the distinct functions of proteoglycans.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous molecules that are critical for signaling mediated by many growth factors, including members of the Wnt, transforming growth factor-beta, Hedgehog, and fibroblast growth factor families, and are essential for cell specification, axon guidance, and the establishment of morphogen gradients. Although the heparan sulfate modifications of HSPGs are critical, there is much to learn about how the protein cores contribute to the specific signaling functions of these cell-surface and matrix molecules. Recent work has demonstrated that glypican-1 and syndecan-1 expressed by tumor cells have specific roles in FGF2 signaling, affecting their responses to this mitogenic stimulus.

Animals↗

Asynchronous progression through the lytic cascade and variations in intracellular viral loads revealed by high-throughput single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth "delayed late" category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

Antigens, Viral↗

Inflammation- and ischemia-induced shedding of venular glycocalyx.

Alterations in the composition of the glycocalyx of venular endothelium in postcapillary venules (rat mesentery) were explored in models of inflammation and ischemia-reperfusion injury. Lectins were covalently linked to fluorescently labeled microspheres (0.1-microm diameter) or directly labeled with FITC. Adhesion of lectins specific for glucose and galactose residues of glycosaminoglycans (GAGs) and other components of the endothelial glycocalyx decreased dramatically after superfusion of the mesentery with the chemoattractant N-formylmethionyl-leucyl-phenylalanine and during reperfusion after 60-min ischemia. These reductions were significantly attenuated by superfusion with pertussis toxin (PTX), suggesting that shedding of glycocalyx was mediated by G proteins. Adhesion of microspheres linked with antibody for syndecan-1, a major proteoglycan to which GAGs are bound, revealed increased labeling as GAGs were lost and permitted greater numbers of spheres to adhere to the protein core, which was not shed. Induction of ischemia by occluding proximal microvessels for 60 min resulted in a 40% increase in galactosaminoglycans and a 15% increase in glucosaminoglycans on the endothelium, which was not inhibited by PTX. Reperfusion of vessels led to a rapid loss of GAGs that was inhibited by pretreatment with PTX, with 40% of galactosaminoglycans and 25% of glucosaminoglycans accumulated being removed by G protein-mediated shedding and the remainder freely convected away by fluid shear. We conclude that the composition of the glycocalyx results from a balance of the rate of biosynthesis of GAGs by the endothelial cell and their shedding, which may be mediated by intracellular and/or membrane-bound proteases or lyases released or activated by G protein signaling.

Animals↗

Circulating sCD138 and some angiogenesis-involved cytokines help to anticipate the disease progression of early-stage B-cell chronic lymphocytic leukemia.

Syndecan-1 (CD138) is a transmembrane heparin sulfate proteoglycan expressed on distinct stages of differentiation of B-lymphoid cells. Its prognostic value in B-cell chronic lymphocytic leukemia (B-CLL) has not been evaluated so far. The serum concentration of sCD138 and some angiogenesis-involved cytokines: vascular endothelial growth factor (VEGF), basis fibroblast growth factor (bFGF), and endostatin were studied in 52 previously untreated patients with B-CLL. We found that bFGF and sCD138 levels were significantly higher in B-CLL patients than in controls. In patients with sCD138 level or endostatin level below the median value the lymphocyte count was higher than in patients with serum level of those cytokines above the median value. In patients with progressive disease bFGF level was significantly higher and sCD138 level significantly lower than in patients with stable one. Moreover, high sCD138 level was associated with longer lymphocyte doubling-free survival, and, on the limit of statistical significance, a high endostatin level was associated with shorter progression-free survival. We conclude that serum sCD138 level is increased in early stage B-CLL patients and may have a positive prognostic value as to the dynamics of the disease.

Adult↗

Analyzing patterns of staining in immunohistochemical studies: application to a study of prostate cancer recurrence.

BACKGROUND: Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. METHODS: Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. RESULTS: In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). CONCLUSIONS: Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis.

Biomarkers, Tumor↗

Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells.

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.

Animals↗

Developmental properties of the Hertwig's epithelial root sheath in mice.

Hertwig's epithelial root sheath (HERS) plays an important role in tooth root formation. In this study, we examined root formation of the first molar in mice, focusing on cell proliferation, cell death, cell migration, and the expression patterns of the signaling molecules, including glycoproteins and proteoglycans between PN8 and PN26. The number of HERS cells decreased during root formation, although HERS retained total length until PN15. The migration of HERS cells did not occur during root formation. Moreover, the immunopositive reaction of laminin beta-3 and syndecan-1 in HERS indicates that both cell adhesion and cell proliferation are essential for HERS development. Bmp-2, Bmp-4, and Msx-2 were expressed in HERS cells during root formation. We also developed an in vitro culture system for investigating the periodontium and suggest that this system provides an excellent vehicle for full exploration, and hence improved understanding, of the development and regeneration of the periodontium. Together, our results provide a comprehensive model describing the morphogenesis of early root development in vertebrates.

Animals↗