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. 2016 Apr 13;30(8):2741–2754. doi: 10.1096/fj.201600261R

Figure 9.

Figure 9.

ECM1 directly associated with PGRN growth factor and inhibited the chondroinductive activity of PGRN. A) ECM1 associated with PGRN directly (solid-phase binding). Microtiter plate coated with PGRN was incubated with various amounts of ECM1 or bovine serum albumin (BSA), and ECM1 bound to PGRN was detected by corresponding antibodies. B) Schematic representation of various ECM1a deletion mutants fused to GST. Amino acid residue numbers are indicated. The predicted molecular weights (kDa) of each recombinant fusion fragment are shown on the right. C) ΔPx/COOH domain of ECM1 is directly associated with PGRN assayed by pull-down assay. Purified ECM1 mutants fused to GST were separated on SDS-PAGE and visualized by Coomassie blue staining. D) PGRN unit E and its linker regions were responsible for binding with ECM1 protein, assayed by yeast-2 hybrid. All motifs of PGRN were isolated and cultured, and yeast-2 hybrid was performed. Schematic diagram of PGRN constructs used to map those of its fragments that bind to ECM1 (left). Galactosidase assays (right). E) ECM1 reduced PGRN localization on surface of C28i2 and rat chondrosarcoma cells chondrocyte progenitor cell lines. F–H) ECM1a inhibited PGRN-stimulated chondrogenesis. Total RNA was prepared from micromass cultures of C3H10T1/2 cells treated with 250 ng/ml recombinant PGRN in the absence or presence of 250 ng/ml recombinant ECM1 for 7 d, and mRNA expression of Col X (F), cartilage oligomeric matrix protein (COMP) (G), aggrecan (H), and GAPDH (serving as an internal control) were examined by RT-PCR. I) A proposed model for the role and regulation of ECM1 in chondrogenesis. ECM1, a downstream mediator of PTHrP, forms a positive regulatory loop with PTHrP and negatively regulates chondrogenesis. In addition, ECM1 also associates with PGRN, a chondrogenic growth factor that binds to TNF receptor (TNFR). Reciprocal inhibition between ECM1 and PGRN may contribute, at least in part, to ECM1-mediated suppression of chondrogenesis. PPR, PTH/PTHrP receptor. Values are means ± sd. *P < 0.05 vs. PBS treatment control group. #P < 0.05 between PGRN alone treatment group and additional use of ECM1.