Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.

Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Proteolytic, cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH > 6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase. (c) 2005 Elsevier Inc. Ail rights reserved.

Macrophages overexpressing tartrate-resistant acid phosphatase show altered profile of free radical production and enhanced capacity of bacterial killing

Activated macrophages and osteoclasts express high amounts of tartrate-resistant acid phosphatase (TRACP, acp5). TRACP has a binuclear iron center with a redox-active iron that has been shown to catalyze the formation of reactive oxygen species (ROS) by Fenton's reaction. Previous Studies Suggest that ROS generated by TRACP may participate in degradation of endocytosed bone matrix products in resorbing osteoclasts and degradation of foreign Compounds during. antigen presentation in activated macrophages. Here we have compared free radical production in macrophages of TRACP overexpressing (TRACP +) and wild-type (WT) mice. TRACP overexpression increased both ROS levels and Superoxide production. Nitric oxide production was increased in activated macrophages or WT mice, but not in TRACP+ mice, Macrophages from TRACP+ mice showed increased capacity or bacterial killing. Recombinant TRACP enzyme was capable of bacterial killing in the presence of hydrogen peroxide. These results suggest that TRACP has an important biological function in immune defense systern.

Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Zoledronic acid preserves bone structure and increases survival but does not limit tumour incidence in a prostate cancer bone metastasis model

Background The bisphosphonate, zoledronic acid (ZOL), can inhibit osteoclasts leading to decreased osteoclastogenesis and osteoclast activity in bone. Here, we used a mixed osteolytic/osteoblastic murine model of bone-metastatic prostate cancer, RM1(BM), to determine how inhibiting osteolysis with ZOL affects the ability of these cells to establish metastases in bone, the integrity of the tumour-bearing bones and the survival of the tumour-bearing mice. Methods The model involves intracardiac injection for arterial dissemination of the RM1(BM) cells in C57BL/6 mice. ZOL treatment was given via subcutaneous injections on days 0, 4, 8 and 12, at 20 and 100 µg/kg doses. Bone integrity was assessed by micro-computed tomography and histology with comparison to untreated mice. The osteoclast and osteoblast activity was determined by measuring serum tartrate-resistant acid phosphatase 5b (TRAP 5b) and osteocalcin, respectively. Mice were euthanased according to predetermined criteria and survival was assessed using Kaplan Meier plots. Findings Micro-CT and histological analysis showed that treatment of mice with ZOL from the day of intracardiac injection of RM1(BM) cells inhibited tumour-induced bone lysis, maintained bone volume and reduced the calcification of tumour-induced endochondral osteoid material. ZOL treatment also led to a decreased serum osteocalcin and TRAP 5b levels. Additionally, treated mice showed increased survival compared to vehicle treated controls. However, ZOL treatment did not inhibit the cells ability to metastasise to bone as the number of bone-metastases was similar in both treated and untreated mice. Conclusions ZOL treatment provided significant benefits for maintaining the integrity of tumour-bearing bones and increased the survival of tumour bearing mice, though it did not prevent establishment of bone-metastases in this model. From the mechanistic view, these observations confirm that tumour-induced bone lysis is not a requirement for establishment of these bone tumours.

Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model

We investigated the potential of using novel zoledronic acid (ZOL)-hydroxyapatite (HA) nanoparticle based drug formulation in a rat model of postmenopausal osteoporosis. By a classical adsorption method, nanoparticles of HA loaded with ZOL (HNLZ) drug formulation with a size range of 100-130 nm were prepared. 56 female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into seven groups and treated with various doses of HNLZ (100, 50 and 25 mu g/kg, intravenous single dose), ZOL (100 mu g/kg, intravenous single dose) and HA nanoparticle (100 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. After three months treatment period, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for trabecular microarchitecture. Sensitive biochemical markers of bone formation and bone resorption in serum were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the therapy with HNLZ drug formulation was more effective than ZOL therapy in OVX rats. Moreover, HNLZ drug therapy preserved the trabecular microarchitecture better than ZOL therapy in OVX rats. Furthermore, the HNLZ drug formulation corrected increase in serum levels of bone-specific alkaline phosphatase, procollagen type I N-terminal propeptide, osteocalcin, tartrate-resistant acid phosphatase 5b and C-telopeptide of type 1 collagen better than ZOL therapy in OVX rats. The results strongly suggest that HNLZ novel drug formulation appears to be more effective approach for treating severe osteoporosis in humans. (C) 2014 Elsevier B.V. All rights reserved.

Effects of Locally Administered Tiludronic Acid on Experimental Periodontitis in Rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Protein phosphatase activities in the serum and saliva of healthy children

The purpose of this study was to investigate the activities of the total acid phosphatase (TAP), tartrate-resistant acid phosphatase (TRAP), low molecular weight protein tyrosine phosphatase (LMW-PTP) and alkaline phosphatase (ALP) enzymes, as well as the possible correlation in the serum and in unstimulated whole saliva of children. Enzymatic activities were measured in pairs of concurrently obtained serum and salivary samples from 32 children in good oral and systemic health (16 of each sex) with a median age of 6.4 ± 3.3 years (range 1.08 – 12.92 years). All collections were made between the hours of 08:00 – 10:00 a.m. We used p-nitrophenyl phosphate as the substrate in the enzymatic assay for TAP, TRAP and LMW-PTP, and thymolphthalein monophosphate as the substrate for ALP. The enzymatic activities of all the studied enzymes were higher in serum than in saliva. The mean of enzymatic activities of serum TAP, TRAP, LMW-PTP and ALP were 36.51 ± 8.21, 23.99 ± 5.73, 11.16 ± 5.65 and 76.50 ± 17.32 U/L, respectively, while the mean salivary TAP, TRAP, LMW-PTP and ALP enzymatic activities were 9.60 ± 5.04, 1.36 ± 0.87, 5.65 ± 3.07 and 4.08 ± 1.83 U/L in this order. The TRAP revealed a positive linear correlation between its activity in the serum and saliva (Spearman r = 0,4685, p < 0,05). We concluded that the salivary TRAP has a potential to be use as biomarkers of pathologies and states that modify its activity in the serum.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets

INTRODUCTION The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55 Angstrom and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases. (C) 2004 Published by Elsevier Inc.

The role of periosteum : a neglected aspect of osteoporosis

Most current studies on the pathogenesis of osteoporosis emphasize the bone metabolic activities occurring on endosteal surfaces, whereas the periosteal aspect is somewhat neglected. In terms of bone physiology, periosteum plays a determining role in de novo cortical bone formation and cortical bone expansion through periosteum is the most efficient way of increasing bone strength against fractures. Despite the important role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular features of periosteum in osteoporosis. This chapter will focus on the major changes occurring in the periosteum of osteoporosis and possible implications of these changes in the pathogenesis of osteoporosis. The changes identified in the periosteum of osteoporosis are mainly located in the metaphyseal compartment, which include: (a) much thicker and more cellular cambial layer; (b) increased number of TRAP (tartrate resistant acid phosphatase), VEGF (vascular endothelial growth factor) cells and the degree of vascularization; and (c) enhanced expression of sympathetic nerve fibers. The structural and cellular changes of osteoporotic periosteum indicate that periosteum plays an important role in the cortical bone resorption in metaphyseal areas and this pathological process may be regulated by the sympathetic nervous system.

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P\0.001). The number of TRAP? osteoclasts in bone resorption pits, VEGF? cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P\0.05), while no significant difference was detected in the number of ALP? cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.

A comparative study of Sr-incorporated mesoporous bioactive glass scaffolds for regeneration of osteopenic bone defects

SUMMARY: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis. While much investigation is focused on preventing disease progression, here we fabricate strontium-containing scaffolds and show that they enhance bone defect healing in the femurs of rats induced by ovariectomy. INTRODUCTION: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis due to its ability to prevent bone loss in osteoporotic patients. Although much emphasis has been placed on using pharmacological agents for the prevention of disease, much less attention has been placed on the construction of biomaterials following osteoporotic-related fracture. The aim of the present study was to incorporate bioactive strontium (Sr) trace element into mesoporous bioactive glass (MBG) scaffolds and to investigate their in vivo efficacy for bone defect healing in the femurs of rats induced by ovariectomy. METHODS: In total, 30 animals were divided into five groups as follows: (1) empty defect (control), (2) empty defects with estrogen replacement therapy, (3) defects filled with MBG scaffolds alone, (4) defects filled with MBG + estrogen replacement therapy, and (5) defects filled with strontium-incorporated mesopore-bioglass (Sr-MBG) scaffolds. RESULTS: The two groups demonstrating the highest levels of new bone formation were the defects treated with MBG + estrogen replacement therapy and the defects receiving Sr-MBG scaffolds as assessed by μ-CT and histological analysis. Furthermore, Sr scaffolds had a reduced number of tartrate-resistant acid phosphatase-positive cells when compared to other modalities. CONCLUSION: The results from the present study demonstrate that the local release of Sr from bone scaffolds may improve fracture repair. Future large animal models are necessary to investigate the future relationship of Sr incorporation into biomaterials.