ascorbic-acid and 3-4-dehydroproline

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

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)-rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP-inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking-antibodies to the FN receptor alpha 5 beta 1 integrin and by the proline analog 3,4-dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up-regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up-regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin alpha 5 beta 1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.

Topics: Adult; Alkaline Phosphatase; Antibodies; Ascorbic Acid; Cells, Cultured; Collagen; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Middle Aged; Proline; Receptors, Fibronectin; Up-Regulation

Osteocalcin is a hormonally regulated calcium-binding protein made almost exclusively by osteoblasts. In normal cells, osteocalcin expression requires ascorbic acid (AA), an essential cofactor for osteoblast differentiation both in vivo and in vitro. To determine the mechanism of this regulation, subclones of MC3T3-E1 preosteoblasts were transiently transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter driving expression of firefly luciferase. AA stimulated luciferase activity 20-fold after 4-5 days. This response was stereospecific to L-ascorbic acid and was only detected in MC3T3-E1 subclones showing strong AA induction of the endogenous osteocalcin gene. Similar results were also obtained in MC3T3-E1 cells stably transfected with the osteocalcin promoter. A specific inhibitor of collagen synthesis, 3,4-dehydroproline, blocked AA-dependent induction of promoter activity, indicating that regulation of the osteocalcin gene requires collagen matrix synthesis. Deletion analysis of the mOG2 promoter identified an essential region for AA responsiveness between -147 and -116 bp. This region contains a single copy of the previously described osteoblast-specific element, OSE2. Deletion and mutation of OSE2 in DNA transfection assays established the requirement for this element in the AA response. Furthermore, DNA-binding assays revealed that MC3T3-E1 cells contain OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to OSE2 is up-regulated by AA treatment. Taken collectively, our results indicate that an intact OSE2 sequence is required for the induction of osteocalcin expression by AA.

Topics: Animals; Ascorbic Acid; Binding Sites; Cell Adhesion Molecules; Cells, Cultured; Collagen; Mice; Nuclear Proteins; Osteoblasts; Osteocalcin; Proline; Promoter Regions, Genetic; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Time Factors; Transfection

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and beta-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per microgram DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1! cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Binding, Competitive; Cell Differentiation; Cells, Cultured; Collagen; Cyclic AMP; DNA; Extracellular Matrix; Gene Expression Regulation; Glycerophosphates; Mice; Osteoblasts; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proline; Proteins; Rats; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; RNA, Messenger

During development and fracture repair, endochondral bone formation is preceded by an orderly process of chondrocyte hypertrophy and cartilage matrix calcification. Analysis of calcifying versus noncalcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel collagen, type X, and decreased synthesis of type II collagen, the major component of cartilage matrix. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo these changes in gene expression and to produce calcified matrix by exposure to ascorbic acid. The mechanism by which ascorbate produces these changes has been examined by analyzing the effect of the vitamin on prehypertrophic chick embryo sternal chondrocytes. Nuclear run-on assays demonstrated that ascorbate alters mRNA levels in chondrocytes by changing the transcription rates. The fact that marked changes in mRNA levels require 1-2 days of ascorbate exposure suggested that the effect of this vitamin on gene transcription may be secondary to other, earlier ascorbate-induced effects. Since cells cultured with ascorbate produce a collagen-enriched matrix, we examined the hypothesis that transcriptional changes were secondary to altered cell-matrix interactions. Chondrocytes were cultured after attachment to tissue culture plastic, in suspension, or on plates coated with collagen type I. Comparison of alkaline phosphatase activity with and without ascorbate addition demonstrated that under all of these conditions, induction of enzyme was dependent on the presence of ascorbate. When plates containing ascorbate-conditioned chondrocyte matrix were used as substrate for naive chondrocytes, the cells continued to require ascorbate for induction of high levels of alkaline phosphatase and type X collagen mRNA. Addition of the hydroxylation inhibitor, 3,4-dehydroproline, caused marked inhibition of collagen secretion as well as accumulation of underhydroxylated collagens within the cells. However, even in the presence of this inhibitor ascorbate was effective in inducing elevated alkaline phosphatase and type X collagen. These results indicate that the ability of ascorbate to induce chondrocyte hypertrophy does not depend on production of a collagen-rich matrix.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Azetidinecarboxylic Acid; Blotting, Northern; Bone Development; Cartilage; Cells, Cultured; Chick Embryo; Collagen; Enzyme Induction; Extracellular Matrix; Gene Expression Regulation; Hydroxyproline; Kinetics; Procollagen; Procollagen-Proline Dioxygenase; Proline; RNA, Messenger; Transcription, Genetic