Figure 3. S1P-suppressed synthesis of syndecan-1 is mediated by S1P₁, heparanase (HPSE), and S1P-induced shedding of syndecan-1 is mediated by S1P₁, HPSE, and matrix metalloprotease (MMP).

Human liver hepatocellular cells (HepG2) were stimulated in the presence or absence of 2 μM S1P for 72 h. (A) S1P increased mRNA expression of S1P₁. HepG2 cells were treated with S1P₁ inhibitor W146 (10 μM), HPSE inhibitor low molecular weight heparin (50 μg/ml), generic MMP inhibitor Ilomastat (GM6601, 10 μM), or GM6001 negative control (NC) for 30 min before S1P stimulation for 72 h. (B) S1P downregulated mRNA transcripts of syndecan-1, which was blocked by W146 and heparin, but was not by Ilomastat. (C) S1P-induced sGAG release from HepG2 cells was eliminated by W146, heparin, and Ilomastat, respectively. sGAG in the culture medium was detected by using 1,9-dimethylmethylene blue (DMMB) assay. (D) S1P decreased syndecan-1 staining (green) but induced translocation of syndecan-1 into the nucleus (DAPI, blue). Confocal images depict a z-plane through the center of the cell nucleus. Scale bar, 10 μm. (E) and (F), a representative image of Western blots (E) with densitometric quantification (F) of syndecan-1 in cell lysates after 30 min exposure of W146, heparin, and Ilomastat before S1P treatment for 72 h. Results are presented as a ratio to GAPDH. (G) and (H), immunofluorescence images (maximum-intensity projections of a confocal z-stack) (G) and the coverage of heparan sulfate (HS, green). Nuclei were counterstained with DAPI (blue) (H). *P < 0.05 as compared with CT; ^#P < 0.05 as compared with S1P (ANOVA).