In fully activated cells, expression of PPAR isoforms was not detected, whereas expression of α-SMA and COL1A2 dramatically increased. Similar to the progression in the expression pattern of these genes and consistent with activated HSCs being a major LDK378 manufacturer source of ADAMTS1, ADAMTS1 mRNA expression was undetectable in quiescent HSCs (days 1-4), enhanced in cultured HSCs (10±0.75-fold increase at day

11) and strongly increased after 4 passages (150- to 200-fold increase; Fig. 2C). In contrast, thrombospondin, previously reported to be present in isolated rat HSCs,22 was expressed early in culture and reached an increase of 11.58±0.24-fold at day 11, but its levels were strongly diminished in myofibroblast-like cells. To avoid interference from thrombospondin activity, human HCSs were routinely used between passages 4 and 10 in all subsequent Enzalutamide order experiments. Up-regulation of ADAMTS1 expression was confirmed by western blotting in both activated HSCs and fibrotic liver tissues relative

to normal livers. The processed 87-kDa ADAMTS1 active form (see Fig. 2D) was recovered mainly in cell extracts and HSC-conditioned media (Fig. 2E) and was clearly induced in liver fibrosis (Fig. 2F). The 110-kDa unprocessed form was only present in cell extracts, and the 65-kDa shorter form was recovered only in conditioned media (Fig. 2E). A major feature of ADAMTS1 is the presence of three thrombospondin type 1 motifs (TSP1), with the proximal TSP1 being separated from the two carboxy-end TSP1 motifs by a “spacer sequence” rich in cysteine residues (Fig. 2D). Next to the proximal TSP1 sequence, we identified a KTFR motif that aligns with the active KRFK sequence of the human thrombospondin TSP1 repeats previously shown to be involved in the interaction with TGF-β (Fig. 3A).23 A tryptophan-rich peptide (WxxW), described as a docking site that promotes the interaction of KRFK sequences with LAP-TGF-β, is also present in the proximal TSP1 motif of ADAMTS1. The WxxW and KxFx motifs are not present in the two carboxy-end TSP1 motifs of ADAMTS1 (not shown). Because the proximal TSP1-containing domain of ADAMTS1 resembles that of 上海皓元 thrombospondin, we asked whether it

might display a structural organization, allowing for interactions with TGF-β (Fig. 3B). An “hhsearch” against the Protein Data Bank (see Supporting Information) identified the following candidate structural templates for ADAMTS1 TSP-like and thrombospondin domains (P values < 10−15): ADAMTS23 (PDB:3ghm); ADAMTS5 (PDB:2rjq); the TSP1 type 1 repeat (PDB:1lsl); F-spondin (PDB:1vex, 1szl); the thrombospondin anonymous protein, Trap (PDB:2bbx); VAP1 (PDB:2ero); Properdin (PDB:1w0r); Catrocollastatin (PDB:2dw0), and the Vitelline membrane outer layer protein I (PDB:1vmo). Except for 2dw0, 2ero, and 1vmo, all matching structural templates have a triple-strand organization, suggesting that this TSP1-like structure is shared by both the TSP1 repeat from thrombospondin and the motif found in ADAMTS1 (Fig. 3C).