A novel chordin-like

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family. These molecules play important roles during many organogenic processes, even though they were originally identified as factors promoting the ectopic formation of cartilage and bone. The concentration of active BMP is controlled in part by inhibitors from three BMP binding protein families: short gastrulation/chordin, noggin and cerberus (reviewed by Balemans and Van Hul, 2002). Among these, chordin and noggin were the first proteins found to inhibit the activity of bound BMPs by preventing interactions with their BMP receptors (Piccolo et al., 1996; Zimmerman et al., 1996). Although noggin is encoded by a single gene in mammals, chordin belongs to a family of proteins that share a cysteine-rich pro-collagen repeat [or chordin-like cysteine-rich repeat (CR)], which is also found in various extracellular matrix proteins (reviewed by Garcia Abreu et al., 2002). Without exception, the homology between chordin family members lies within their CRs. The chordin polypeptide contains four CRs, of which the first and the third (CR1 and CR3) are responsible for BMP binding (Larrain et al., 2000). Binding of chordin to BMP4 is specific and tight (Piccolo et al., 1996). Proteolysis by Tolloid (or BMP1), which liberates CR1 and CR4 from chordin, is required to release bound BMP4 (Piccolo et al., 1997; Scott et al., 1999). The importance of CR for BMP interactions has been strengthened by the recent finding that connective tissue growth factor functions as a BMP-binding inhibitor, and that its single CR domain is essential for this activity (Abreu et al., 2002). We previously described a small chordin-like secreted protein, CHL1 (for chordin-like 1, re-designated from CHL), a novel BMP-binding inhibitor with three CRs (Nakayama et al., 2001). CHL1 was isolated originally from mouse bone marrow stromal cells. Interestingly, CHL1 expression 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.