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Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

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Resolving polyP distribution by spectral isolation of DAPI 580 nm emission and attenuation by alkaline phosphatase (ALP) treatment.(A) Control growth plate section; (B) a different control growth plate section (higher magnification); (C) ALP-treated (+ALP) growth plate section. First column, green: 430 nm emission (DAPI-DNA) Second column, red: 580 nm emission (DAPI-polyP). The third column is an overlay of the 430 and 580 nm images to show the spatial distribution of polyP that occurred primarily in the hypertrophic zone of control sections but was absent in ALP-treated sections. The spectral analyses in the last column show profiles for the various ROI outlined in the smaller inset image. Growth plate regions analyzed included the region above the hypertrophic matrix (red ROI), in the hypertrophic matrix (green ROI), and in the bone (blue ROI). The emission profile of each control (−ALP) hypertrophic matrix (green ROI) shows a shift to a higher wavelength emission compared to the other −ALP regions, suggesting the presence of polyP. All regions analyzed in +ALP sections showed an emission profile similar to that of DAPI-DNA, suggesting polyP was hydrolyzed from the hypertrophic matrix after exposure to active ALP. All scale bars represent 150 µm.
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pone-0005634-g005: Resolving polyP distribution by spectral isolation of DAPI 580 nm emission and attenuation by alkaline phosphatase (ALP) treatment.(A) Control growth plate section; (B) a different control growth plate section (higher magnification); (C) ALP-treated (+ALP) growth plate section. First column, green: 430 nm emission (DAPI-DNA) Second column, red: 580 nm emission (DAPI-polyP). The third column is an overlay of the 430 and 580 nm images to show the spatial distribution of polyP that occurred primarily in the hypertrophic zone of control sections but was absent in ALP-treated sections. The spectral analyses in the last column show profiles for the various ROI outlined in the smaller inset image. Growth plate regions analyzed included the region above the hypertrophic matrix (red ROI), in the hypertrophic matrix (green ROI), and in the bone (blue ROI). The emission profile of each control (−ALP) hypertrophic matrix (green ROI) shows a shift to a higher wavelength emission compared to the other −ALP regions, suggesting the presence of polyP. All regions analyzed in +ALP sections showed an emission profile similar to that of DAPI-DNA, suggesting polyP was hydrolyzed from the hypertrophic matrix after exposure to active ALP. All scale bars represent 150 µm.

Mentions: Figure 5 compares representative individual scans and overlays of emissions at a wavelength indicative of DAPI-DNA (430 nm, green) and DAPI-polyP (580 nm, red) for the control (−ALP; Figure 5A,B) and ALP-treated (+ALP; Figure 5C) murine vertebral body sections. When we displayed the DAPI fluorescence images collected at 580 nm with respect to 430 nm, we found a unique spatial distribution for the two wavelength ranges (Figure 5, overlay column). Thus, the DAPI emission above 580 nm may be a good index of polyP distribution. The spectral analyses on the right show representative profiles for the growth plate regions above the hypertrophic matrix (resting zone, red outline in inset), within the hypertrophic matrix (green outline), and within the bone (blue outline). The emission profiles of the hypertrophic matrix in control sections (Figure 5A,B) showed a shift to longer wavelengths compared to the other regions, suggesting the presence of polyPs. An overlay of all control profiles over the hypertrophic matrix region (Figure 6A) features a long tail of appreciable DAPI fluorescence extending beyond 580 nm.


Control of vertebrate skeletal mineralization by polyphosphates.

Omelon S, Georgiou J, Henneman ZJ, Wise LM, Sukhu B, Hunt T, Wynnyckyj C, Holmyard D, Bielecki R, Grynpas MD - PLoS ONE (2009)

Resolving polyP distribution by spectral isolation of DAPI 580 nm emission and attenuation by alkaline phosphatase (ALP) treatment.(A) Control growth plate section; (B) a different control growth plate section (higher magnification); (C) ALP-treated (+ALP) growth plate section. First column, green: 430 nm emission (DAPI-DNA) Second column, red: 580 nm emission (DAPI-polyP). The third column is an overlay of the 430 and 580 nm images to show the spatial distribution of polyP that occurred primarily in the hypertrophic zone of control sections but was absent in ALP-treated sections. The spectral analyses in the last column show profiles for the various ROI outlined in the smaller inset image. Growth plate regions analyzed included the region above the hypertrophic matrix (red ROI), in the hypertrophic matrix (green ROI), and in the bone (blue ROI). The emission profile of each control (−ALP) hypertrophic matrix (green ROI) shows a shift to a higher wavelength emission compared to the other −ALP regions, suggesting the presence of polyP. All regions analyzed in +ALP sections showed an emission profile similar to that of DAPI-DNA, suggesting polyP was hydrolyzed from the hypertrophic matrix after exposure to active ALP. All scale bars represent 150 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683572&req=5

pone-0005634-g005: Resolving polyP distribution by spectral isolation of DAPI 580 nm emission and attenuation by alkaline phosphatase (ALP) treatment.(A) Control growth plate section; (B) a different control growth plate section (higher magnification); (C) ALP-treated (+ALP) growth plate section. First column, green: 430 nm emission (DAPI-DNA) Second column, red: 580 nm emission (DAPI-polyP). The third column is an overlay of the 430 and 580 nm images to show the spatial distribution of polyP that occurred primarily in the hypertrophic zone of control sections but was absent in ALP-treated sections. The spectral analyses in the last column show profiles for the various ROI outlined in the smaller inset image. Growth plate regions analyzed included the region above the hypertrophic matrix (red ROI), in the hypertrophic matrix (green ROI), and in the bone (blue ROI). The emission profile of each control (−ALP) hypertrophic matrix (green ROI) shows a shift to a higher wavelength emission compared to the other −ALP regions, suggesting the presence of polyP. All regions analyzed in +ALP sections showed an emission profile similar to that of DAPI-DNA, suggesting polyP was hydrolyzed from the hypertrophic matrix after exposure to active ALP. All scale bars represent 150 µm.
Mentions: Figure 5 compares representative individual scans and overlays of emissions at a wavelength indicative of DAPI-DNA (430 nm, green) and DAPI-polyP (580 nm, red) for the control (−ALP; Figure 5A,B) and ALP-treated (+ALP; Figure 5C) murine vertebral body sections. When we displayed the DAPI fluorescence images collected at 580 nm with respect to 430 nm, we found a unique spatial distribution for the two wavelength ranges (Figure 5, overlay column). Thus, the DAPI emission above 580 nm may be a good index of polyP distribution. The spectral analyses on the right show representative profiles for the growth plate regions above the hypertrophic matrix (resting zone, red outline in inset), within the hypertrophic matrix (green outline), and within the bone (blue outline). The emission profiles of the hypertrophic matrix in control sections (Figure 5A,B) showed a shift to longer wavelengths compared to the other regions, suggesting the presence of polyPs. An overlay of all control profiles over the hypertrophic matrix region (Figure 6A) features a long tail of appreciable DAPI fluorescence extending beyond 580 nm.

Bottom Line: Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium.The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals.When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

ABSTRACT

Background: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal findings/methodology: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/significance: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Show MeSH
Related in: MedlinePlus