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Originally published In Press as doi:10.1074/jbc.C300430200 on February 19, 2004

J. Biol. Chem., Vol. 279, Issue 16, 15715-15718, April 16, 2004
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Syndecan-2 Regulates Transforming Growth Factor-{beta} Signaling*

Ligong Chen, Carmen Klass{ddagger}, and Anne Woods§

From the Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the {ddagger}Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30303

Received for publication, September 22, 2003 , and in revised form, January 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta}) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-{beta} signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-{beta}-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2{Delta}S) promoted TGF-{beta} binding and S2 core protein ectodomain directly bound TGF-{beta}. Transfection with S2 increased the amounts of type I and type II TGF-{beta} receptors (T{beta}RI and T{beta}RII), whereas S2{Delta}S was much less effective. In contrast, S2{Delta}S dramatically increased the level of type III TGF-{beta} receptor (T{beta}RIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with T{beta}RI or T{beta}RII. This is a novel mechanism of control of TGF-{beta} action that may be important in fibrosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta})1 is a member of the TGF-{beta} superfamily (1). There are three mammalian TGF-{beta} isoforms, with TGF-{beta}1 being the most abundant in tissues (2). TGF-{beta} regulates cell proliferation, differentiation, adhesion, development, tissue repair, immunosuppression, and tumor suppression (3–7). It promotes fibrosis through increasing matrix proteins and metalloproteinase inhibitors and decreasing metalloproteinases (5). The exact sequence leading to divergent TGF-{beta} downstream signals is not yet determined (8, 9), but there is a consensus that TGF-{beta} signals through a complex of type I, II, and III receptors (T{beta}RI, T{beta}RII, and T{beta}RIII). Upon binding to ligands, T{beta}RII transphosphorylates T{beta}RI and T{beta}RIII (betaglycan), propagating downstream signaling. Betaglycan is a "part-time" cell surface proteoglycan that binds TGF-{beta} and transfers TGF-{beta} to T{beta}RII. Its core protein binds TGF-{beta}, and its GAG chains are not needed (10–12). Indeed, GAG modification of betaglycan may inhibit TGF-{beta} signaling (13). The cytoplasmic domain of betaglycan is essential for downstream signaling (8). It associates with autophosphorylated T{beta}RII and promotes the formation of the T{beta}RI·T{beta}RII complex. Following phosphorylation by T{beta}RII, betaglycan dissociates from the active T{beta}RI·T{beta}RII complex. The cytoplasmic domain of betaglycan contains a PDZ (post-synaptic density-95, disks large, zonula occludens-1)-domain binding motif, whose interaction with the PDZ-domain containing protein GIPC (GAIP-interacting protein, C terminus) leads to increased betaglycan expression (9).

Syndecan-2 (S2) is one of four mammalian members of a transmembrane proteoglycan family (14). Syndecans act as co-receptors for growth factor binding, cell-matrix interactions, and cell-cell interactions (15–18). They have divergent ectodomains and highly homologous transmembrane domains, and their cytoplasmic domains have two regions of homology (C1 and C2) flanking V regions unique to each syndecan. S2 interacts with matrix proteins such as laminin (19) and fibronectin (20), and its cytoplasmic V region (Fig. 1) controls matrix assembly at the cell surface (21). This may be due to oligomerization-dependent V region phosphorylation2 (22). Cells expressing an S2 construct that is truncated in the V region (S2{Delta}S) lack matrix deposition (21, 23). The V region of S2 also controls left-right asymmetry during Xenopus development through the actions of TGF-{beta} family members (23), with parallel phosphorylation requirements (24). Interestingly, S2 has similar motifs to betaglycan (Fig. 1) including the GAG attachment sites, a possible serine phosphorylation site (Ser-ser-ala-ala; SSAA) and a C-terminal PDZ-domain binding motif that binds GIPC. Since both fibrosis and determination of left-right asymmetry involve TGF-{beta} family members (5, 23), we investigated whether S2 and TGF-{beta} act in concert in fibrosis.



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  		FIG. 1.
Schematic structures of syndecan-2, S2{Delta}S, and betaglycan. Cytoplasmic domain residues are shown. C1, V, and C2 regions of S2 are indicated. PDZ-domain binding residues are capitalized. s represents putative phosphorylation sites.

 


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—General chemicals, Tween, and coverslips and cultureware were from Sigma, Bio-Rad, and Fisher, respectively. TGF-{beta}1 and biotinylated TGF-{beta}1 were from R & D Systems (Minneapolis, MN). RNAzolTM B was from Leedo Medical Laboratories (Houston, TX). pcDNA3 vector was obtained from Invitrogen. Antibodies against S2 (T-17), T{beta}RI (V-22), T{beta}RII (L-21), T{beta}RIII (C-20), and donkey anti-goat IgG-horseradish peroxidase (HRP) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polycolonal anti-S2 (R1891), monoclonal anti-S4 (150.9), and anti-fibronectin have been described previously (14, 25, 26). Protein-G-Sepharose, blocking agent for detection of biotinylated proteins, and streptavidin-HRP were obtained from Amersham Biosciences. Fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat anti-rabbit or anti-mouse IgG were purchased from Cappel (Durham, NC). Heparitinase I (EC 4.2.2.8 [EC] ) and chondroitinase ABC (EC 4.2.2.4 [EC] ) were from Seikagaku America (Falmouth, MA). UltraLink immobilized streptavidin gel and NHS-Sulfo-Biotin were from Pierce.

Immunohistochemical Analysis of S2 Expression in Renal Sections— Frozen sections from control and diabetic type II patients were incubated with anti-S2 or anti-S4 (45 min, 37 °C), washed with phosphate-buffered saline (PBS) (3 x 15 min), incubated with FITC-conjugated goat anti-rabbit or goat anti-mouse (45 min, 37 °C), washed with PBS (3 x 15 min), and photographed using a Nikon Optiphot microscope. Equivalent exposure, development, and scanning procedures were used for digital images.

Northern and Immunoblotting Analyses of Syndecan-2 Expression— Renal papillary fibroblasts (RPF) cells were cultured in 50% Dulbecco's modified Eagle's medium + 50% Ham's F-12 with 5% fetal bovine serum. Subconfluent cultures were serum-starved (7 h) and treated with TGF-{beta}1 (10 ng/ml) with retreatment at 24 h. Total RNA was isolated with RNAzolTM B, and 30 µg were used for Northern analysis (14), using the partial transmembrane domain plus the cytoplasmic domain of rat S2 as the 5'-end-labeled probe. For immunoblotting, cells were lysed into SDS sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (10% (w/v)) and transferred to nitrocellulose membranes. Membranes were incubated with goat anti-S2 (1:1000) followed by donkey anti-goat IgG-HRP (1:1000), and antibodies were visualized by enhanced chemiluminescence (ECL).

Analysis of Fibronectin Matrix Deposition—Cells were treated with TGF-{beta}1 as above for 48 h, fixed, and stained with anti-fibronectin, followed by FITC-conjugated goat anti-rabbit IgG (1:50). Levels of matrix deposition were semiquantified using camera exposure time.

Cell Surface Binding of TGF-{beta}—Confluent 6-well plate cultures were incubated (27) with 400 pM biotinylated TGF-{beta}1 + 100-fold unlabeled TGF-{beta}1. In some experiments, cells were pretreated with 5 milliunits of heparitinase I (37 °C, 2 h), with repeated enzyme addition at 1 h. Proteins were resolved by SDS-PAGE (15% (w/v)), transferred to nitrocellulose membranes, blocked (5% blocking agent in PBS-0.1% Tween), and bound biotinylated TGF-{beta}1 was visualized by incubation with streptavidin-HRP (1:1000) followed by ECL.

Binding Assays—Recombinant S2 ectodomain was prepared as described previously (14). RPF (T-75 flask) were treated with heparitinase and chondroitinase as described previously (14), followed by addition of 12 ml of lysis buffer (0.5% Triton X-100 in 25 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine HCl, 10 µg/ml leupeptin, and 10 mM N-ethylmaleimide). Streptavidin gel (20 µl) was washed twice with 1 ml of 0.1% Triton X-100 in lysis buffer and incubated with biotinylated TGF-{beta}1 (250 ng, 4 °C, 1 h). Following three washes with 1 ml of lysis buffer, S2 ectodomain (0.5 µg) or RPF cell lysate was incubated with the gel (4 °C, 2 h). Gels were washed three times, and bound material was eluted with SDS sample buffer (30 µl), resolved by SDS-PAGE (10% (w/v)), and detected by R1891 (1:2000), followed by goat anti-rabbit-HRP (1:2000) and ECL.

Immunoblotting of T{beta}RI, T{beta}RII, and T{beta}RIII—For immunoblotting of T{beta}RI and T{beta}RII, confluent cultures were lysed into SDS sample buffer. For immunoblotting of T{beta}RIII, the GAG chains were removed as described previously (14). Proteins were resolved by SDS-PAGE, 10% (w/v) for T{beta}RI and T{beta}RII, 3–10% (w/v) for T{beta}RIII, and transferred to nitrocellulose membranes. Receptors were visualized by blotting with anti-T{beta}RI (1:1000 in 0.1% Tween, 0.1% BSA, 5% nonfat milk in Tris-buffered saline (TBS) for 2 h), T{beta}RII (1:1000 in the same buffer as for T{beta}RI for 1h), or T{beta}RIII (1:1000 in PBS for 1.5 h), followed by goat anti-rabbit IgG-HRP (1:2000 in 0.1% Tween, 0.1% BSA, 1% nonfat milk in TBS for 1 h) or donkey anti-goat IgG-HRP (1:1000 in 0.1% Tween, 0.1% BSA, 1% nonfat milk in PBS for 1 h) and ECL.

Cell Surface Biotinylation and Immunoprecipitation—Confluent cultures (T-75 flask) were washed with cold PBS three times, incubated with NHS-Sulfo-Biotin (2.5 mg) in PBS (5 ml, 30 min, 4 °C), washed with cold PBS three times, and scraped into lysis buffer (5 ml). The cell number of parallel cultures was used to confirm equal loading. After centrifugation (10,000 rpm, 10 min, 4 °C), the supernatant was incubated (1 h, 4 °C) with protein-G-Sepharose beads preblocked with 5% fetal bovine serum. Beads were removed by centrifugation (400 x g, 10 min, 4 °C) and the supernatant incubated with fresh preblocked beads and 5 µg/ml antibody (2 h, 4 °C). Beads were washed three times with lysis buffer, three times with PBS, and bound proteins were eluted by boiling with SDS sample buffer (5 min), subjected to SDS-PAGE, and blotted with streptavidin-HRP and ECL. Membranes were stripped and blotted with antibodies against S2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
When renal sections from age-matched controls (Fig. 2A, left panel) were compared with those from type II diabetic patients (Fig. 2A, second panel), S2 was increased in the tubulointerstitium. In contrast, despite some increase in the glomerulus, syndecan-4 (right two panels) was not dramatically increased in the tubulointerstitium. Similar results were obtained with streptozotocin-induced diabetic rats (data not shown). TGF-{beta} increases S2 expression in several cell types (28, 29). We, therefore, tested whether the increase in S2 was due to TGF-{beta}, a known mediator of fibrosis (5). However, S2 mRNA or protein levels were not increased (Fig. 2B and data not shown) in RPF cells treated with TGF-{beta} at concentrations sufficient to increase fibronectin matrix in these (Fig. 2C) and other cells (30–32).



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  		FIG. 2.
Syndecan-2 mediates TGF-{beta} function. A, S2 and S4 labeling in normal and diabetic kidney sections. Note a lack of interstitial labeling (stars) in normal kidney but labeling of a nerve (arrowhead) and blood vessel (arrow). No significant differences were seen with S4. Bar in left panel = 50 µm. Bars in right three panels = 100 µm. B, S2 levels ± TGF-{beta} treatment were analyzed by Northern analysis and densitometry. Relative levels of S2 to GAPDH mRNA are shown. C, anti-fibronectin labeling in vector- (left), S2{Delta}S- (middle) and S2- (right panels) transfected RPF cells in the presence (bottom) or absence (top) of TGF-{beta} treatment. Arrowheads represent cells lacking fibrils; arrows represent fibrils. Bar = 10 µm.

 
We then tested whether S2 can mediate TGF-{beta}-induced increased matrix deposition. Immunofluorescent labeling indicated that overexpression of S2 in RPF increased fibronectin matrix deposition with more fibrils/cell and larger fibrils (arrows) than in cells transfected with vector only (Fig. 2C). This difference was not statistically significant in a semiquantitative assay using exposure time. Construct expression levels vary among cells, although immunoblotting (data not shown) indicated transfection with S2 or S2{Delta}S increased S2 levels 2.5-fold overall. In contrast, expression of S2{Delta}S decreased basal fibronectin deposition (Fig. 2C), compared with cells transfected with vector only (102 ± 11 versus 76 ± 6.0, p < 0.001), and several cells (arrowheads) lacked fibrils. This suggests that S2 cytoplasmic domain regulates matrix deposition in RPF cells, as it does in CHO-K1 cells (21). Differences in matrix deposition were exaggerated after TGF-{beta} treatment. The percentage decrease of exposure time after TGF-{beta} treatment parallels increased matrix deposition. Treatment of vector-only transfected cells with TGF-{beta} (Fig. 2C) increased matrix deposition (18% ± 9.7% decrease in exposure time). Increased matrix deposition in S2 cells was exaggerated (21% ± 7.0%), but S2{Delta}S cells showed (Fig. 2C) less increase (14% ± 9.1%).

These results suggest that S2 regulates TGF-{beta}-mediated matrix increase. Transfection of cells with either S2 or S2{Delta}S increased surface binding of TGF-{beta} over that to cells transfected with empty vector (Fig. 3, A and B). Cleavage of HS chains reduced TGF-{beta} binding (Fig. 3B), suggesting that some binding is through HS chains. Surprisingly, recombinant S2 ectodomain bound biotinylated TGF-{beta}1, and TGF-{beta}1 could capture S2 from RPF cell lysates (Fig. 3, C and D). Thus, the core protein of both S2 and S2{Delta}S binds TGF-{beta}, and binding is independent of the cytoplasmic domain.



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  		FIG. 3.
Syndecan-2 binds TGF-{beta}. A, cell surface binding of TGF-{beta} to vector-, S2-, or S2{Delta}S-transfected RPF cells. Lanes 1–3, treatment with 40 nM non-biotinylated TGF-{beta}1 + 400 pM biotinylated TGF-{beta}1 (B-TGF-{beta}1). Lanes 4–6, treatment with 400 pM biotinylated TGF-{beta}1. B, densitometric analysis of three binding experiments. Data are shown as percentage of bound biotinylated TGF-{beta}1 compared with vector-transfected cells ± S.D. H indicates heparitinase pretreatment. Differences between S2 and S2{Delta}S and vector-only cells were statistically significant (p < 0.01). C and D, biotinylated TGF-{beta}1 on streptavidin beads was used to pull down recombinant S2 ectodomain (C, lane 2) and S2 core protein from RPF cell lysate (D, lane 2) as shown by S2 immunoblotting. Lanes 1, of input; lanes 3, the same experiments as in lanes 2 except that beads in the absence of biotinylated TGF-{beta}1 were used.

 
Immunoblotting of total cell lysates demonstrated that S2{Delta}S slightly, and S2 dramatically, increased protein levels of T{beta}RI and T{beta}RII (Fig. 4, A, B, D, and E). The expression of T{beta}RIII, betaglycan, was increased in S2{Delta}S cells but decreased in S2 cells (Fig. 4, C and F). When cell surface receptor levels were monitored by biotinylation followed by immunoprecipitation (Fig. 5, A–C), these were consistent with the total levels. In Fig. 5C, the lowest species is coprecipitated S2, as confirmed in Fig. 5D.



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  		FIG. 4.
Syndecan-2 and S2{Delta}S differentially regulate the expression of TGF-{beta} type I, II, and III receptors. A–C, immunoblotting of T{beta}RI (A), T{beta}RII (B), and T{beta}RIII (C) in vector-, S2-, or S2{Delta}S-transfected RPF total cell lysates. D–F, densitometric analysis of results from three individual experiments. Data are shown as percentage of receptor level to that in vector transfected cells ± S.D.; *, p < 0.025; **, p < 0.01.

 



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  		FIG. 5.
Cell surface TGF-{beta} receptor levels and association with syndecan-2. A–C, cell surface biotinylation, immunoprecipitation of T{beta}RI (A), T{beta}RII (B), or T{beta}RIII (C), followed by streptavidin-HRP. D, the membrane in C was stripped and immunoblotted with rabbit anti-S2. E is a repeat of C with increased loading and longer time exposure.

 
The regulation of the expression of TGF-{beta} receptors by S2 may be through formation of a complex between S2 and the receptor(s). Syndecan-2 was not detected in T{beta}RI or T{beta}RII immunoprecipitates (data not shown) but was detected in T{beta}RIII immunoprecipitates (Fig. 5D). Although S2{Delta}S cells expressed the highest levels of betaglycan (Fig. 4C), less S2 coprecipitated with betaglycan from S2{Delta}S cells than from vector-only transfected cells, and that detected may be endogenous full-length S2. Thus, the cytoplasmic domain of S2 may be needed for association with betaglycan. Low amounts of coprecipitated S2 were detected from S2 cells, probably due to the decreased level of betaglycan in S2 cells (Fig. 4C). To confirm differential association of betaglycan with S2{Delta}S and S2, further coprecipitation experiments were performed with increased loading on SDS-PAGE and longer time exposure on the film (Fig. 5E).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Syndecans can act as co-receptors for growth factors (16–18) by delivery of ligands to their signaling receptors and the formation of a ternary complex (18, 33). Most growth factor binding has been thought to be through the glycosaminoglycan chains (16–18). Here, for the first time, we report that S2 can bind and regulate the signaling of TGF-{beta}, and the binding is through the core protein of the ectodomain, with the HS GAGs having only a minor role. The binding is independent of the cytoplasmic domain. However, the cytoplasmic domain of S2 is crucial in the regulation of TGF-{beta} receptor levels and in association of S2 with betaglycan.

Total and surface levels of T{beta}RI and T{beta}RII were dramatically increased by S2 but not by S2{Delta}S. The cell surface level of receptors is balanced by rates of synthesis, cleavage, and internalization-dependent down-regulation and recycling. There are conflicting reports whether TGF-{beta} up- or down-regulates its receptor expression (27, 34–36). Additionally, other S2 ligands, such as FGF2 and GM-CSF (granulocyte-macrophage colony-stimulating factor), can up-regulate the expression of T{beta}RI and T{beta}RII (37, 38). S2 and S2{Delta}S may differentially regulate cell surface degradation of TGF-{beta} receptors; T{beta}RIII can be cleaved at the cell surface (39), although cleavage of T{beta}RI and T{beta}RII was not detected (35). Finally, S2 and S2{Delta}S may differentially regulate endocytosis-dependent degradation and recycling of TGF-{beta} receptors. The distinct effects of S2 and S2{Delta}S on betaglycan levels are intriguing. Effects on betaglycan (increased by S2{Delta}S but not by S2) may be explained by similarities between S2 and betaglycan cytoplasmic domains. Both contain a PDZ-domain binding motif (Fig. 1) that binds GIPC (9, 40). GIPC binding up-regulates betaglycan levels on the cell surface by preventing proteosome degradation (9). If S2 competes for GIPC binding, less GIPC will be available for betaglycan in S2 cells but more in S2{Delta}S cells. All three receptors may be required for efficient receptor down-regulation (36). Decreased cell surface betaglycan in S2 cells may then reduce down-regulation of T{beta}RI and T{beta}RII. Whatever the mechanism, the finding that S2 can regulate the cell surface levels of TGF-{beta} receptors adds a new level of complexity to TGF-{beta} action.

Despite increased TGF-{beta} binding and betaglycan level in S2{Delta}S cells, TGF-{beta} did not increase matrix deposition in S2{Delta}S cells, suggestive of defective downstream signaling. Both betaglycan and S2 cytoplasmic domain contain a SSAA sequence (Fig. 1). Phosphorylation of betaglycan cytoplasmic domain is needed for downstream signaling (8), and, although the site has not been determined, this can be by T{beta}RII. The SSAA sequence in S2 can be phosphorylated if not oligomerized (22), and phosphorylation is needed for both matrix assembly2 and the regulation of left-right asymmetry in Xenopus embryos (23, 24). Whether S2 and betaglycan compete for the same kinase(s) is not known, but overexpression of S2 may promote oligomerization, as it does with syndecan-4 (41), and thus reduce its phosphorylation potential. Finally, we found that betaglycan coprecipitates with S2, and the cytoplasmic domain of S2 is needed for this association. Whether this is a direct interaction is now being determined. There may be two distinct signaling mechanisms, involving either S2 or betaglycan, with coprecipitation due to common binding to TGF-{beta}. While much remains to be determined, it is clear that, as is the case with betaglycan (8), cytoplasmic domain truncation of S2 has no effect on TGF-{beta} binding but reduces downstream effects. In summary, the data presented here demonstrate a mechanism of TGF-{beta} regulation by syndecan-2. Since TGF-{beta} is a major fibrogenic agent, this highlights a possible role of syndecan-2 in fibrosis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK54605 (to A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama at Birmingham, THT 946, 1530 3rd Ave. S, Birmingham, AL 35294-0006. Tel.: 205-934-1548; Fax: 205-934-7029; E-mail: anwoods{at}uab.edu.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; T{beta}RI, T{beta}RII, and T{beta}RIII, type I, II, and III TGF-{beta} receptors; V region, variable region; S2, syndecan-2; S4, syndecan-4; S2{Delta}S, S2 truncated in the V region; GAG, glycosaminoglycan; HS, heparan sulfate; PDZ, post-synaptic density-95, disks large, zonula occludens-1; GIPC, GAIP-interacting protein, C terminus; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; RPF, renal papillary fibroblasts; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BSA, bovine serum albumin. Back

2 C. M. Klass, L. Chen, and A. Woods, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Grupp (University of Goetingen, Germany) for providing the renal papillary fibroblasts.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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