Haiou Pan

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2

BACKGROUND AIMS Human bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases. METHODS All stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining. RESULTS Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes. CONCLUSIONS The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell-based therapy for cartilage-related diseases.

Effects of tumor necrosis factor α on proliferation and expression of differentiated phenotypes in rabbit costal chondrocytes in culture

SummaryTumor necrosis factor α (TNFα) decreased the synthesis of glycosaminoglycan (GAG) in rabbit costal chondrocytes in culture, but did not stimulate the release of GAG from cell layers. Like chondrocytes cultured in control medium, chondrocytes cultured in the presence of TNFα produced putative “cartilage-specific” proteoglycans identified by density gradient centrifugation under dissociative conditions. Although TNFα decreased the synthesis of the proteoglycans, it did not change their monomeric size, which is a marker of cartilage phenotypes. Moreover, TNFα did not affect the responsiveness to parathyroid hormone, insulin-like growth factor I, or transforming growth factor β, which is known to stimulate GAG synthesis in cultured chondrocytes. TNFα decreased the alkaline phosphatase activity in the chondrocytes dose dependently. On the other hand, it stimulated their DNA synthesis slightly, but significantly. The stimulatory effect of TNFα on DNA synthesis was potentiated by fibroblast growth factor, epidermal growth factor, and fetal bovine serum. These findings suggest that in the presence of hormones and growth factors, TNFα promotes the proliferation of chondrocytes while suppressing their further differentiation at the stage of synthesis of cartilage-specific proteoglycans.

Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A

Membrane-bound transferrin-like protein (MTf), a glycosylphosphatidylinositol-anchored protein, is expressed at high levels in many tumors and in several fetal and adult tissues including cartilage and the intestine, as well as in the amyloid plaques of Alzheimers disease, although its role remains unknown. MTf is one of the major concanavalin A-binding proteins of the cell surface. In this study, we examined the effects of anti-MTf antibodies and concanavalin A on cell shape and gene expression, using cultures of chondrocytes and MTf-overexpressing ATDC5 and C3H10T1/2 cells. In cultures expressing MTf at high levels, concanavalin A induced cell-shape changes from fibroblastic to spherical cells, whereas no cell-shape changes were observed with wild-type ATDC5 or C3H10T1/2 cells expressing MTf at very low levels. The cell-shape changes were associated with enhanced proteoglycan synthesis and expression of cartilage-characteristic genes, including aggrecan and type II collagen. Some anti-MTf antibodies mimicked this action of concanavalin A, whereas other antibodies blocked the lectin action. The findings suggest that the crosslinking of MTf changes the cell shape and induces chondrogenic differentiation. MTf represents the first identification of a plant lectin receptor involved in cell-shape changes and the differentiation of animal cells.

Effects of Concanavalin A on Chondrocyte Hypertrophy and Matrix Calcification

Resting chondrocytes do not usually undergo differentiation to the hypertrophic stage and calcification. However, incubating these cells with concanavalin A resulted in 10-100-fold increases in alkaline phosphatase activity, binding of 1,25(OH)2-vitamin D3, type X collagen synthesis, 45Ca incorporation into insoluble material, and calcium content. On the other hand, other lectins tested (including wheat germ agglutinin, lentil lectin, pea lectin, phytohemagglutinin-L, and phytohemagglutinin-E) marginally affected alkaline phosphatase activity, although they activate lymphocytes. Methylmannoside reversed the effect of concanavalin A on alkaline phosphatase within 48 h. Concanavalin A did not increase alkaline phosphatase activity in articular chondrocyte cultures. In resting chondrocyte cultures, succinyl concanavalin A was as potent as concanavalin A in increasing alkaline phosphatase activity, the incorporation of [35S]sulfate, D-[3H]glucosamine, and [3H]serine into proteoglycans, and the incorporation of [3H]serine into protein, although concanavalin A, but not succinyl concanavalin A, induced a rapid change in the shape of the cells from flat to spherical. These findings suggest that concanavalin A induces a switch from the resting, to the growth-plate stage, and that this action of concanavalin A is not secondary to changes in the cytoskeleton. Chondrocytes exposed to concanavalin A may be useful as a novel model of endochondral bone formation.