We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently,CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.

We readily produced recombinant pro-macrophage stimulating protein in a mammalian expression system, but it was only weakly active after proteolytic activation. Active macrophage stimulating protein is a disulfide-bonded heterodimer, but in our hands, the subunits of recombinant macrophage stimulating protein were mostly not disulfide bonded. 3 Ala macrophage stimulating protein were fully disulfide linked, and the mutant macrophage stimulating protein had 10 -20-fold higher specific activity than the wild type recombinant macrophage stimulating protein.Macrophage-stimulating protein (MSP) 1 was originally purified from human serum as a protein that enhances the chemotactic response of murine peritoneal macrophages to the C5a fraction of complement (1). In a screen for proteins that contain kringle domains, Degen and co-workers (2) identified a cDNA that was most closely related to hepatocyte growth factor (HGF) and hence named it HGF-like (HGF-L). MSP and HGF-L were later shown to be the same molecule (3). MSP and HGF (4) are plasminogen-related growth factors and appear to exert their biological effects as ligands for the receptor protein tyrosine kinases RON (5, 6) and MET (7), respectively. The murine orthologue of human RON is also known as STK (8, 9). Like plasminogen, these growth factors require proteolytic activation for biological activity, which occurs between homologous Arg-Val residues and generates heterodimeric disulfidebonded subunits (10). The ␣ subunit contains four kringle domains, whereas the ␤ subunit contains an inactive serine protease domain (SPD). It is not established which protease is responsible for the in vivo activation of MSP, but several proteases, such as human plasma kallikrein, are reported to efficiently activate MSP in vitro (10). The biological role of the RON/MSP cognate receptor/ligand pair is still unclear. Experimental strategies to address this issue include analysis of the expression pattern of RON in situ, 2 and a survey of the effects of MSP in a variety of in vitro bioassays. Because the latter requires a steady supply of MSP, we began producing recombinant MSP. Our initial attempts to produce recombinant MSP generated preparations with a dramatically reduced specific activity (ϳ50-fold) as compared with material that was purified from a nonrecombinant source. The following studies were undertaken in attempts to determine the underlying mechanism for the reduced activity and to investigate how the activity might be restored to levels observed with naturally produced protein. (11)) was a gift of Professor Sandra Degen, University of Cincinnati. The Cys 672 3 Ala MSP mutant was constructed by polymerase chain reaction from the MSP cDNA with a mutant primer containing a change from TG to GC at nucleotides 2024 and 2025 (5Ј-CACAACGCCTGGGTCCTGGAAG-3Ј) and a downstream primer (5Ј-CTGGCAACTAGAAGGCACAGTCG-3Ј) complementary to vector pCDNA3 (Invitrogen, San Diego, CA). All contructs were verified by DNA sequencing. MATERIALS AND METHODS Construction of MSP cDNAs-The human MS...