Identification of tristetraprolin as a factor that modulates the stability of the TAFI transcript through binding to the 3'-untranslated region.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen encoded by the human gene CPB2. TAFI constitutes a molecular link between coagulation and fibrinolysis, and between coagulation and inflammation. The 3'-untranslated region (UTR) of the human CPB2 mRNA plays a key role in regulating CPB2 mRNA abundance, but the exact mechanisms that mediate this regulation are largely unexplored. OBJECTIVES: To pinpoint cis-acting elements in the CPB2 3'-UTR that act as stability determinants and to identify protein factors binding to these sites. METHODS: We constructed a series of plasmids encoding mRNAs containing rabbit ß-globin sequences (as a reporter) fused to sequences of the CPB2 3'-UTR (encompassing 5' and internal deletions). These plasmids were transfected into HepG2 (human hepatoma) cells and the stability of the fusion transcripts measured. We performed a series of gel mobility shift analyses using RNA probes encompassing putative (in)stability elements. RESULTS: We identified one element conferring stability and three elements conferring instability. Supershift assays identified the protein bound to the site between the second and third polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP site abolished TTP binding in gel mobility shift assays and also stabilized ß-globin/CPB2 fusion transcripts. TTP knockdown stabilized the fusion transcript containing the TTP site, but not a fusion transcript in which this site was mutated. CONCLUSIONS: Our findings are indicative of a role for TTP in constitutive, and perhaps regulated, control of CPB2 mRNA stability and hence abundance.

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy.

OBJECTIVE: This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy. METHOD: In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-ß (TGFß) following 21 days of culture in either growth or chondrogenic media. RESULTS: In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFß. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets. CONCLUSION: The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.

Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair.

OBJECTIVE: Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone morphogenetic protein 7 (BMP-7) induces cartilage differentiation, we hypothesized that the addition of the morphogen would improve the repair tissue generated by microfracture. We determined the effects of these two treatments alone and in combination on the quality and quantity of repair tissue formed in a model of full-thickness articular cartilage injury in adolescent rabbits. DESIGN: Full-thickness defects were made in the articular cartilage of the patellar grooves of forty, 15-week-old rabbits. Eight animals were then assigned to (1) no further treatment (control), (2) microfracture, (3) BMP-7, (4) microfracture with BMP-7 in a collagen sponge (combination treatment), and (5) microfracture with a collagen sponge. Animals were sacrificed after 24 weeks at 39 weeks of age. The extent of healing was quantitated by determining the thickness and the surface area of the repair tissue. The quality of the repair tissue was determined by grading specimens using the International Cartilage Repair Society Visual Histological Assessment Scale. RESULTS: Compared to controls, BMP-7 alone increased the amount of repair tissue without affecting the quality of repair tissue. Microfracture improved both the quantity and surface smoothness of repair tissue. Compared to either single treatment, the combination of microfracture and BMP-7 increased both the quality and quantity of repair tissue. CONCLUSIONS: Microfracture and BMP-7 act synergistically to stimulate cartilage repair, leading to larger amounts of repair tissue that more closely resembles native hyaline articular cartilage.

Association of the transmembrane TGF-alpha precursor with a protein kinase complex.

A variety of growth factors including transforming growth factor-alpha (TGF-alpha) are synthesized as transmembrane precursors. The short cytoplasmic domain of the transmembrane TGF-alpha precursor lacks any apparent motif associated with signal transduction. However, the sequence conservation of this cytoplasmic domain and its abundance of cysteine residues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest a biological function. In this study, we showed that transmembrane TGF-alpha was rapidly internalized after interaction with a specific antibody and that this internalization was greatly decreased when the COOH-terminal 31 amino acids were removed. Chemical cross-linking experiments revealed two associated proteins of 86 and 106 kD which coimmunoprecipitated with the TGF-alpha precursor. The association of p86 was dependent on the presence of the COOH-terminal cytoplasmic 31 amino acids of the TGF-alpha precursor, whereas p106 still remained associated when this segment was deleted. In addition, p106 was tyrosine-phosphorylated and exposed on the cell surface. The protein complex associated with transmembrane TGF-alpha displayed kinase activities towards tyrosine, serine, and threonine residues. These activities were not associated with transmembrane TGF-alpha when the COOH-terminal segment was truncated. The association of a protein kinase complex with transmembrane TGF-alpha may provide the basic elements for a "reverse" mode of signaling through the cytoplasmic domain of this growth factor, which may lead to two-directional communication during ligand-receptor interaction.