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

Isolated gonadotropin-releasing hormone (GnRH) deficiency, including Kallmann's syndrome (KS) and idiopathic hypogonadotropic hypogonadism (IHH), is a congenital disorder, which is characterized by a functional deficit in hypothalamic GnRH secretion. Despite recent advances in the understanding of the pathogenesis of the X-linked form of KS as the identification of the KAL gene (Xp22.3), the genetic basis of the sporadic form in female patients remains unclear. Although most searches for mutations in X chromosome have been reported in males, the newly recognized phenomenon of inheritance, such as genomic imprinting and uniparental disomy, raises the possibility of a female phenotype in the X- linked genetic defect. Here, the molecular study of the coding region of the KAL gene (exon 5 to 14) in 10 unrelated females with KS (n=6) or IHH (n=4) is reported. None of the subjects had familial histories of delayed puberty or hypogonadism. Samples from 4 healthy, unrelated female volunteers were used for identification of polymorphisms. PCR of the 10 exons of the KAL gene was performed on genomic DNA. The PCR products of the 10 exons were subject to single strand conformation polymorphism (SSCP) analysis to identify possible mutations. In an SSCP analysis of the amplified fragments (fragment size: 147 to 302bp), no mutations or polymorphisms were found in any of the 10 patients and 4 controls. In conclusion, it is unlikely that KAL gene mutations are a clinically significant cause of sporadic GnRH deficiency in female patients, indicating the existence of defects in unidentified genes that result in the expression of the phenotypes in females.
Adolescent ; Adult ; DNA Mutational Analysis ; Extracellular Matrix Proteins/*genetics ; Female ; Gonadorelin/*deficiency ; Human ; Kallmann Syndrome/*genetics/metabolism ; Nerve Tissue Proteins/*genetics ; Phenotype ; Polymorphism, Single-Stranded Conformational ; Support, Non-U.S. Gov't