Noboru Yamaji

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

The Fibronectin Extra Domain A Activates Matrix Metalloproteinase Gene Expression by an Interleukin-1-dependent Mechanism

The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3.6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III11 or III12, scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1α and IL-1β, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH2 or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH2terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue.

A Kinase Domain-truncated Type I Receptor Blocks Bone Morphogenetic Protein-2-induced Signal Transduction in C2C12 Myoblasts

Members of the transforming growth factor (TGF)-β superfamily bind the transmembrane serine/threonine kinase complex consisting of type I and type II receptors. Their intracellular signals are propagated via respective type I receptors. Bone morphogenetic protein (BMP)-2, a member of the TGF-β superfamily, induces ectopic bone formation when implanted into muscular tissues. Two type I receptors (BMPR-IA and BMPR-IB) have been identified for BMP-2. We have reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts their differentiation pathway into that of osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994) J. Cell Biol. 127, 1755–1766). In the present study, we examined the involvement of functional BMP-2 type I receptors in signal transduction in C2C12 cells, which expressed mRNA for BMPR-IA, but not for BMPR-IB in Northern blotting. TGF-β type I receptor (TβR-I) mRNA was also expressed in C2C12 cells. Subclonal cell lines of C2C12 that stably expressed a kinase domain-truncated BMPR-IA (ΔBMPR-IA) differentiated into myosin heavy chain-expressing myotubes but not into alkaline phosphatase (ALP)-positive cells, even in the presence of BMP-2. In contrast, the differentiation of the ΔBMPR-IA-transfected C2C12 cells into myotubes was suppressed by TGF-β1, as in the parental C2C12 cells. BMP-2 did not efficiently suppress the mRNA expression of muscle-specific genes such as muscle creatine kinase, MyoD, and myogenin, nor did it induce the expression of ALP mRNA in the ΔBMPR-IA-transfected C2C12 cells. In contrast, TGF-β1 inhibited mRNA expression of the muscle-specific genes in those cells. When wild-type BMPR-IA was transiently transfected into the ΔBMPR-IA-transfected C2C12 cells, a number of ALP-positive cells appeared in the presence of BMP-2. Transfection of wild-type BMPR-IB or TβR-I failed to increase the number of ALP-positive cells. These results suggest that the BMP-2-induced signals, which inhibit myogenic differentiation and induce osteoblast differentiation, are transduced via BMPR-IA in C2C12 myoblasts.