Non-specific Alkaline Phosphatase Research Articles

Genetic engineering a large animal model of human hypophosphatasia in sheep

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

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.

Tissue nonspecific alkaline phosphatase promotes calvarial progenitor cell cycle progression and cytokinesis via Erk1,2.

Bone growth is dependent upon the presence of self-renewing progenitor cell populations. While the contribution of Tissue Nonspecific Alkaline Phosphatase (TNAP) enzyme activity in promoting bone mineralization when expressed in differentiated bone forming cells is well understood, little is known regarding the role of TNAP in bone progenitor cells. We previously found diminished proliferation in the calvarial MC3T3E1 cell line upon suppression of TNAP by shRNA, and in calvarial cells and tissues of TNAP-/- mice. These findings indicate that TNAP promotes cell proliferation. Here we investigate how TNAP mediates this effect. Results show that TNAP is essential for calvarial progenitor cell cycle progression and cytokinesis, and that these effects are mediated by inorganic phosphate and Erk1/2. Levels of active Erk1/2 are significantly diminished in TNAP deficient cranial cells and tissues even in the presence of inorganic phosphate. Moreover, in the absence of TNAP, FGFR2 expression levels are high and FGF2 rescues phospho-Erk1/2 levels and cell cycle abnormalities to a significantly greater extent than inorganic phosphate. Based upon the data we propose a model in which TNAP stimulates Erk1/2 activity via both phosphate dependent and independent mechanisms to promote cell cycle progression and cytokinesis in calvarial bone progenitor cells. Concomitantly, TNAP feeds back to inhibit FGFR2 expression. These results identify a novel mechanism by which TNAP promotes calvarial progenitor cell renewal and indicate that converging pathways exist downstream of FGF signaling and TNAP activity to control craniofacial skeletal development.

Supplemented Alkaline Phosphatase Supports the Immune Response in Patients Undergoing Cardiac Surgery: Clinical and Computational Evidence

Alkaline phosphatase (AP) is an enzyme that exhibits anti-inflammatory effects by dephosphorylating inflammation triggering moieties (ITMs) like bacterial lipopolysaccharides and extracellular nucleotides. AP administration aims to prevent and treat peri- and post-surgical ischemia reperfusion injury in cardiothoracic surgery patients. Recent studies reported that intravenous bolus administration and continuous infusion of AP in patients undergoing coronary artery bypass grafting with cardiac valve surgery induce an increased release of liver-type “tissue non-specific alkaline phosphatase” (TNAP) into the bloodstream. The release of liver-type TNAP into circulation could be the body's way of strengthening its defense against a massive ischemic insult. However, the underlying mechanism behind the induction of TNAP is still unclear. To obtain a deeper insight into the role of AP during surgery, we developed a mathematical model of systemic inflammation that clarifies the relation between supplemented AP and TNAP and describes a plausible induction mechanism of TNAP in patients undergoing cardiothoracic surgery. The model was validated against clinical data from patients treated with bovine Intestinal AP (bIAP treatment) or without AP (placebo treatment), in addition to standard care procedures. We performed additional in-silico experiments adding a secondary source of ITMs after surgery, as observed in some patients with complications, and predicted the response to different AP treatment regimens. Our results show a strong protective effect of supplemented AP for patients with complications. The model provides evidence of the existence of an induction mechanism of liver-type tissue non-specific alkaline phosphatase, triggered by the supplementation of AP in patients undergoing cardiac surgery. To the best of our knowledge this is the first time that a quantitative and validated numerical model of systemic inflammation under clinical treatment conditions is presented.

The Evolutionarily Conserved Cassette Exon 7b Drives ERG's Oncogenic Properties

The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.

Alkaline phosphatase: a potential biomarker for stroke and implications for treatment.

Stroke is the fifth leading cause of death in the U.S., with more than 100,000 deaths annually. There are a multitude of risks associated with stroke, including aging, cardiovascular disease, hypertension, Alzheimer's disease (AD), and immune suppression. One of the many challenges, which has so far proven to be unsuccessful, is the identification of a cost-effective diagnostic or prognostic biomarker for stroke. Alkaline phosphatase (AP), an enzyme first discovered in the 1920s, has been evaluated as a potential biomarker in many disorders, including many of the co-morbidities associated with stroke. This review will examine the basic biology of AP, and its most common isoenzyme, tissue nonspecific alkaline phosphatase (TNAP), with a specific focus on the central nervous system. It examines the preclinical and clinical evidence which supports a potential role for AP in stroke and suggests potential mechanism(s) of action for AP isoenzymes in stroke. Lastly, the review speculates on the clinical utility of AP isoenzymes as potential blood biomarkers for stroke or as AP-targeted treatments for stroke patients.

Hybrid compounds from chalcone and 1,2-benzothiazine pharmacophores as selective inhibitors of alkaline phosphatase isozymes

Chalcones and 1,2-benzothiazines are two important classes of bioactive compounds, each scaffold endowed with diverse pharmacological activities. Combining both of these pharmacophores in a single molecule was aimed to yield multi-modal agents. Herein, we report a series of hybrid compounds 3a–3o derived from chalcones and 1,2-benzothiazine cores. They were synthesized from commercially available sodium saccharin, and the resulting 1,2-benzothiazine-derived ketone was then condensed with aromatic aldehydes in an aldol condensation to obtain the respective chalcones. The compounds were characterized using different analytical techniques including FT-IR, NMR spectroscopy, mass spectrometry and X-ray crystallography. Some synthesized chalcones revealed potent and/or selective inhibitory properties towards alkaline phosphatase isozymes transiently expressed in COS-7 cells. A detailed structure-activity and selectivity study was carried out with regard to the effect of different substituents at ortho-, meta- and para-positions of the phenyl residue. Compound 3c was the most effective human intestinal alkaline phosphatase (h-IAP) inhibitor (IC50 value of 1.04 μM), while it was not active against human tissue non-specific alkaline phosphatase (h-TNAP) isozyme. In contrast, 3i was a selective inhibitor of h-TNAP with IC50 values of 0.25 ± 0.01 μM. The possible binding interactions of the most effective inhibitors of h-TNAP and h-IAP were obtained from molecular docking studies.

Neurological symptoms in Hypophosphatasia.

Hypophosphatasia (HPP) is an inborn error of metabolism due to deficiency of tissue non-specific alkaline phosphatase (TNSALP). It is traditionally characterized by rickets in children and osteomalacia in adults, along with fractures, tooth loss, and muscle pain. Neurological symptoms and mental health diagnoses have not been widely reported, and we therefore report their prevalence in a cohort of patients with HPP. A retrospective chart review was performed on a series of 82 HPP patients. Patient charts were reviewed to identify the possible presence and onset of 13 common neurological symptoms. Median age was 36years (2 to 79). Seventeen had adult onset HPP (> 18years) and 65 had pediatric onset HPP (< 18years). Median time from symptom onset to HPP diagnosis was 8years (0 to 67). Seventy-four percent had a family history of bone disease, while 17% had a family history of neurologic disease. Bone problems occurred in 89%, dental problems in 77%, and muscle problems in 66%. Fatigue occurred in 66%, headache in 61%, sleep disturbance in 51%, gait change in 44%, vertigo in 43%, depression in 39%, anxiety in 35%, neuropathy in 35%, and hearing loss in 33%. The extra-skeletal manifestations of HPP, specifically neurological symptoms, have not been previously well documented. However, mental health diagnoses and neurological symptoms such as headache and sleep disturbance occur commonly in patients with HPP. The recognition of non-traditional symptoms in HPP may improve patient satisfaction, preempt costly evaluation and misdiagnosis, and may lead to further treatment options.

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.

Synthesis of Extracellular Pyrophosphate Increases in Vascular Smooth Muscle Cells During Phosphate-Induced Calcification.

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia.

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone and/or dental mineralization, and decreased serum alkaline phosphatase (ALP) activity. ALPL, the only gene related with HPP, encodes tissue non-specific ALP (TNSALP). Few studies were carried out in ALPL gene mutations in the Chinese population with HPP. The purpose of the present study is to elucidate the clinical and genetic characteristics of HPP in five unrelated Chinese families and two sporadic patients. Ten clinically diagnosed HPP patients from five unrelated Chinese families and two sporadic patients and fifty healthy controls were genetically investigated. All 12 exons and exon–intron boundaries of the ALPL gene were amplified by PCR and directly sequenced. The laboratory and radiological investigations were conducted simultaneously in these HPP ten patients. A 3D model of the TNSALP was used to predict the dominant negative effect of identified missense mutations. Three odonto, three childhood, and four adult types of HPP were clinically diagnosed. Ten mutations were identified in five unrelated Chinese families and two sporadic patients, including eight missense mutations and two frameshift mutations. Of which, four were novel: one frameshift mutation (p.R138Pfsx45); three missense mutations (p.C201R, p.V459A, p.C497S). No identical mutations and any other new ALPL mutations were found in unrelated 50 healthy controls. Our study demonstrated that the ALPL gene mutations are responsible for HPP in these Chinese families. These findings will be useful for clinicians to improve understanding of this heritable bone disorder.

МОРФОЛОГИЧЕСКАЯ ОЦЕНКА ЭФФЕКТИВНОСТИ ЭЛЕКТРОХИМИЧЕСКОГО МЕТОДА С ПРИМЕНЕНИЕМ НАНОТЕХНОЛОГИЧЕСКИХ БИОСЕНСОРОВ ДЛЯ ВЫЯВЛЕНИЯ ЩЕЛОЧНОЙ ФОСФАТАЗЫ В БИОПТАТАХ КОЛОРЕКТАЛЬНЫХ КАРЦИНОМ

Objective: To evaluate (morphologically) the effectiveness of the electrochemical method with biosensors applying to study the content of ALP in biopsies of tumors and intact colon mucosa. Materials and methods: Using the electrochemical method with nanotechnological biosensors, the concentration of alkaline phosphatase in biopsy specimens of colorectal carcinomas and intact colon mucosa, obtained from endoscopy from 24 patients were examined. Based on the results of a subsequent histological and immunohistochemical study of biopsy specimens, the effectiveness of the electrochemical method was evaluated. Results: The average value of the current, obtained in the study of fresh tumor biopsies was 0.0492 ± 0. 0007 μA, in case of biopsy specimens of the intact colon mucosa was 0.1197 ± 0.019 μA. Similar results were obtained in the study of biopsies, fixed in formalin: tumor tissue - 0.033 ± 0.0005 μA, intact mucosa - 0.0596 ± 0.0008 μA. The histological structure of tumors corresponded to adenocarcinoma of various degrees of differentiation. A positive correlation of the results of electrochemical and histological / immunohistochemical studies was observed in all patients. Immunohistochemical study of biopsy specimens with antibodies to non-specific alkaline phosphatase revealed high expression of enzyme in the cell membranes of the glandular crypt’s colon epithelium and a weak “background” staining of the cytoplasm and cell membranes of tumor cells. Conclusion: An electrochemical study with biosensors can be used to assess the alkaline phosphatase content in tumor biopsies and intact colon mucosa. Differences in the content of alkaline phosphatase in the tissue of colon adenocarcinoma and the unmodified mucosa are statistically significant. The existing differences in the study of fresh and fixed in formalin biopsy samples indicate the advisability of non-fixed material using.

Hydrolysis of Extracellular Pyrophosphate increases in post-hemodialysis plasma

Vascular calcification (VC) is associated with significant morbidity and mortality of dialysis patients. Previous studies showed an association between loss of plasma pyrophosphate and VC. Moreover, loss of pyrophosphate occurs during dialysis in this population, suggesting that therapeutic approaches that prevent reduction of plasma pyrophosphate levels during dialysis could improve the quality of life of dialysis patients. This study found that pyrophosphate hydrolysis was 51% higher in post- than pre-dialysis plasma. Dialysis sessions modified the kinetic behavior of alkaline phosphatase, increasing its Vmax and reducing its Km, probably due to the elimination of uremic toxins during dialysis. At least 75% of alkaline phosphatase activity in human plasma was found to depend on a levamisole-sensitive enzyme probably corresponding to tissue non-specific alkaline phosphatase (TNAP). Dialysis increased total plasma protein concentration by 14% and reduced TNAP enzyme by 20%, resulting in an underestimation of pyrophosphate hydrolysis in post-dialysis plasma. Levamisole inhibited TNAP activity (IC50, 7.2 µmol/L), reducing pyrophosphate hydrolysis in plasma and increasing plasma pyrophosphate availability. Alkaline phosphatase is also found in many tissues and cells types; therefore, our results in plasma may be indicative of changes in phosphatase activity in other locations that collectively could contribute significantly to pyrophosphate hydrolysis in vivo. In conclusion, these findings demonstrate that dialysis increases pyrophosphate hydrolysis, which, taken together with previously reported increases in alkalization and calcium ion levels in post-dialysis plasma, causes VC and could be prevented by adding calcification inhibitors during dialysis.

Exploration of carboxy pyrazole derivatives: Synthesis, alkaline phosphatase, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase inhibition studies with potential anticancer profile

In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different aryl hydrazines. The products, which can be regarded as 1H-pyrazol-1-yl-one analogues (3a-f, 3g-o, 4a-c, 5a-b) and represent drug like molecules along with well-developed structure–activity relationships, were obtained in good to excellent yield. The structures of synthesized compounds were charcterized on the basis of FT-IR, 1H NMR, 13C NMR and mass spectroscopic data. Considering alkaline phosphatases (APs), nucleotide pyrophosphatases/phosphodiesterases (NPPs) and nucleoside triphosphate diphosphohydrolase as the molecular targets, the effects of these synthesized compounds were investigated on different isozymes of APs, NPPs and NTPDases. The data revealed that the synthesized compounds inhibited both enzymes but most of them inhibited tissue non-specific alkaline phosphatase (TNAP) more selectively. The antitumor activity results indicated that the synthesized derivatives have strong inhibitory effects on the growth of selected cell lines from different tissues such as breast, bone marrow and cervix (MCF-7, K-562 and Hela) but with varying intensities. Moreover the binding mode of interactions were explained on the basis of molecular docking and in-silico studies.

Incidence of Mutations in the ALPL, GGPS1, and CYP1A1 Genes in Patients With Atypical Femoral Fractures.

ABSTRACTAtypical femoral fractures (AFFs) are uncommon and often related to prolonged bisphosphonate (BP) treatment. Isolated cases have been linked to mutations of tissue nonspecific alkaline phosphatase (ALPL). Moreover, mutations in the geranylgeranyl pyrophosphate synthase (GGPPS) gene, which can be inhibited by BPs, and in the enzyme of the cytochrome P450 superfamily (CYP1A1), related to the metabolism of several drugs, have also been associated with AFF development. Our aim was to analyze the incidence of ALPL, GGPS1, and CYP1A1 gene mutations in patients with AFFs and their clinical characteristics. Seventeen women with AAFs were included. All patients underwent Sanger sequencing of the ALPL, GGPS1, and CYP1A1 genes, analyzing the presence of mutations and polymorphisms in these genes. The clinical characteristics of the patients, previous treatments, ALP substrates (vitamin B6 and phosphoethanolamine), bone turnover markers, and bone mass were also analyzed. Three of 17 patients (17.6%) presented heterozygous mutations in the ALPL (p.Gly288Ala) or CYP1A1 (p.Arg136His, p.Val409Ile) genes. Only the patient with the ALPL mutation presented increased ALP substrates. Patients with CYP1A1 variants had glucocorticoid‐induced osteoporosis. All patients were previously treated with BPs during 85.5 ± 38 months, and nearly 50% were also treated with glucocorticoids. The AFF was bilateral in 35% of cases. In conclusion, ALPL and CYP1A1 mutations may be related to the development of AFF in patients treated with BPs. The evaluation of ALP substrates in patients with low ALPL levels allows the identification of patients with hypophosphatasia. The role of CYP1A1 mutations in AFF needs further study. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Augmented Fibroblast Growth Factor-23 Secretion in Bone Locally Contributes to Impaired Bone Mineralization in Chronic Kidney Disease in Mice.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic disorder of mineral and bone metabolism caused by CKD. Impaired bone mineralization together with increased bony secretion of fibroblast growth factor-23 (FGF23) are hallmarks of CKD-MBD. We recently showed that FGF23 suppresses the expression of tissue nonspecific alkaline phosphatase (TNAP) in bone cells by a Klotho-independent, FGF receptor-3-mediated signaling axis, leading to the accumulation of the mineralization inhibitor pyrophosphate. Therefore, we hypothesized that excessive FGF23 secretion may locally impair bone mineralization in CKD-MBD. To test this hypothesis, we induced CKD by 5/6 nephrectomy in 3-month-old wild-type (WT) mice and Fgf23−/−/VDRΔ/Δ (Fgf23/VDR) compound mutant mice maintained on a diet enriched with calcium, phosphate, and lactose. Eight weeks postsurgery, WT CKD mice were characterized by reduced bone mineral density at the axial and appendicular skeleton, hyperphosphatemia, secondary hyperparathyroidism, increased serum intact Fgf23, and impaired bone mineralization as evidenced by bone histomorphometry. Laser capture microdissection in bone cryosections showed that both osteoblasts and osteocytes contributed to the CKD-induced increase in Fgf23 mRNA abundance. In line with our hypothesis, osteoblastic and osteocytic activity of alkaline phosphatase was reduced, and bone pyrophosphate concentration was ~2.5-fold higher in CKD mice, relative to Sham controls. In Fgf23/VDR compound mice lacking Fgf23, 5/6-Nx induced secondary hyperparathyroidism and bone loss. However, 5/6-Nx failed to suppress TNAP activity, and bone pyrophosphate concentrations remained unchanged in Fgf23/VDR CKD mice. Collectively, our data suggest that elevated Fgf23 production in bone contributes to the mineralization defect in CKD-MBD by auto-/paracrine suppression of TNAP and subsequent accumulation of pyrophosphate in bone. Hence, our study has identified a novel mechanism involved in the pathogenesis of CKD-MBD.

2559 Role of tissue non-specific alkaline phosphatase (TNAP) in promoting the survival of acute myeloid leukemia (AML) cells within the bone marrow microenvironment

OBJECTIVES/SPECIFIC AIMS: Treatment of acute myeloid leukemia (AML) is challenging, as apoptosis-resistant AML cells often persist within the bone marrow microenvironment despite chemotherapy. The overall goal of our laboratory is to identify and ultimately target the bone marrow factors that protect AML cells. METHODS/STUDY POPULATION: Using cell cultures, we previously reported that SDF-1 (CXCL12), an abundant bone marrow chemokine, induces apoptosis of isolated CXCR4+ AML cells, including freshly isolated bone marrow-derived AML cells from approximately one-third of AML patients. However, co-culture of AML cells with differentiating osteoblasts protected AML cells from apoptosis. RESULTS/ANTICIPATED RESULTS: Histone deacetylase inhibitors (HDACi) abrogated the ability of osteoblasts to protect AML cells and altered expression of matrix mineralization genes including tissue nonspecific alkaline phosphatase (TNAP). A different drug, cyclosporine A (CSA), similarly inhibited osteoblast-mediated protection of AML cells and reduced TNAP expression. Specifically targeting osteoblast TNAP via siRNA was sufficient to prevent osteoblasts from protecting AML cells in co-cultures. In addition, we are targeting TNAP enzymatically. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that targeting TNAP may be useful in AML treatment to render the bone marrow microenvironment more hostile to leukemic cell survival.

LncRNA HOTAIR Inhibits Mineralization in Osteoblastic Osteosarcoma Cells by Epigenetically Repressing ALPL.

HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.

Distinctive inhibition of alkaline phosphatase isozymes by thiazol-2-ylidene-benzamide derivatives: Functional insights into their anticancer role.

In the recent years, the role of alkaline phosphatase (AP) isozymes in the cause of neoplastic diseases such as breast, liver, renal, and bone cancer has been confirmed and, thus they represent a novel target for the discovery of anticancer drugs. In this study different derivatives of thiazol-2-ylidene-benzamide were evaluated for their potential to inhibit alkaline phosphatase (AP) isozymes. Their anticancer potential was assessed using human breast cancer (MCF-7), bone-marrow cancer (K-562), and cervical cancer (HeLa) cell lines in comparison to normal cells from baby hamster kidney BHK-21. The results suggested that in comparison to other derivatives, compounds 2i, 2e, and 2a showed more sensitivity towards human tissue non-specific alkaline phosphatase (h-TNAP). Among these, 2″-chloro-N-(3-(4'-fluorophenyl)-4-methylthiazol-2(3H)-ylidene) benzamide (2e) was found as the most potent and selective inhibitor for h-TNAP with an IC50 value of 0.079 ± 0.002 μM. Moreover, a significant correlation was observed between the enzyme inhibition profile and cytotoxic data. The compounds exhibiting maximum anticancer potential also induced maximum apoptosis in the respective cell lines. Furthermore, the DNA interaction studies exhibited the non-covalent mode of interaction with the herring sperm-DNA. Molecular docking studies also supported the in vitro inhibitory activity of potent compounds. Our findings suggested that potent and selective inhibitors might be useful candidates for the treatment or prevention of those diseases associated with the higher level of AP. Moreover, the study can be useful for the researcher to explore more molecular mechanisms of such derivatives and their analogues with the exact findings.

Ultrastructure and biological function of matrix vesicles in bone mineralization.

Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca2+) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO43-) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO43- monomers: the prevention of crystal growth is blocked, and PO43- monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO43- in the vesicles. Accumulation of Ca2+ and PO43- inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle's membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.