TNAP activity is localized at critical sites of retinal neurotransmission across various vertebrate species

Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.     

Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)     

Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified—one tissue-nonspecific (TNAP) and three tissue-specific—named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.     

Tissue-nonspecific alkaline phosphatase promotes axonal growth of hippocampal neurons

Axonal growth is essential for establishing neuronal circuits during brain development and for regenerative processes in the adult brain. Unfortunately, the extracellular signals controlling axonal growth are poorly understood. Here we report that a reduction in extracellular ATP levels by tissue-nonspecific alkaline phosphatase (TNAP) is essential for the development of neuritic processes by cultured hippocampal neurons. Selective blockade of TNAP activity with levamisole or specific TNAP knockdown with short hairpin RNA interference inhibited the growth and branching of principal axons, whereas addition of alkaline phosphatase (ALP) promoted axonal growth. Neither activation nor inhibition of adenosine receptors affected the axonal growth, excluding the contribution of extracellular adenosine as a potential hydrolysis product of extracellular ATP to the TNAP-mediated effects. TNAP was colocalized at axonal growth cones with ionotropic ATP receptors (P2X₇ receptor), whose activation inhibited axonal growth. Additional analyses suggested a close functional interrelation of TNAP and P2X₇ receptors whereby TNAP prevents P2X₇ receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore inhibition of P2X₇ receptor reduced TNAP expression, whereas addition of ALP enhanced P2X₇ receptor expression. Our results demonstrate that TNAP, regulating both ligand availability and protein expression of P2X₇ receptor, is essential for axonal development.     

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.     

Tricyclic coumarin sulphonate derivatives with alkaline phosphatase inhibitory effects: in vitro and docking studies

Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was evaluated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC₅₀?=?0.38?±?0.01?μM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC₅₀?=?0.45?±?0.02?μM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.     

Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment

In mammalian alkaline phosphatase (AP) dimers, the N-terminus of one monomer embraces the other, stretching toward its active site. We have analyzed the role of the N-terminus and its microenvironment in determining the enzyme stability and catalysis using human placental (PLAP) and tissue-nonspecific AP (TNAP) as paradigms. Deletion of nine amino acid (aa) residues in PLAP reduced its AP activity and heat stability, while deletion of 25 aa resulted in an inactive enzyme. In turn, deletion of five and nine N-terminal aa in TNAP reduced and abolished AP activity, respectively. The N-terminal aa deletions in both isozymes affected the rate of substrate catalysis (k(cat)), with an only minor effect on the Michaelis constant (K(m)) explained by decelerated intramolecular transition rates in the active site. Arg370 in PLAP, and the corresponding Arg374 in TNAP, critically control the structure and function of the enzymes, but the Glu6-Arg370 bond predicted by the PLAP crystal structure appeared to be irrelevant with respect to PLAP stability or catalysis. Structural disruption was also noted in [R374A]TNAP, [Delta5]TNAP, [Delta9]TNAP, and [Delta25]TNAP using a panel of 19 anti-TNAP antibodies illustrating the structural role of the N-terminus. Our data reveal that the N-terminal alpha-helical folding is more crucial for the structural stability of the second monomer in TNAP than in PLAP. The correct folding of the N-terminus and of interacting loops in its immediate environment is essential for overall structural integrity and for execution of intramolecular transitions during enzyme catalysis. These findings provide a mechanistic interpretation for loss-of-function mutations of N-terminal TNAP residues in cases of hypophosphatasia.     

Identification of pulmonary surfactant that bears intestinal-type and tissue-nonspecific-type alkaline phosphatase in endotoxin-induced rat bronchoalveolar fluid

The characteristics of lipopolysaccharide (LPS)-induced alkaline phosphatase (AP) isozymes on the various pulmonary surfactant subtypes were investigated. We used continuous sucrose-gradient centrifugation to separate surfactant into subtypes. The density of each surfactant subtype isolated from LPS-instilled rats was greater than that of the subtypes from the control rats; and the proportion of light surfactant was lower, thereby decreasing the ratio of light to heavy surfactant. The results of an inhibition study revealed the main AP isozyme in bronchoalveolar fluid (BAF) to be tissue-nonspecific AP (TNAP), but some of the activity was characteristic of intestinal-type AP (IAP). IAP, in addition to TNAP and surfactant-associated protein A (SP-A), was detected on heavy surfactant, and LPS induced both APs. To examine the expression of IAP in the lungs, we prepared primers to detect the cDNAs of two types of rat IAP mRNA, IAP-I and -II, and amplified their cDNAs. LPS instillation induced IAP-I mRNA, but not IAP-II mRNA or TNAP mRNA. Immunohistochemical localization of IAP and TNAP revealed reaction products for both in type II cells. The present study thus demonstrated that, in rats, type II cells produce both IAP and TNAP and that these surfactants bearing AP isozymes are secreted into the alveolar space following induction by intratracheal instillation of LPS.     

Structural evidence of functional divergence in human alkaline phosphatases

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family.     

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.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.     

Immunolocalization of tissue non-specific alkaline phosphatase in mice

 Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes. Accepted: 18 October 1996     

Tissue nonspecific alkaline phosphatase is activated via a two-step mechanism by zinc transport complexes in the early secretory pathway

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.     

Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.     

Plasma membrane homing of tissue nonspecific alkaline phosphatase under the influence of 3-hydrogenkwadaphnin, an antiproliferative agent from Dendrostellera lessertii

Several mammalian enzymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) structure. These include acetylcholinesterase, alkaline phosphatase (AP) and 5'-nucleotidase among other enzymes. Recently, it has been reported that these membrane enzymes can be released into the serum by the GPI-dependent phospholipase D under various medical disturbances such as cancer and/or by chemical and physical manipulation of the biological systems. Treatment of MCF-7 cells with two consecutive effective concentrations of 3-hydrogenkwadaphnin (3-HK, 3 nM) for 48 h enhanced membrane AP activity by almost 330% along with a 40% reduction in the AP activity of the cell culture medium. In addition, our data indicate that 3-HK is capable of inducing mainly the tissue-nonspecific alkaline phosphatase (TNAP) isoenzyme, along with enhancing its thermostability. These findings, besides establishing a correlation between the antiproliferative activity of 3-HK and the extent of plasma membrane AP activity, might assist in the development of new diagnostic tools for following cancer medical treatments.     

Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.