Tissue-nonspecific Alkaline Phosphatase Protein Research Articles

Osteoblast-lineage calcium/calmodulin-dependent kinase 2 delta and gamma regulates bone mass and quality.

Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

The role of endomembrane homeostasis in vascular calcification

Abstract Background Cardiovascular diseases are a leading cause of mortality, with cardiovascular calcification as a prominent predictor and contributor. Microcalcifications in thin fibrous caps of atherosclerotic plaques create biomechanical instability, leading to plaque vulnerability. Recent work indicated that cellular-derived extracellular vesicles (EVs) are pivotal for microcalcification nucleation. EVs can originate from the endolysosomal system, and FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), a lipid kinase, plays a key role in the endolysosomal maturation. Purpose We hypothesize that alterations in endomembrane homeostasis will modulate EV cargo and, thereby, vascular calcification. Methods and Results In calcified arteries and vascular smooth muscle cells (SMCs), we observed increased PIKfyve expression. Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs (-40%) and recovered by Apilimod, a small molecule PIKfyve inhibitor. Apilimod prevented matrix mineralization and collagen network formation in calcifying SMCs (Fig. 1A) accompanied by reduced pro-collagen 1A1 secretion (-90%). Apilimod inhibited tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA, protein and activity level. PIKfyve silencing using siRNA showed similar results. Furthermore, Apilimod caused a dose-dependent increase of EV release assessed by nanoparticle tracking analysis (Fig. 1B). Analysis of the EV cargo demonstrated that Apilimod reduces TNAP protein abundance and activity (-60%) - a characteristic cargo that may determine EV calcification potential. Apilimod-induced EVs exhibited lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Next, we performed transcriptome and kinome analyses to discover the underlying mechanism of PIKfye-mediated SMC calcification. These data revealed a link to adipocyte-like differentiation and SMC phenotype specifying pathways. Validation supported increased expression of adipogenic transcription factors (CEBPA, PPARG),genes of the cholesterol and fatty acid metabolism pathways (FABP3, CD36) and enhanced fatty acid (C12) uptake (Fig. 1A) by Apilimod; while SMC-specific markers (ACTA2, SM22) and key SMC transcription factors (GATA6, MYOCD) were downregulated. Finally, in Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks, Apilimod application for 5 weeks increased PI3P levels (1.6-fold) and decreased vascular calcification (Fig. 1C), but did not alter plaque size or collagen content in atherosclerotic plaques. Conclusion Disrupting endolysosomal maturation with Apilimod promotes the release of EVs with reduced calcification potential and induces a phenotypic adaption towards adipocyte-like SMCs, causing reduced SMC calcification. Anti-arteriosclerotic effects of Apilimod in vivo remain to be further investigated.Figure

Characterization of Genetic Variants of Uncertain Significance for the ALPL Gene in Patients With Adult Hypophosphatasia.

Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23_Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype (p-value <0.001). Structurally, p.His379Asn variant resulted in the loss of two Zn2+ binding sites in the protein dimer which may greatly affect ALP activity. In summary, we identified three novel ALPL gene mutations associated with adult HPP. The correct identification and characterization of new variants and the subsequent study of their phenotype will allow the establishment of genotype-phenotype relationships that facilitate the management of the disease as well as making it possible to individualize treatment for each specific patient. This would allow the therapeutic approach to HPP to be personalized according to the unique genetic characteristics and clinical manifestations of each patient.

TNAP upregulation is a critical factor in Tauopathies and its blockade ameliorates neurotoxicity and increases life-expectancy

Tauopathies are a family of neurodegenerative diseases characterized by the presence of abnormally hyperphosphorylated Tau protein. Several studies have proposed that increased extracellular Tau (eTau) leads to the spread of cerebral tauopathy. However, the molecular mechanisms underlying eTau-induced neurotoxicity remain unclear. Previous in vitro studies reported that the ecto-enzyme tissue-nonspecific alkaline phosphatase (TNAP) dephosphorylate eTau at different sites increasing its neurotoxicity. Here, we confirm TNAP protein upregulation in the brains of Alzheimer's patients and found a similar TNAP increase in Pick's disease patients and P301S mice, a well-characterized mouse model of tauopathies. Interestingly, the conditional overexpression of TNAP causes intracellular Tau hyperphosphorylation and aggregation in cells neighbouring those overexpressing the ectoenzyme. Conversely, the genetic disruption of TNAP reduced the dephosphorylation of eTau and decreased neuronal hyperactivity, brain atrophy, and hippocampal neuronal death in P301S mice. TNAP haploinsufficiency in P301S mice prevents the decreased anxiety-like behaviour, motor deficiency, and increased memory capacity and life expectancy. Similar results were observed by the in vivo pharmacological blunting of TNAP activity. This study provides the first in vivo evidence demonstrating that raised TNAP activity is critical for Tau-induced neurotoxicity and suggest that TNAP blockade may be a novel and efficient therapy to treat tauopathies.

Pathophysiology of hypophosphatasia and the potential role of asfotase alfa.

Hypophosphatasia (HPP) is an inherited systemic bone disease that is characterized by bone hypomineralization. HPP is classified into six forms according to the age of onset and severity as perinatal (lethal), perinatal benign, infantile, childhood, adult, and odontohypophosphatasia. The causative gene of the disease is the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed ubiquitously, and its physiological role is apparent in bone mineralization. A defect in bone mineralization can manifest in several ways, including rickets or osteomalacia in HPP patients. Patients with severe forms suffer from respiratory failure because of hypoplastic chest, which is the main cause of death. They sometimes present with seizures due to a defect in vitamin B6 metabolism resulting from the lack of alkaline phosphatase activity in neuronal cells, which is also lethal. Patients with a mild form of the disease exhibit rickets or osteomalacia and a functional defect of exercise. Odontohypophosphatasia shows only dental manifestations. To date, 302 mutations in the ALPL gene have been reported, mainly single-nucleotide substitutions, and the relationships between phenotype and genotype have been partially elucidated. An established treatment for HPP was not available until the recent development of enzyme replacement therapy. The first successful enzyme replacement therapy in model mice using a modified human TNAP protein (asfotase alfa) was reported in 2008, and subsequently success in patients with severe form of the disease was reported in 2012. In 2015, asfotase alfa was approved in Japan in July, followed by in the EU and Canada in August, and then by the US Food and Drug Administration in the USA in October. It is expected that therapy with asfotase alfa will drastically change treatments and prognosis of HPP.

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.

The effects of tissue-non-specific alkaline phosphatase gene therapy on craniosynostosis and craniofacial morphology in the FGFR2C342Y/+ mouse model of Crouzon craniosynostosis.

Craniosynostosis, the premature fusion of cranial bones, has traditionally been described as a disease of increased bone mineralization. However, multiple mouse models of craniosynostosis display craniosynostosis simultaneously with diminished cranial bone volume and/or density. We propose an alternative hypothesis that craniosynostosis results from abnormal tissue mineralization through the downregulation of tissue-non-specific alkaline phosphatase (TNAP) enzyme downstream of activating mutations in FGFRs. Neonatal Crouzon (FGFRC342Y/+) and wild-type (FGFR+/+) mice were injected with lentivirus to deliver a recombinant form of TNAP. Mice were sacrificed at 4 weeks postnatal. Serum was collected to test for alkaline phosphatase (AP), phosphorus, and calcium levels. Craniofacial bone fusion and morphology were assessed by micro-computed tomography. Injection with the TNAP lentivirus significantly increased serum AP levels (increased serum AP levels are indicative of efficient transduction and production of the recombinant protein), but results were variable and dependent upon viral lot and the litter of mice injected. Morphological analysis revealed craniofacial form differences for inferior surface (p=0.023) and cranial height (p=0.014) regions between TNAP lentivirus-injected and vehicle-injected Crouzon mice. With each unit increase in AP level, the odds of lambdoid suture fusion decreased by 84.2% and these results came close to statistical significance (p=0.068). These results suggest that TNAP deficiency may mediate FGFR2-associated craniosynostosis. Future studies should incorporate injection of recombinant TNAP protein, to avoid potential side effects and variable efficacy of lentiviral gene delivery.

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.

The ANKH ΔE490 Mutation in Calcium Pyrophosphate Dihydrate Crystal Deposition Disease (CPPDD) Affects Tissue Non-specific Alkaline Phosphatase (TNAP) Activities

ANKH (human homolog of progressive ankylosis) regulates inorganic pyrophosphate (PPi) transport. Dominant ANKH mutations were detected in at least five multiplex families with calcium pyrophosphate dihydrate crystal deposition disease (CPPPD). The objective of this study is to assess the functional consequences of one CPPDD-associated ANKH mutation (ΔE490) in chondrogenic ATDC5 cells. Stable ATDC5 transfectants bearing myc-tagged constructs of wild-type ANKH, mutant ANKH (ΔE490) and neo controls were generated. Upon ITS (insulin, transferrin and selenium) induction, expression of chondrocyte markers including alkaline phosphatase activity in the various transfectants was assessed. The ANKH ΔE490- transfectants had low alkaline phosphatase activities throughout ITS treatment due to lower TNAP protein expression and the presence of intracellular low-molecular-weight inhibitors. Our results suggest that the interplay of ANKH and TNAP activities is tightly regulated.

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)

Expression of tissue non‐specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations

Background Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. Methods The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Results Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Conclusion Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation. © 1996 Wiley-Liss, Inc.

Expression of tissue non-specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations.

Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation.