Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures

Nanci, Antonio; Zalzal, S.; Gotoh, Y.; McKee, Marc D.

doi:10.1002/(sici)1097-0029(19960201)33:2<214::aid-jemt11>3.0.co;2-x

Cited by 83 publications

(61 citation statements)

References 47 publications

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“…As a control for our methods for demonstrating the presence of primary cilia, we used cilia expressing renal tubular derived MadinDarby canine kidney cells as a positive control. These cells have been shown to express cilia by immunohistochemical analysis using an antibody to ␣-tubulin (52) and by scanning electron microscopy (48). Both confluent MC3T3-E1 osteoblast and MLO-Y4 osteocyte-like cell lines (Fig.…”

Section: Evidence For Expression Of Pkd1 and Pkd2 And The Presence Ofsupporting

confidence: 91%

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Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

Xiao

Zhang

Mahlios

et al. 2006

Journal of Biological Chemistry

232

259

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We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1 m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1 m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1 m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1 m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblastrelated genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1 m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1 m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

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Section: Evidence For Expression Of Pkd1 and Pkd2 And The Presence Ofsupporting

confidence: 91%

“…After dehydration, the coverslips were mounted on a stub, sputtered with gold-palladium, and viewed with the FEI/Philips XL30 Field emission environmental scanning electron microscope (48).…”

Section: Measurement Of Alkaline Phosphatase Activity and Mineralizatmentioning

confidence: 99%

Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

Xiao

Zhang

Mahlios

et al. 2006

Journal of Biological Chemistry

232

259

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“…Opn binds directly to hydroxyapatite crystal surfaces both in vivo (64) and in vitro (34), and dephosphorylation of Opn by Tnap prevents much of its mineral binding and crystal growth inhibiting activity (56,65,66) (Fig. 5A).…”

Section: Discussionmentioning

confidence: 89%

“…5A). Our high resolution, immunogold localization of Opn at crystal growth sites in the extracellular matrix of MC3T3-E1 osteoblast cultures is consistent with previous data demonstrating a similar, electron microscopic localization of Opn in primary rat osteoblast cultures (34). Dose-dependent inhibition of calcification in vitro by the addition of exogenous Opn has been shown previously for vascular smooth muscle cell cultures (58).…”

Section: Discussionmentioning

confidence: 99%

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Addison

Azari

Sørensen

et al. 2007

Journal of Biological Chemistry

333

267

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Inorganic pyrophosphate (PP i ) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP i . This inhibition could be reversed by Tnap, but not if PP i was bound to mineral. PP i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP i , hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP i inhibits Tnap-mediated P i release from ␤-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

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“…Osteogenic cells were isolated by sequential trypsin/ collagenase digestion of calvarial bone from newborn (2-4 days) Wistar rats (Charles River Canada, St-Constant, Quebec, Canada), as described previously. 55,56 Cells were seeded in six-well plates (70 000 cells/well) and 24-well plates (20 000 cells/well) on glass coverslips. Cells were grown for 3 days in MEM with Earle's salts (MEM Earle's) supplemented with 10% FBS and 1% penicillinstreptomycin (Invitrogen).…”

Section: Cell Isolation Of Primary Calvaria-derived Osteogenic Cellsmentioning

confidence: 99%

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector

et al. 2006

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Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.

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Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures

Cited by 83 publications

References 47 publications

Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector

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