Impaired Calcification Around Matrix Vesicles of Growth Plate and Bone in Alkaline Phosphatase-Deficient Mice

Abstract

The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.

Association of a TNAP haplotype with ankylosing spondylitis

“…Several human and mouse diseases with excessive or insufficient bone formation have been related to defects in, respectively, the phosphodiesterases (such as nucleotide pyrophosphatase/phosphodiesterase 1), which produce PPi (11,12), and the phosphatases (such as tissue-nonspecific alkaline phosphatase [TNAP]), which degrade PPi (13)(14)(15). The homozygous ank (progressive ankylosis) mouse has a loss-of-function nonsense mutation in the ank gene that codes for a regulator of PPi export, and these mice develop a condition characterized by pathologic calcium apatite crystal deposition in the synovial and subsynovial spaces, followed by chondro-osteophyte formation and eventual bony ankylosis of the affected joints (16).…”

Calcification of Advanced Atherosclerotic Lesions in the Innominate Arteries of ApoE-Deficient Mice

“…ALP activity has been shown inside matrix vesicles shed from chondrocytes and most likely works to increase the availabil- ity of inorganic phosphate (P i ) needed for hydroxyapatite crystal growth. 30 Increases in ALP activity is a marker of the transition to an osteogenic phenotype by smooth muscle cells in vitro. ALP activity is increased after exposure of smooth muscle cells to inflammatory factors.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Accordingly, mutations in the TNAP gene that cause impairment or loss of functional activity lead to hypophosphatasia in humans [486,487]. Similarly, TNAP knockout mice (Akp2−/−) accumulate extracellular PP i , reveal hypomineralization [488][489][490][491], and can serve as a model for the infantile form of hypophosphatasia [492]. The defect can be eliminated by subcutaneous injections of soluble TNAP targeted to mineralizing tissue [493,494] or by lentiviral application of a bone-targeted form of TNAP [495].…”