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ABSTRACT: Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization.
Proceedings of the National Academy of Sciences 12/2012; · 9.68 Impact Factor
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ABSTRACT: To systematically identify novel gene functions essential for osteogenesis and skeletal mineralization, we performed a forward genetic mutagenesis screen in zebrafish and isolated a mutant that showed delayed skeletal mineralization. Analysis of the mutant phenotype in an osterix:nuclear-GFP transgenic background demonstrated that mutants contain osterix-expressing osteoblasts comparable to wild-type embryos. Positional cloning revealed a premature stop mutation in the macrophage-stimulating protein (msp) gene, predicted to result in a biologically inactive protein. Analysis of the embryonic expression pattern for the receptor for Msp, Ron, shows specific expression in the corpuscles of Stannius, a teleost-specific organ that produces stanniocalcin, a pivotal hormone in fish calcium homeostasis. Knockdown of Ron resulted in identical phenotypes as observed in msp mutants. Msp mutant embryos could be rescued by excess calcium. Consistent with a role for Msp/Ron in calcium homeostasis, calcium-regulating factors, such as pth1, pth2, stc1l, and trpv5/6 were significantly affected in msp mutant larvae. While Msp and Ron have previously been shown to play a critical role in a wide variety of biological processes, we introduce here the Msp/Ron signaling axis as a previously unappreciated player in calcium homeostasis and embryonic skeletal mineralization.-Huitema, L. F. A., Renn, J., Logister, I., Gray, J. K., Waltz, S. E., Flik, G., Schulte-Merker, S. Macrophage stimulating protein and calcium homeostasis in zebrafish.
The FASEB Journal 07/2012; 26(10):4092-101. · 5.71 Impact Factor
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ABSTRACT: Calcium is an essential ion serving a multitude of physiological roles. Aside from its role as a second messenger, it is an essential component of the vertebrate bone matrix. Efficient uptake and storage of calcium are therefore indispensable for all vertebrates. Transient receptor potential family, vanilloid type (TRPV)5 and TRPV6 channels are known players in transcellular calcium uptake, but the exact contribution of this pathway is unclear. We used forward genetic screening in zebrafish (Danio rerio) to identify genes essential in bone formation and identified a lethal zebrafish mutant (matt-und-schlapp) with severe defects in bone formation, including lack of ossification of the vertebral column and craniofacial structures. Mutant embryos show a 68% reduction in calcium content, and systemic calcium homeostasis is disturbed when compared with siblings. The phenotype can be partially rescued by increasing ambient calcium levels to 25 mM. We identified the mutation as a loss-of-function mutation in the single orthologue of TRPV5 and 6, trpv5/6. Expression in HEK293 cells showed that Trpv5/6 is a calcium-selective channel capable of inward calcium transport at physiological concentrations whereas the mutant channel is not. Taken together, this study provides both genetic and functional evidence that transcellular epithelial calcium uptake is vital to sustain life and enable bone formation.
The FASEB Journal 06/2011; 25(9):3197-207. · 5.71 Impact Factor
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ABSTRACT: During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures ( approximately 40 microm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.
Biochimica et Biophysica Acta 10/2007; 1774(9):1108-17. · 4.66 Impact Factor
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ABSTRACT: Mineralization is an essential requirement for normal skeletal development, but under certain pathological conditions organs like articular cartilage and cardiovascular tissue are prone to unwanted mineralization. Recent findings suggest that the mechanisms regulating skeletal mineralization may be similar to those regulating pathological mineralization. In general, three forms of cell-mediated mineralization are recognized in an organism: intramembranous ossification, endochondral ossification and pathological mineralization. This review summarizes recent work that tried to elucidate how cell-mediated mineralization is initiated and regulated. To explain mineralization, several theories have been proposed. One theory proposes that mineralization is initiated within matrix vesicles (MVs). A second, not mutually exclusive, theory proposes that phosphate induces apoptosis, and that apoptotic bodies nucleate crystals composed of calcium and phosphate. A third theory suggests that mineralization is mediated by certain non-collagenous proteins, which associate with the extracellular matrix. Regardless of the way mineralization is initiated, the organism also actively inhibits mineralization by specific proteins and removal of an inhibitor may also induce mineralization. Although many studies greatly contributed to a better understanding of the mechanisms regulating cell-mediated mineralization, many questions remain about the mechanisms that trigger cell-mediated mineralization and how this process is regulated. Further investigation is necessary to develop in the future novel therapeutic strategies to prevent pathological mineralization.
Frontiers in Bioscience 02/2007; 12:2631-45. · 3.52 Impact Factor
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ABSTRACT: Sodium nitroprusside (SNP) is a nitric oxide (NO) donor drug, which is therapeutically used as a vasodilating drug in heart transplantations. In our previous study it was found that SNP at a concentration of 100 microM inhibited mineralization in a cell culture system, indicating that the beneficial effects of this drug may also include inhibition of vascular calcification. The aim of this study was to investigate which bioactive compounds generated from SNP inhibit mineralization. ATDC5 cells were grown for 14 days and mineralization was induced by addition of 5 mM phosphate for 24 h. Mineralization was determined by staining precipitated calcium with an alizarin red stain. It was found that the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine were not able to inhibit mineralization and NO scavengers could not antagonize the inhibiting effect of SNP on mineralization. The iron chelator deferoxamine (200 microM) antagonized the inhibiting effect on mineralization mediated by SNP and ammonium iron sulfate inhibited mineralization in a dose-dependent manner (10-100 microM). Furthermore, iron ions (30 microM) were detected to be released from SNP in the cell culture. These data show that the iron moiety of sodium nitroprusside, rather than nitric oxide inhibits mineralization.
European Journal of Pharmacology 09/2006; 542(1-3):48-53. · 2.52 Impact Factor
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ABSTRACT: During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures (∼ 40 μm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.
Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics.
