Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.
Animals ; Bone Morphogenetic Proteins/metabolism ; Cartilage/*cytology/metabolism ; *Cell Differentiation ; Chondrocytes/cytology/enzymology ; Extracellular Matrix Proteins/deficiency/*metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Integrin alpha5/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Mice ; Neoplasm Proteins/deficiency/*metabolism ; Osteogenesis ; Protein Binding ; Signal Transduction ; Smad Proteins/metabolism

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.
Animals ; Collagen ; chemistry ; metabolism ; Humans ; Integrins ; metabolism ; Magnetic Resonance Spectroscopy ; Molecular Dynamics Simulation ; Peptides ; chemistry ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Signal Transduction