Bone Acidic Glycoprotein-75 Delineates the Extracellular Sites of Future Bone Sialoprotein Accumulation and Apatite Nucleation in Osteoblastic Cultures

Midura, Ronald J.; Wang, Aimin; Lovitch, Dinah; Law, Douglas; Powell, Kimerly; Gorski, Jeff P.

doi:10.1074/jbc.m312409200

Cited by 73 publications

(76 citation statements)

References 58 publications

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“…It is important to note that the decreases observed in the blocking experiments were less than that observed when inhibiting phosphorylation of these proteins with the CK2 inhibitors or dephosphorylation with phosphoprotein phosphatases. This implies that other phosphoproteins, e.g., matrix extracellular phosphoprotein (MEPE), DMP1, and bone acidic glycoprotein (BAG-75), all expressed by chondrocytes [58][59][60][61] may be involved in cartilage calcification. These will either have to be examined in future studies when antibodies that cross react with chick are available or in cultures from a different species for which such antibodies are available.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…We propose that such large protein inhibitors of calcification may paradoxically promote mineralization of the collagen fibril by selectively inhibiting apatite growth everywhere but within the fibril. Other proteins that are too large to penetrate the fibril may nucleate mineral formation as follows: proteins such as bone sialoprotein (44,45) and the recently discovered serum nucleator of collagen calcification (26) as well as large structures such as matrix vesicles (46). We propose that such proteins generate apatite crystal nuclei outside of the collagen fibril, and that some of these small crystals can then diffuse into the interior of the fibril and grow.…”

Section: Test Molecule Massmentioning

confidence: 99%

The Size Exclusion Characteristics of Type I Collagen

Toroian

Lim

Price

2007

Journal of Biological Chemistry

210

110

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The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.

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“…Immunogold labeling has shown that both BSP and OPN appear at mineralization foci (Figure 10 A) and thereafter pack in the spaces between collagen fibrils (Figure 10 B). Although BSP has been reported to accumulate and colocalize with bone acidic glycoprotein-75 at sites of mineral nucleation Midura et al 2004), a recent report revealed only slight mineral deficit in BSP knockout mice (Malaval et al 2008). The presence of lectin cytochemistry and immunolabeling for BSP and OPN have further revealed that locally, within a same bone tissue there are areas that can be either poor or enriched in NCPs (Nanci 1999).…”

Section: Distribution Of Noncollagenous Matrix Proteins In Bonementioning

confidence: 99%

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

Nanci¹,

Wazen²,

Nishio³

et al. 2009

Eur J Histochem

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The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of "biochemistry on section", to correlate composition with structure. For cytochemical characterizations, including immunolabeling, our laboratory has opted for colloidal gold labelings and pioneered their application to calcified tissues because they yield high spatial resolution and are quantitative. Over the years, this approach has been applied to identify and map various NCPs in bone and teeth and, in this review of our work, we will emphasize some selected studies that highlight it application to also achieve functional information.

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Bone Acidic Glycoprotein-75 Delineates the Extracellular Sites of Future Bone Sialoprotein Accumulation and Apatite Nucleation in Osteoblastic Cultures

Cited by 73 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

The Size Exclusion Characteristics of Type I Collagen

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

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