13 research outputs found
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
Sucrose-density gradient analysis of DRMs obtained from a [<sup>35</sup>S]sulfate labeled rat parathyroid cell line.
<p>Rat parathyroid cells (2×10<sup>7</sup>) were metabolically labeled with [<sup>35</sup>S]sulfate, and subjected to a preparation of DRMs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Fractions (200 µl each) were collected and counted for radioactivity after removal of free [<sup>35</sup>S]sulfate. A. Graph shows total radioactivity (○) and protein concentration (•) detected in each fraction. <i>Inset</i>, the same experimental data plotted in an expanded scale, emphasizing the presence of <sup>35</sup>S-labeled material in low-density fractions. B. Graphic representation of specific radioactivity calculated for each fraction. The specific activity was expressed as total radioactivity per amount of the total protein material. DRM fractions were characterized by high specific activity suggesting enrichment with [<sup>35</sup>S]sulfate labeled molecules.</p
Identification of HSPGs expressed by a rat parathyroid cell line.
<p>A. RT-PCR analysis of PTr cells using syndecan-specific primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a> for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.</p
Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line.
<p>DRMs were prepared from confluent PTr cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).</p
Characterization of DRM fractions isolated from a parathyroid cell line.
<p>Confluent PTr cells were subjected to DRMs preparation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Concentrated fractions were analyzed for presence of HSPGs by WB. They were also examined for the presence of ganglioside GM1 by the binding of cholera toxin B (CTB) subunit and sphingomyelin (SM) by HPTLC analysis. A. Staining with 3G10 antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were used for analysis. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. B. DRM markers, Lyn and Giα showed a broad distribution, but partially overlapped with low-density fractions containing HSPGs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Successful preparation was confirmed by immunostaining for TfR, which was found mainly in the bottom fractions. C. An equal aliquot (2 µl) of each fraction was dot-blotted onto PVDF membrane and stained with HRP-conjugated CTB subunit. Numbers correspond to the fraction number; L, original lysate before sucrose-density gradient ultracentrifugation. D. Lipids extracted from sucrose density gradient fractions (fractions 2–11) were developed on the HPTLC plate and stained with 3% cupric acetate and 8% phosphoric acid. Chol, cholesterol; PE, phosphoethanolamine; PC, phosphocholine; SM, sphingomyelin. Non-specific staining due to the presence of traces of detergent in samples is marked with (*). E. Graphic representation of distribution of HSPGs, ganglioside GM1 and SM in DRM fractions. Low-density fractions containing HSPGs showed enrichment with SM. Density of bands detected in WB and HPTLC was measured with ImageJ and expressed in arbitrary units. Graph shows distribution of ganglioside GM1 (○); HSPGs (▴) and SM (▪).</p
TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals.
Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-β (TGF-β), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-β2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-β2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-β2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-β and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema
Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks
The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks