ascorbic-acid and arginyl-glycyl-aspartic-acid

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Topics: 3T3 Cells; Animals; Antibodies, Monoclonal; Ascorbic Acid; Cell Differentiation; Collagen; Collagenases; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Kinetics; Matrix Metalloproteinase 13; Mice; Oligopeptides; Osteocalcin; Osteopontin; Proline; Recombinant Proteins; Sialoglycoproteins; Transfection

Chondrocyte terminal differentiation is associated with cellular hypertrophy increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro studies indicated that ascorbic acid-induced chondrocyte alkaline phosphatase activity was inhibited by the addition of both collagen and proteoglycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phosphatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation. In addition, supplementation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vitamin D receptor number. Ascorbic acid-treated chondrocytes maintained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unaltered. These results indicate that ascorbic acid promotes both chondrocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor-dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte terminal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Cartilage; Cell Differentiation; Cells, Cultured; Chickens; Chondrocytes; Collagen; Extracellular Matrix; Growth Plate; Iron Chelating Agents; Lipid Peroxidation; Oligopeptides; Proteoglycans; Receptors, Calcitriol; Vitamin D; Vitamin E

The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).

Topics: Alkylation; Androstadienes; Ascorbic Acid; Cells, Cultured; Chloroquine; Endocytosis; Fibroblasts; Heparin; Humans; Integrins; Lysosomes; Oligopeptides; Proteoglycans; Receptors, Vitronectin; Vitronectin; Wortmannin