Tissue-nonspecific Alkaline Phosphatase mRNA Research Articles

Adenosine Attenuates Aortic Smooth Muscle Cell Calcification through A<sub>3</sub> Adenosine Receptor

Vascular calcification is a typical feature of atherosclerosis and is associated with adverse cardiovascular events such as myocardial infarction and stroke. Several studies have suggested that adenosine, an ATP metabolite may function as an endogenous regulator of arterial calcification. However, its effects on vascular smooth muscle cell calcification have not been clarified. In this study, we investigated the inhibitory effects of adenosine on vascular calcification in vitro by utilizing the culture of human aortic smooth muscle cells (HASMCs). Osteoblastic differentiation of HASMCs was induced by the treatment with oncostatin M and osteogenic differentiation medium. Adenosine and its metabolically stable analogue, 2-chloroadenosine (CADO) significantly reduced matrix mineralization and alkaline phosphatase (ALP) activities in HASMCs. The mRNA expression of tissue non-specific alkaline phosphatase (TNAP) was down-regulated by adenosine and CADO, but the mRNA expression of other osteoblastic differentiation markers, such as Runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP)-II, was not significantly affected by these two reagents. Among the adenosine receptor (AR) subtype-selective agonists used, only IB-MECA (A3 AR-selective agonist) significantly decreased in vitro mineralization and ALP activities in HASMCs, but not with CCPA (A1 AR-selective agonist), CGS21680 (A2a AR-selective agonist), or BAY60-6583 (A2b AR-selective agonist). Importantly, IB-MECA also down-regulated expression of TNAP mRNA. Finally, knockdown of A3 AR, but not A1 AR, A2a AR, or A2b AR, significantly reversed the inhibitory actions of adenosine, CADO, or IB-MECA on in vitro calcification and ALP activities in HASMCs. These data suggest that adenosine attenuates HASMC calcification through A3 AR.

Liver-type of tissue non-specific alkaline phosphatase is induced during mouse bone and tooth cell differentiation

Background and objective: Tissue non-specific alkaline phosphatase (TNSALP) contains two types—bone- and liver-type—which are produced from the same gene due to differences in splicing. These two differ in their promoter, but the amino acid sequences of the mature proteins are identical. In this study, we examined the relationship between the two types of TNSALP expression and osteoblast differentiation.Design: Gene expression of the two types of TNSALP was observed by reverse transcription-polymerase chain reaction. MC3T3-NM4 was sub-cloned from an established mouse osteoblastic cell line in which osteoblast characters do not appear without dexamethasone. The C2C12 mouse myoblastic cell line, which can be induced to osteoblasts with bone morphogenic protein 2, and organ-cultured tooth germs were also used in this work.Results: The gene expression of liver-type TNSALP was observed in only MC3T3-NM4 activated by dexamethasone. For C2C12, the gene expression of bone-type TNSALP was observed even in non-induced conditions where myotubes were formed, whereas the liver-type TNSALP mRNA was only expressed when C2C12 differentiated into osteoblasts by bone morphogenic protein 2. Furthermore, in the organ-cultured tooth germs, the liver-type TNSALP mRNA was expressed according to differentiation of tooth germs.Conclusion: These results suggest that the liver-type TNSALP mRNA is induced according to differentiation of bone and tooth.

Experimental acute pancreatitis is enhanced in mice with tissue nonspecific alkaline phoshatase haplodeficiency due to modulation of neutrophils and acinar cells

Tissue nonspecific alkaline phosphatase (TNAP) has a well established role in bone homeostasis and in hepatic/biliary conditions. In addition, TNAP is expressed in the inflamed intestine and is relevant to T and B lymphocyte function. TNAP KO mice are only viable for a few days, but TNAP+/− haplodeficient mice are viable. Acute pancreatitis was induced by repeated caerulein injection in WT and TNAP+/− mice. TNAP+/− mice presented an increased expression of Cxcl2, Ccl2, Selplg (P-selectin ligand), Il6 and Il1b in the pancreas. Freshly isolated acinar cells showed a dramatic upregulation of Cxcl1, Cxcl2, Ccl2, Il6, Selpg or Bax in both pancreatitis groups. TNAP+/− cells displayed a 2-fold higher expression of Cxcl2, and a smaller increase in Il6. These findings could be partly replicated by in vitro treatment of primary acinar cells with caerulein. Furthermore, the proinflammatory effect on acinar cells could be partially reproduced in wild type cells treated with the TNAP inhibitor levamisole. TNAP mRNA levels were also markedly upregulated by pancreatitis in acinar cells. Neutrophil infiltration (MRP8+ cells) and activation (IL-6 and TNF production in LPS treated primary neutrophils) were increased in TNAP+/− vs WT mice. Neutrophil depletion greatly attenuated inflammation, indicating that this cell type is mainly responsible for the higher inflammatory status of TNAP+/− mice. In conclusion, our results show that altered TNAP expression results in heightened pancreatic inflammation, which may be explained by an augmented response of neutrophils and by a higher sensitivity of acinar cells to caerulein injury.

Specific knock-down of tissue non-specific alkaline phosphatase mRNA levels inhibits intracellular lipid accumulation in 3T3-L1 and HepG2 cells.

The use of non-specific inhibitors of tissue non-specific alkaline phosphatase (TNSALP) in pre-adipocytes blocks intracellular lipid accumulation. TNSALP is also expressed in hepatocytes, which are known to accumulate lipid in a similar manner to pre-adipocytes. The purpose of this study was to use specific silencing of TNSALP mRNA, using short interfering (si) RNA, to investigate the role of TNSALP in intracellular lipid accumulation in 3T3-L1 and HepG2 cells. Cellular activity of TNSALP was measured using an automated colorimetric assay, and intracellular lipid accumulation was determined using the lipid-specific dye, Oil Red O. Cells were transfected with siRNA directed against TNSALP mRNA, and expression of the TNSALP gene was determined at selected time points postinduction of lipid droplet formation. Expression of the TNSALP gene was inhibited by a maximum of 88±1.9% (P<0.005 vs. control) 11days after initiation of lipid droplet formation in the 3T3-L1 cells and 80±8.9% (P<0.05 vs. control) after 4days in the HepG2 cells. This led to significant inhibition of both TNSALP activity and intracellular lipid accumulation in both cell lines. These data demonstrates that TNSALP plays an important role in the control of lipid droplet formation in both pre-adipocyte and hepatocyte cell lines.

Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

Allopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro.

Interactive roles of CD73 and tissue nonspecific alkaline phosphatase in the renal vascular metabolism of 5'-AMP.

CD73 metabolizes extracellular 5'-AMP to adenosine; yet recent experiments in brain tissue suggest that CD73 is not required for the metabolism of 5'-AMP to adenosine because of tissue nonspecific alkaline phosphatase (TNAP), which like CD73 is a GPI-anchored ecto-enyzme with 5'-nucleotidase activity. Because adenosine importantly regulates renovascular function, we investigated whether both TNAP and CD73 are involved in the renovascular metabolism of 5'-AMP. To test this, we examined in isolated, perfused mouse kidneys the metabolism of 5'-AMP (applied to the lumen of the renal vasculature via intrarenal artery administration) to adenosine by measuring renal venous levels of 5'-AMP, adenosine, and inosine (adenosine metabolite) by mass spectrometry. In one study, we compared 5'-AMP metabolism in naive CD73+/+ (wild-type, n = 16) vs. CD73-/- (knockout, n = 16) kidneys; and in a second study, we compared 5'-AMP metabolism in CD73+/+ (n = 9) vs. CD73-/- (n = 8) kidneys pretreated with levamisole (1 mmol/l; TNAP inhibitor). In naive kidneys, 5'-AMP increased renal venous 5'-AMP, adenosine, and inosine, and these responses were similar in CD73+/+ vs. CD73-/- kidneys. Levamisole per se did not inhibit renovascular 5'-AMP metabolism; however, in the presence of levamisole, 5'-AMP increased renal venous 5'-AMP threefold more in CD73-/- vs. CD73+/+ kidneys and knockout of CD73 inhibited 5'-induced adenosine and inosine by 81 and 86%, respectively. TNAP mRNA, protein, and activity were similar in CD73+/+ vs. CD73-/- kidneys. In conclusion, CD73 and TNAP play interactive roles to metabolize luminally applied 5'-AMP in the renal vasculature such that inhibition of both is required to inhibit the production of adenosine.

Serotonergic 5-HT2B Receptor Controls Tissue-nonspecific Alkaline Phosphatase Activity in Osteoblasts via Eicosanoids and Phosphatidylinositol-specific Phospholipase C

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT(2B)R) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT(2B)R and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT(2B)R and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT(2B)R intrinsic activity or shRNA-mediated 5-HT(2B)R knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT(2B)R couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT(2B)R controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT(2B)R-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT(2B)R-null mice exhibit defects in TNAP activity.

The Mechanism of Mineralization and the Role of Alkaline Phosphatase in Health and Disease

Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. Physiological mineralization occurs in hard tissues, whereas pathological calcification occurs in soft tissues. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. This is followed by propagation of hydroxyapatite into the extracellular matrix and its deposition between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization. Inorganic pyrophosphate, pyridoxal phosphate, and phosphoethanolamine are thought to be the physiologic substrates of TNAP. These accumulate in the event of TNAP deficiency, e.g., in cases of hypophosphatasia. The gene encoding TNAP is mapped to chromosome 1, consists of 12 exons, and possesses regulatory motifs in the 5'-untranslated region. Inhibition of TNAP enzymatic activity suppresses TNAP mRNA expression and mineralization in vitro. Hypophosphatasia is an inherited systemic bone disease characterized by hypomineralization of hard tissues. The phenotype of hypophosphatasia is varied. To date, more than 200 mutations in the TNAP gene have been reported. Knockout mice mimic the phenotypes of severe hypophosphatasia. Among the mutations in the TNAP gene, c.1559delT is frequent in the Japanese population. This frameshift mutation results in the expression of an abnormally long protein that is degraded in cells. DNA-based prenatal diagnosis using chorionic villus sampling has been developed, but requires thorough genetic counseling. Although hypophosphatasia is untreatable at present, the recent success of enzyme replacement therapy offers promise. The problems presented by impaired mineralization in age-related chronic diseases, such as pathologic calcification and decreasing physiological mineralization are growing in importance. Strategies for preventing pathologic calcification using TNAP and NPP1 are in development. A nutrigenomic approach, based on the relationship between TNAP gene polymorphism and bone mineral density, is also discussed.

Upregulation of alkaline phosphatase and pyrophosphate hydrolysis: Potential mechanism for uremic vascular calcification

Pyrophosphate is a potent inhibitor of medial vascular calcification where its level is controlled by hydrolysis via a tissue-nonspecific alkaline phosphatase (TNAP). We sought to determine if increased TNAP activity could explain the pyrophosphate deficiency and vascular calcification seen in renal failure. TNAP activity increased twofold in intact aortas and in aortic homogenates from rats made uremic by feeding adenine or by 5/6 nephrectomy. Immunoblotting showed an increase in protein abundance but there was no increase in TNAP mRNA assessed by quantitative polymerase chain reaction. Hydrolysis of pyrophosphate by rat aortic rings was inhibited about half by the nonspecific alkaline phosphatase inhibitor levamisole and was reduced about half in aortas from mice lacking TNAP. Hydrolysis was increased in aortic rings from uremic rats and all of this increase was inhibited by levamisole. An increase in TNAP activity and pyrophosphate hydrolysis also occurred when aortic rings from normal rats were incubated with uremic rat plasma. These results suggest that a circulating factor causes pyrophosphate deficiency by regulating TNAP activity and that vascular calcification in renal failure may result from the action of this factor. If proven by future studies, this mechanism will identify alkaline phosphatase as a potential therapeutic target.

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.

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.

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)

Tissue-non-specific alkaline phosphatase mRNA expression and alkaline phosphatase activity following application of retinoic acid in cultured human dental pulp cells

Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10 −7, 10 −6, 10 −5 M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.

The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

Bovine enamel organ cells express tissue non-specific alkaline phosphatase mRNA.

Alkaline phosphatase (AP) is expressed at high levels in all mineralizing tissues, and the isoform identified in developing enamel has biochemical properties similar to that found in bone. While the bone AP is referred to as the liver/bone/kidney or tissue non-specific (TNS) form, other APs are highly specific for tissue of expression. To determine unequivocally the AP isoform made by enamel organ cells, we constructed a fetal bovine enamel organ cDNA library, which yielded eight AP cDNA clones. In each case, the DNA sequence was homologous to the partial cDNA reported for bovine kidney AP (Garattini et al., 1987). It is concluded that enamel organ cells express the TNS-AP isoform. The extended 3' untranslated region of the cDNA has considerable homology to human TNS-AP, and the conservation of sequence suggests that the 3' end may have a role in post-transcriptional regulation of expression.