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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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Related in: MedlinePlus

Emission spectra of DAPI-DNA and DAPI-polyP.(A) Emission spectra of DAPI-DNA obtained from murine brain section. Note position of maximum intensity at 460 nm, intensity at 430 nm, and intensity at 520 nm. The intensity at 430 nm is used as a proxy for the contribution of the DAPI-DNA curve to the convoluted DAPI-DNA-polyP spectra. (B) Emission spectrum of DAPI-polyP obtained from synthetic polyP. Note position of maximum intensity near 520 nm and minimal intensity at 430 nm. The intensity at 520 nm is used as a proxy for the contribution of the polyP to the convoluted DAPI-DNA-polyP spectra in bone sections. Fluorescence above 580 nm is exclusively due to DAPI-polyP emission and was used for imaging purposes in Figures 2 and 5.
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pone-0005634-g010: Emission spectra of DAPI-DNA and DAPI-polyP.(A) Emission spectra of DAPI-DNA obtained from murine brain section. Note position of maximum intensity at 460 nm, intensity at 430 nm, and intensity at 520 nm. The intensity at 430 nm is used as a proxy for the contribution of the DAPI-DNA curve to the convoluted DAPI-DNA-polyP spectra. (B) Emission spectrum of DAPI-polyP obtained from synthetic polyP. Note position of maximum intensity near 520 nm and minimal intensity at 430 nm. The intensity at 520 nm is used as a proxy for the contribution of the polyP to the convoluted DAPI-DNA-polyP spectra in bone sections. Fluorescence above 580 nm is exclusively due to DAPI-polyP emission and was used for imaging purposes in Figures 2 and 5.

Mentions: The labeled sections were excited with a multiphoton laser (Milennia XS – Tsunami, SpectraPhysics, CA) at ∼780 nm, in accordance with the suggested DAPI excitation wavelengths by Neu et al. [78]). The multiphoton laser was directly coupled to a Leica SP2 confocal scanning microscope system. Wavelength scans (xyλ) were acquired in 20 nm bins (400–700 nm) that allowed spectral analysis of the emission spectra. The DAPI-DNA complex emission peak was detected and identified based on its 460–465 nm peak emission, and the DAPI-polyP complex peak based on its 520–580 nm emission (Figure 10, solid lines). We assumed that emissions at 580 nm represented DAPI-polyP emissions minimally convoluted with DAPI-DNA ones (Figure 10, red dashed line); we therefore used the 580 nm emissions to represent DAPI-polyP emission regions (for example, in Figures 2, 5). The DAPI-DNA emissions for Figure 10A were collected from a murine brain section, while Figure 10B depicts the emission spectra from a solution of 10 µg/mL sodium polyphosphate (Type 28, Sigma-Aldrich) and 10 µg/mL DAPI in TRIS (0.2 M, pH 9). Spectral scans were analyzed with Leica LCS or Leica Lite® software. Mathematical subtraction or addition of spectral emission curves (Figure 6C, dashed lines) was re-normalized so that the peak intensity equaled 0.5 before plotting.


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)

Emission spectra of DAPI-DNA and DAPI-polyP.(A) Emission spectra of DAPI-DNA obtained from murine brain section. Note position of maximum intensity at 460 nm, intensity at 430 nm, and intensity at 520 nm. The intensity at 430 nm is used as a proxy for the contribution of the DAPI-DNA curve to the convoluted DAPI-DNA-polyP spectra. (B) Emission spectrum of DAPI-polyP obtained from synthetic polyP. Note position of maximum intensity near 520 nm and minimal intensity at 430 nm. The intensity at 520 nm is used as a proxy for the contribution of the polyP to the convoluted DAPI-DNA-polyP spectra in bone sections. Fluorescence above 580 nm is exclusively due to DAPI-polyP emission and was used for imaging purposes in Figures 2 and 5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683572&req=5

pone-0005634-g010: Emission spectra of DAPI-DNA and DAPI-polyP.(A) Emission spectra of DAPI-DNA obtained from murine brain section. Note position of maximum intensity at 460 nm, intensity at 430 nm, and intensity at 520 nm. The intensity at 430 nm is used as a proxy for the contribution of the DAPI-DNA curve to the convoluted DAPI-DNA-polyP spectra. (B) Emission spectrum of DAPI-polyP obtained from synthetic polyP. Note position of maximum intensity near 520 nm and minimal intensity at 430 nm. The intensity at 520 nm is used as a proxy for the contribution of the polyP to the convoluted DAPI-DNA-polyP spectra in bone sections. Fluorescence above 580 nm is exclusively due to DAPI-polyP emission and was used for imaging purposes in Figures 2 and 5.
Mentions: The labeled sections were excited with a multiphoton laser (Milennia XS – Tsunami, SpectraPhysics, CA) at ∼780 nm, in accordance with the suggested DAPI excitation wavelengths by Neu et al. [78]). The multiphoton laser was directly coupled to a Leica SP2 confocal scanning microscope system. Wavelength scans (xyλ) were acquired in 20 nm bins (400–700 nm) that allowed spectral analysis of the emission spectra. The DAPI-DNA complex emission peak was detected and identified based on its 460–465 nm peak emission, and the DAPI-polyP complex peak based on its 520–580 nm emission (Figure 10, solid lines). We assumed that emissions at 580 nm represented DAPI-polyP emissions minimally convoluted with DAPI-DNA ones (Figure 10, red dashed line); we therefore used the 580 nm emissions to represent DAPI-polyP emission regions (for example, in Figures 2, 5). The DAPI-DNA emissions for Figure 10A were collected from a murine brain section, while Figure 10B depicts the emission spectra from a solution of 10 µg/mL sodium polyphosphate (Type 28, Sigma-Aldrich) and 10 µg/mL DAPI in TRIS (0.2 M, pH 9). Spectral scans were analyzed with Leica LCS or Leica Lite® software. Mathematical subtraction or addition of spectral emission curves (Figure 6C, dashed lines) was re-normalized so that the peak intensity equaled 0.5 before plotting.

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