Adenosine 5′‐triphosphate promotes mineralization in differentiating chick limb‐bud mesenchymal cell cultures

Boskey, Adele L.; Doty, Stephen B.; Binderman, Itzhak

doi:10.1002/jemt.1070280605

Cited by 32 publications

(22 citation statements)

References 58 publications

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“…As previously reported [18][19][20][21][22][23][24][25][26], the mesenchymal cells differentiated in culture and formed alcian blue positive chondrocyte nodules. At day 14 (figure 1a), the cultures began to show trace amounts of von Kossa positive staining.…”

Section: Characterization Of Culturessupporting

confidence: 82%

“…Chick limb-bud mesenchymal cells plated in high-density micromass cultures differentiate to form a mineralizable matrix that resembles the growth plate in the chick [18][19][20][21][22][23][24][25][26]. Our earlier studies using 2,3-diphosphoglycerate and heparin, both non-specific inhibitors of CK2, showed inhibition of mineralization when protein phosphorylation was blocked [18].…”

Section: Introductionmentioning

confidence: 95%

“…Kinase and Phosphatase Activities-To define concentrations of inhibitors for analysis, a series of pilot experiments were performed in which a wide range of concentrations for each inhibitor starting with those reported in the literature [18,21,[33][34][35][36][37] were added either continuously from day 5, 7, 9 or 11, or once (acutely) at day 5,7,9,11, or 14. Where treatments caused visible cell death and cultures lifted off the culture dish no further analysis was done.…”

Section: Studies Of Effects Of Inhibitingmentioning

confidence: 99%

“…Accumulation of 45 Ca per micromass spot was calculated and data normalized to the accumulation in the mineralizing control culture at day 21 or 23. While historically we had corrected for 45 Ca uptake by the non-mineralizing controls [18][19][20][21][22][23][24][25][26], in initial experiments it was noted that this correction caused a difference of less than 1%; hence data are presented without this correction. However it should be noted that a single set of experiments for each inhibitor or blocking agent was performed to validate the fact that the controls treated with the agent in question were not binding more calcium than the untreated controls.…”

Section: Studies Of Effects Of Inhibitingmentioning

confidence: 99%

“…It is our hypothesis that it is the phosphorylation of the anionic matrix proteins that is important, rather than the ability of enzymes such as alkaline phosphatase to release inorganic phosphate from these proteins. To test this hypothesis mineralization was monitored in a culture system that mimics endochondral ossification [19][20][21][22][23][24][25][26]. The cultures were challenged with inhibitors of CK2 and of phosphoprotein phosphatases, and with blocking antibodies to selected phosphorylated proteins.…”

Section: Introductionmentioning

confidence: 99%

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Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Boskey

Doty

Kudryashov

et al. 2008

Bone

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Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45 Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating the phosphatases were, in part, providing a source of inorganic phosphate.To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

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Section: Characterization Of Culturessupporting

confidence: 82%

Section: Introductionmentioning

confidence: 95%

Section: Studies Of Effects Of Inhibitingmentioning

confidence: 99%

Section: Studies Of Effects Of Inhibitingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Boskey

Doty

Kudryashov

et al. 2008

Bone

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Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

et al. 1997

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In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10(-10) M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-1R microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on 45Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization.

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Type I collagen influences cartilage calcification: An immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

et al. 2000

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Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.

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Adenosine 5′‐triphosphate promotes mineralization in differentiating chick limb‐bud mesenchymal cell cultures

Cited by 32 publications

References 58 publications

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

Type I collagen influences cartilage calcification: An immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

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