Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum
ABSTRACT Mouse serum alkaline phosphatase (ALP) is frequently measured and interpreted in mammalian bone research, however; little is known about the circulating ALPs in mice and their relation to human ALP isozymes and isoforms. Mouse ALP was extracted from liver, kidney, intestine, and bone from vertebra, femur and calvaria tissues. Serum from mixed strains of wild-type (WT) mice and from individual ALP knockout strains were investigated, i.e., Alpl(-/-) (a.k.a. Akp2 encoding tissue-nonspecific ALP or TNALP), Akp3(-/-) (encoding duodenum-specific intestinal ALP or dIALP), and Alpi(-/-) (a.k.a. Akp6 encoding global intestinal ALP or gIALP). The ALP isozymes and isoforms were identified by various techniques and quantified by high-performance liquid chromatography. Results from the WT and knockout mouse models revealed identical bone-specific ALP isoforms (B/I, B1, and B2) as found in human serum, but in addition mouse serum contains the B1x isoform only detected earlier in patients with chronic kidney disease and in human bone tissue. The two murine intestinal isozymes, dIALP and gIALP, were also identified in mouse serum. All four bone-specific ALP isoforms (B/I, B1x, B1, and B2) were identified in bone tissues from mice, in good correspondence with those found in human bones. All mouse tissues, except liver and colon, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Histochemical staining, Northern and Western blot analysis confirmed undetectable ALP expression in liver tissue. ALP activity staining showed some positive staining in the bile canaliculi for BALB/c and FVB/N WT mice, but not in C57Bl/6 and ICR mice. Taken together, while the main source of ALP in human serum originates from bone and liver, and a small fraction from intestine (<5%), mouse serum consists mostly of bone ALP, including all four isoforms, B/I, B1x, B1, and B2, and two intestinal ALP isozymes dIALP and gIALP. We suggest that the genetic nomenclature for the Alpl gene in mice (i.e., ALP liver) should be reconsidered since murine liver has undetectable amounts of ALP activity. These findings should pave the way for the development of user-friendly assays measuring circulating bone-specific ALP in mice models used in bone and mineral research.
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ABSTRACT: Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl(-/-) mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl(-/-) mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl(-/-) mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10-20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by microCT analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl(-/-) mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than non-transgenic controls and osteoblasts from [Col1a1-Tnap(+/-) ; Alpl(-/-) ] mice are able to mineralize to the level of Alpl(+/-) heterozygous osteoblasts, while Alpl(-/-) osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl(-/-) mice are comprised of phosphorylated forms of OPN while WT and [Col1a1-Tnap(+/-) ; Alpl(-/-) ] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more phosphorylated than non-transgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl(-/-) bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S(174) FQVS(178) DEQY(182) PDAT(186) DEDLT(191) )SHMK and FRIp(S(299) HELES(304) S(305) S(306) S(307) )EVN, with one non-localized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the pro-mineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN. © 2013 American Society for Bone and Mineral Research.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 07/2013; 28(7). DOI:10.1002/jbmr.1901 · 6.59 Impact Factor
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ABSTRACT: Surface functionalization of bioactive glasses with the enzyme alkaline phosphatase (ALP) was studied in order to combine the inorganic properties of bioactive glass (bioactivity) with the biological properties of the biomolecule (mineralization). Two different bioactive glasses (SCNA and CEL2), with different degree of bioactivity, were used as substrates for the functionalization. ALP was grafted either by direct bonding or via an organic coupling agent. The cellular response to surface-modified bioactive glass was investigated using human osteosarcoma cells (MG63, osteoblast-like). Biocompatibility of all modified surfaces was verified. Of note, the more reactive glass (CEL2), modified with ALP, showed improved mineralization ability.Applied Surface Science 06/2014; 313:372-381. DOI:10.1016/j.apsusc.2014.06.001 · 2.54 Impact Factor