Human Tissue-nonspecific Alkaline Phosphatase Research Articles

Efficient one-pot synthesis of 5-perfluoroalkylpyrazoles by cyclization of hydrazone dianions.

A highly selective and efficient method for the synthesis of 5-trifluoromethylated and 5-perfluoroalkylated pyrazoles has been developed which relies on the cyclization of hydrazine dianions with ethyl perfluorocarboxylates. The pyrazoles prepared were evaluated as potential inhibitors of alkaline phosphatases, namely human tissue non-specific alkaline phosphatase (h-TNAP) and tissue specific intestinal alkaline phosphatase (IAP). Most pyrazole derivatives inhibited h-IAP more markedly than h-TNAP and had minor effects on nucleotide pyrophosphatase/phosphodiesterases. Therefore, the compounds appear as potential selective inhibitors of h-IAP.

Synthesis, characterization and biological evaluation of N-(2,3-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-4-yl)benzamides

Comparison of active site residues of modelled h-GCAP with template h-PLAP.

Pathophysiological role of vascular smooth muscle alkaline phosphatase in medial artery calcification.

Medial vascular calcification (MVC) is a pathological phenomenon that causes vascular stiffening and can lead to heart failure; it is common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases. These conditions share the common feature of tissue-nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X-linked manner. Hemizygous overexpressor male mice (Tagln-Cre(+/-) ; Hprt(ALPL) (/Y) or TNAP-OE) show extensive vascular calcification, high blood pressure, and cardiac hypertrophy, and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln-Cre(+/-) ; Hprt(ALPL) (/-) ) female TNAP-OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP-OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug-like pharmacokinetic characteristics. TNAP-OE mice were treated with the prototypical TNAP inhibitor SBI-425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle-treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target.

Molecular Evolution of the Tissue-nonspecific Alkaline Phosphatase Allows Prediction and Validation of Missense Mutations Responsible for Hypophosphatasia

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.

Clinical Aspects of Hypophosphatasia: An Update

Hypophosphatasia (HPP) is a heterogeneous rare inborn error of bone and mineral metabolism caused by mutations in the ALPL gene encoding the isoenzyme, tissue-nonspecific alkaline phosphatase (TNAP). These mutations result in a decreased level of TNAP activity and increased levels of its substrates, including inorganic pyrophosphate, pyridoxal-5′-phosphate and phosphoethanolamine. Clinical presentations are highly variable, ranging from stillbirth and absence of mineralization in severe disease to mild dental problems or osteopenia in adulthood. Further clinical symptoms include defective bone mineralization with bone deformities, recurrent fractures, chronic non-bacterial osteomyelitis, craniosynostosis, neonatal seizures, nephrocalcinosis, muscular hypotonia, failure to thrive and dental abnormalities with premature exfoliation of teeth and caries. Prognosis is very poor in severe perinatal forms with most patients dying from pulmonary complications of their skeletal disease but patients with mild phenotypes (adult form or Odonto-HPP) usually do not have a limitation in their life expectancy. Although TNAP is a ubiquitous enzyme, mostly known for its crucial role during mineralization of bone and teeth, its exact biological role in different human organs is still unclear, and the pathophysiology of symptoms due to TNAP deficiency in HPP are not understood in detail. Since inflammation and tissue destruction of the musculoskeletal system may occur in HPP, TNAP may also play an important role in controlling inflammatory processes. Recent investigations provide evidence that TNAP is also essentially involved in the development of the central nervous system and might contribute to multiple functions of the human brain. HPP can be diagnosed on clinical, biochemical and radiological criteria, and genetic testing confirms the diagnosis and is useful for genetic counseling. Since clinical symptoms are highly variable, patients should be followed up by a multidisciplinary team having experience in HPP treatment. Up to now, no curative treatment of HPP is available. Therefore, symptomatic treatment in particular with regard to pain, seizures and other metabolic phenomena is most important. However, recently, enzyme replacement therapy with a bone-targeted recombinant human TNAP molecule has been reported to improve bone mineralization, respiratory function and physical activity in severely affected infants with HPP, and further clinical trials are ongoing. Hopefully, this and other new therapeutic strategies may improve the prognosis and quality of life of patients with HPP and may contribute to our understanding of bone metabolism in general.

In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

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 to 20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by micro-computed tomography (µCT) 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 nontransgenic controls and osteoblasts from [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice are able to mineralize to the level of Alpl⁺/⁻ heterozygous osteoblasts, whereas 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 whereas wild-type (WT) and [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more dephosphorylated than nontransgenic 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(S¹⁷⁴FQVS¹⁷⁸DEQY¹⁸²PDAT¹⁸⁶DEDLT¹⁹¹)SHMK and FRIp(S²⁹⁹HELES³⁰⁴S³⁰⁵S³⁰⁶S³⁰⁷)EVN, with one nonlocalized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the promineralization 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.

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Enzyme Replacement Therapy Prevents Dental Defects in a Model of Hypophosphatasia

Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2 -/- ) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B6-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2 -/- mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD10). sALP-FcD10 prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin – a marker of acellular cementum – was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2 -/- mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life – where the majority of tooth root development occurs, including acellular cementum formation – could prevent the accelerated tooth loss seen in individuals with HPP.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.

ENB-0040, a bone-targeted human recombinant tissue nonspecific alkaline phosphatase (TNSALP) fusion protein, in the treatment of infants and adults with hypophosphatasia

ENB-0040, a bone-targeted human recombinant tissue nonspecific alkaline phosphatase (TNSALP) fusion protein, in the treatment of infants and adults with hypophosphatasia

Bone-Targeted Replacement Therapy for Hypophosphatasia

Bone-Targeted Replacement Therapy for Hypophosphatasia

The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by beta-glycerophosphate and NaH(2)PO(4) in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited beta-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5'-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH(2)PO(4)-induced mineralization. PFA completely inhibited NaH(2)PO(4)-induced mineralization and NPP1 enzymatic activation, and partly inhibited beta-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of beta-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by beta-glycerophosphate or NaH(2)PO(4), suggesting that the effects of these phosphates may be indirect.

A Novel Glycosylphosphatidylinositol-Anchored Alkaline Phosphatase Dwells in the Hepatic Duct of the Pearl Oyster, Pinctada fucata

Alkaline phosphatases are ubiquitous enzymes involved in many important biological processes. Mammalian tissue-nonspecific alkaline phosphatase (TNAP) has long been thought to play an important role in bone mineralization. In this study, we identified a full-length cDNA encoding a potential alkaline phosphatse from pearl oyster Pinctada fucata by RT-PCR and RACE and designated the encoded protein as PFAP. The sequence of PFAP shares an overall similarity of 67% with that of human TNAP. Prediction and analysis of its secondary and tertiary structure revealed that the PFAP contains two mammalian-specific regions, the crown domain, involved in collagen binding, and the calcium binding domain, which hint its potential ability to participate in biomineralization. RT-PCR and in situ hybridization showed that the PFAP mRNA distributes specifically in the hepatic duct of the digestive diverticula. These findings implied its possible role in calcium absorption and transportation. In vivo, PFAP could be specifically released by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting it is glycophosphatidylinositol-anchored to the plasma membrane. Therefore, a human growth hormone-PFAP fusion was constructed to locate the cleavage/attachment site. Immunofluorescent labeling and immunoblotting showed that Asn-477 is the cleavage/attachment site and the 25-residue peptide COOH-terminal to Asn-477 is removed during glycophosphatidylinositol anchoring. This research will hopefully pave the way to illustrate the role PFAP plays in calcium transportation related to pearl biomineralization.

High Level Expression of Tissue-Nonspecific Alkaline Phosphatase in the Milk of Transgenic Rabbits

Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses.

Zinc Transport Complexes Contribute to the Homeostatic Maintenance of Secretory Pathway Function in Vertebrate Cells

Zinc transporters play important roles in a wide range of biochemical processes. Here we report an important function of ZnT5/ZnT6 hetero-oligomeric complexes in the secretory pathway. The activity of human tissue-nonspecific alkaline phosphatase (ALP) expressed in ZnT5(-)ZnT7(-/-) cells was significantly reduced compared with that expressed in wild-type cells as in the case of endogenous chicken tissue-nonspecific ALP activity. The inactive human tissue-nonspecific ALP in ZnT5(-)ZnT7(-/-) cells was degraded by proteasome-mediated degradation without being trafficked to the plasma membrane. ZnT5(-)ZnT7(-/-) cells showed exacerbation of the unfolded protein response as did the wild-type cells cultured under a zinc-deficient condition, revealing that both complexes play a role in homeostatic maintenance of secretory pathway function. Furthermore, we showed that expression of ZnT5 mRNA was up-regulated by the endoplasmic reticulum stress in various cell lines. The up-regulation of the hZnT5 transcript was mediated by transcription factor XBP1 through the TGACGTGG sequence in the hZnT5 promoter, and this sequence was highly conserved in the ZnT5 genes of mouse and chicken. These results suggest that zinc transport into the secretory pathway is strictly regulated for the homeostatic maintenance of secretory pathway function in vertebrate cells.

Posttranscriptional modulation of the human tissue–nonspecific alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D 3 in MG-63 osteoblastic osteosarcoma cells

Although tissue-nonspecific alkaline phosphatase (TNSALP) plays a key role in bone mineralization and 1,25-dihydroxyvitamin D 3 is an important dietary regulator of bone mineralization, relationships between TNSALP and vitamin D have not been well characterized. We studied the effect of 1,25-dihydroxyvitamin D 3 on the expression of the human TNSALP gene in a cell culture system. We tested several osteoblastic osteosarcoma cell lines but found that only MG-63 cells responded to 1,25-dihydroxyvitamin D 3. When MG-63 cells were cultured for 48 hours with 1,25-dihydroxyvitamin D 3 in a serum-free medium, vitamin D treatment prevented a decrease in alkaline phosphatase activity observed in nontreated cells. Real-time reverse transcriptase polymerase chain reaction showed that 1,25-dihydroxyvitamin D 3 caused conserved expression of TNSALP messenger RNA (mRNA) levels compared with nontreated cells. Despite this conserved expression of TNSALP mRNA levels, luciferase assays revealed that reporter plasmid containing a vitamin D response element-like sequence in the upstream region of the human TNSALP gene did not activate transcription. However, mRNA stability assay using 5,6-dichlorobenzimidazole 1- β- d-ribofuranoside as a transcription inhibitor showed that 1,25-dihydroxyvitamin D 3 increased the half-life of TNSALP mRNA approximately 2.4-fold. Thus, 1,25-dihydroxyvitamin D 3 modulated posttranscriptionally the human TNSALP activity and the mRNA expression by increasing mRNA stability in MG-63 osteoblastic osteosarcoma cells.

Enhancement of drug delivery to bone: Characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of l-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.

Effects of phosphates on the expression of tissue-nonspecific alkaline phosphatase gene and phosphate-regulating genes in short-term cultures of human osteosarcoma cell lines

We studied the effects of phosphates on the expression of the human tissue-nonspecific alkaline phosphatase (TNSALP) gene and phosphate-regulating genes in short-term cultures of human osteoblastic osteosarcoma cell lines. When human osteosarcoma cell lines, SaOS-2, MG-63, and U(2)OS were cultured with 10 mM inorganic sodium dihydrogenphosphate, 10 mM beta-glycerophosphate, 250 microM pyridoxal phosphate, or 100 microM inorganic pyrophosphate, enzymatic activity of alkaline phosphatase began to increase at 72 h after addition of sodium dihydrogenphosphate and beta-glycerophosphate in SaOS-2 cells. Pyridoxal phosphate and pyrophosphate did not induce alkaline phosphatase activity. U(2)OS cells slightly reacted to beta-glycerophosphate, but MG-63 cells did not react on exposure to phosphates. In SaOS-2 cells, TNSALP mRNA measured by real-time RT-PCR reached a peak level at 72 h after the addition of beta-glycerophosphate. PHEX and MEPE mRNAs were also induced by beta-glycerophosphate. These results suggest that TNSALP, PHEX and MEPE were concordantly induced by beta-glycerophosphate on mineralisation.

Regulation of the human tissue-nonspecific alkaline phosphatase gene expression by all- trans-retinoic acid in SaOS-2 osteosarcoma cell line

While tissue-nonspecific alkaline phosphatase (TNSALP) is a well-known indicator of bone formation and all- trans-retinoic acid a key regulator of that process, the relationship between TNSALP and retinoic acid has not yet been clearly described. The aim of the present study was therefore to clarify the mechanism by which retinoic acid modulates expression of TNSALP. After culturing SaOS-2 human osteoblastic osteosarcoma cells in the presence or absence of 10 −6 M all- trans-retinoic acid, real-time RT-PCR confirmed that retinoic acid up-regulates expression of TNSALP mRNA. Notably, this time-dependent induction of TNSALP expression was accompanied by a corresponding increase in detected catalytic activity of the enzyme. When we then isolated the 5′-upstream region of the human TNSALP gene and carried out luciferase assays with a set of deletion mutants, we found that the promoter region contains a retinoic acid response element-like motif. Moreover, electrophoretic mobility shift assays showed that the nuclear extract bound to the motif. It thus appears that retinoic acid regulates the expression of human TNSALP via a retinoic acid response element in the genes promoter region.

Structural Evidence for a Functional Role of Human Tissue Nonspecific Alkaline Phosphatase in Bone Mineralization

The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Ménez, A., and Stura, E. A. (2001) J. Biol. Chem, 276, 9158-9165). The degree of homology allowed us to build a reliable TNAP model to investigate the relationship between mutations associated with hypophosphatasia and their probable consequences on the activity or the structure of the enzyme. The mutations are clustered within five crucial regions, namely the active site and its vicinity, the active site valley, the homodimer interface, the crown domain, and the metal-binding site. The crown domain and the metal-binding domain are mammalian-specific and were observed for the first time in the PLAP structure. The crown domain contains a collagen binding loop. A synchrotron radiation x-ray fluorescence study confirms that the metal in the metal-binding site is a calcium ion. Several severe mutations in TNAP occur around this calcium site, suggesting that calcium may be of critical importance for the TNAP function. The presence of this extra metal-binding site gives new insights on the controversial role observed for calcium.