Showing papers on "Alkaline phosphatase published in 2010"

Physiological role of alkaline phosphatase explored in hypophosphatasia

Abstract: Hypophosphatasia (HPP) is the instructive rickets or osteomalacia caused by loss-of-function mutation(s) within TNSALP, the gene that encodes the "tissue nonspecific" isoenzyme of alkaline phosphatase (TNSALP). HPP reveals a critical role for this enzyme in skeletal mineralization. Increased extracellular levels of pyridoxal 5'-phosphate and inorganic pyrophosphate (PP(i)) demonstrate that TNSALP is a phosphomonoester phosphohydrolase and a pyrophosphatase that hydrolyzes much lower concentrations of natural substrates than the artificial substrates of laboratory assays. Clearly, TNSALP acts at physiological pH and "alkaline phosphatase" is a misnomer. Aberrations of vitamin B(6) metabolism in HPP revealed that TNSALP is an ectoenzyme. PP(i) excesses cause chondrocalcinosis and sometimes arthropathy. The skeletal disease is due to PP(i) inhibition of hydroxyapatite crystal growth extracellularly so that crystals form within matrix vesicles but fail to enlarge after these structures rupture. Trials of alkaline phosphatase replacement therapy for HPP suggest that TNSALP functions at the level of skeletal tissues.

Intestinal alkaline phosphatase: multiple biological roles in maintenance of intestinal homeostasis and modulation by diet

Abstract: The diverse nature of intestinal alkaline phosphatase (IAP) functions has remained elusive, and it is only recently that four additional major functions of IAP have been revealed. The present review analyzes the earlier literature on the dietary factors modulating IAP activity in light of these new findings. IAP regulates lipid absorption across the apical membrane of enterocytes, participates in the regulation of bicarbonate secretion and of duodenal surface pH, limits bacterial transepithelial passage, and finally controls bacterial endotoxin-induced inflammation by dephosphorylation, thus detoxifying intestinal lipopolysaccharide. Many dietary components, including fat, protein, and carbohydrate, modulate IAP expression or activity and may be combined to sustain a high level of IAP activity. In conclusion, IAP has a pivotal role in intestinal homeostasis and its activity could be increased through the diet. This is especially true in pathological situations (e.g., inflammatory bowel diseases) in which the involvement of commensal bacteria is suspected and when intestinal AP is too low to detoxify a sufficient amount of bacterial lipopolysaccharide.

Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

Abstract: In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.

Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

Abstract: Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 µM) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

Shear stress induces osteogenic differentiation of human mesenchymal stem cells.

Abstract: Aim: To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. Materials & methods: hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. Results: Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. Conclusion: These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting ...

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification

Abstract: Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-κB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.

Ferritin ferroxidase activity: a potent inhibitor of osteogenesis.

Abstract: Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor alpha-1 (CBF-alpha1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-alpha1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin.

Acute toxicity of ferric oxide and zinc oxide nanoparticles in rats.

Abstract: We investigated the toxic effects of inhalation exposure to ferric oxide (Fe2O3) and zinc oxide (ZnO) nanoparticles in rats. Male Wistar rats were consecutively treated with Fe22O3 at 8.5 mg/kg body weight and ZnO nanoparticles at 2.5 mg/kg body weight, twice daily for 3 days. Content of Fe2O3 and ZnO in tissues, biochemical parameters in serum, and hispathological examinations were analyzed at 12 h and 36 h after the 3 day treatment. In the Fe2O3-treated group, iron (Fe) content in liver and lung tissues was significantly increased at 36 h. In the ZnO-treated group, zinc (Zn) content in liver tissues was significantly increased at 12 h and further increased at 36 h. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), and lactate dehydrogenase (LDH) in both nanoparticle-exposed groups were significantly decreased compared to the unexposed controls. Histopathological examination showed that both types of nanoparticles caused severe damage in liver and lung tissues. Although this damage progressed in both liver and lung throughout the postexposure period, no significant elevation of serum enzyme activities was observed in response to either nanoparticle type.

The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase.

Abstract: We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.

The effect of bone morphogenetic protein-2, bone morphogenetic protein-7, parathyroid hormone, and platelet-derived growth factor on the proliferation and osteogenic differentiation of mesenchymal stem cells derived from osteoporotic bone.

Abstract: Introduction: It has been previously shown that in patients with osteoporosis, mesenchymal stem cell (MSC) growth rate and osteogenic potential is decreased contributing to inferior fracture consolidation. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2), BMP-7, parathyroid hormone (PTH), and platelet-derived growth factor (PDGF) on proliferation and osteogenic differentiation of MSCs derived from patients with osteoporosis. Materials and Methods: Trabecular bone was obtained from 10 patients (four males, mean age 76 years) with lower extremity osteoporotic fractures. MSCs were isolated by enzymatic digestion. Functional assays of proliferation and osteogenic differentiation were performed under the influence of a wide range of concentrations of BMP-2, BMP-7, PTH, and PDGF-BB. Proliferation was assessed using CFU-F and XTT assays. Osteogenic differentiation was assessed by alkaline phosphatase activity and total calcium production. Results: MSC proliferation was found to be stimulated by supplementation with BMP-7 and PDGF-BB, whereas BMP-2 and PTH had little effect. The largest increase in proliferation rate was observed after administration 100 ng/mL of BMP-7. All four molecules induced alkaline phosphatase activity and calcium production in growing osteoblasts with a dose-dependent effect noted. BMP-2 and BMP-7 at their highest studied concentration (100 ng/mL) produced a threefold increase in the osteogenic potential of MSCs. Conclusion: BMP-7, BMP-2, PTH, and PDGF-BB were observed to have a positive effect on osteogenic differentiation of MSCs. BMP-7 and PDGF-BB (in high doses) could be considered most potentially advantageous because they enhance both proliferation and osteogenic differentiation of MSCs derived from elderly osteoporotic bone.

Determination of the minimal melatonin exposure required to induce osteoblast differentiation from human mesenchymal stem cells and these effects on downstream signaling pathways.

Abstract: The purpose of this study was to determine the critical time periods of melatonin treatment required to induce human mesenchymal stem cells (hAMSCs) into osteoblasts and to determine which osteogenic genes are involved in the process. The study design consisted of adding melatonin for different times (2, 5, 10, 14 or 21 days) toward the end of a 21-day treatment containing osteogenic (OS+) medium or at the beginning of the 21-day treatment and then withdrawn. The results show that a 21-day continuous melatonin treatment was required to induce both alkaline phosphatase (ALP) activity and calcium deposition and these effects were mediated through MT₂Rs. Functional analysis revealed that peak ALP levels induced by melatonin were accompanied by attenuation of melatonin-mediated inhibition of forskolin-induced cAMP accumulation. Immunoprecipitation and western blot analyses, respectively, showed that MT₂R/β-arrestin scaffolds complexed to Gi, MEK1/2 and ERK1/2 formed in these differentiated hAMSCs (i.e., when ALP levels were highest) where ERK1/2 resided primarily in the cytosol. It is hypothesized that these complexes form to modulate the subcellular localization of ERK1/2 to affect osteogenic gene expression. Using real-time RT-PCR, chronic melatonin exposure induced the expression of osteogenic genes RUNX-2, osteocalcin and BMP-2, through MT₂Rs. No melatonin-mediated changes in the mRNA expression of ALP, BMP-6 or in the oxidative enzymes MtTFA, PGC-1α, Polγ, NRF-1, PDH, PDK and LDH occurred. These data show that a continuous 21-day melatonin exposure is required to induce osteoblast differentiation from hAMSCs through the formation of MT₂R/Gi/β-arrestin/MEK/ERK1/2 complexes to induce osteogenesis.

A Role for N‐Cadherin in the Development of the Differentiated Osteoblastic Phenotype

Abstract: Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.

Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase.

Abstract: Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear....

Early Weaning Reduces Small Intestinal Alkaline Phosphatase Expression in Pigs

Abstract: Expression of the small intestinal alkaline phosphatase (IAP) is enterocyte differentiation dependent and plays essential roles in the detoxification of pathogenic bacterial lipopolysaccharide endotoxin, maintenance of luminal pH, organic phosphate digestion, and fat absorption. This study was conducted to examine the effect of early weaning on adaptive changes in IAP digestive capacity (V(cap)) and IAP gene expression compared with suckling counterparts in pigs at ages 10-22 d. Weaning decreased (P < 0.05) IAP enzyme affinity by 26% and IAP maximal enzyme activity by 22%, primarily in the jejunal region, with the jejunum expressing 84-86% of the whole gut mucosal IAP V(cap) [mol/(kg body weight.d)]. The majority (98%) of the jejunal mucosal IAP maximal activity was associated with the apical membrane and the remaining (2%) existed as the intracellular soluble IAP. Weaning reduced the abundance of the 60-kDa IAP protein associated with the proximal jejunal apical membrane by 64% (P < 0.05). Furthermore, weaning reduced (P < 0.05) the relative abundance of the proximal jejunal IAP mRNA by 58% and this was in association with decreases (P < 0.05) in the abundances of cytoplasmic (27%) and nuclear (29%) origins of IAP caudal-associated homeobox transcription factor 1. In conclusion, early weaning decreased small intestinal IAP V(cap), IAP catalytic affinity, and IAP gene expression, and this may in part contribute to the susceptibility of early-weaned piglets to increased occurrence of enteric diseases and growth-check.

The effects of fluoride on osteoblast progenitors in vitro.

Abstract: The number of discrete, three-dimensional bone nodules formed in vitro from a class of osteoprogenitor cells present in fetal rat calvaria cell populations (RC cells) is linearly related to the number of cells plated, implying that this system functions as a colony assay for the expression of osteoprogenitor cells. To determine the effect of fluoride on the expression of these cells, primary RC cells were grown for periods of up to 21 days in alpha-MEM (minimal essential medium) containing 5-15% heat-inactivated fetal bovine serum (FBS), 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and NaF at concentrations from 10 microM to 5 mM. The continuous presence of NaF resulted in an increase in the number of bone nodules with maximal response occurring at 500 microM (p less than 0.001). A similar response at 500 microM NaF was observed also with regard to alkaline phosphatase activity. NaF levels up to 500 microM did not affect the growth of the mixed RC cell population, however, higher concentrations (1 mM) significantly reduced cell numbers (p less than 0.001) suggestive of cytotoxicity. Plating efficiency tests for colony formation in the presence of 0.5 to 2 mM NaF showed that the decreases in nodule formation observed at concentrations above 500 microM correlated with cytotoxicity. NaC1 at 1 mM had no effect on nodule formation, alkaline phosphatase activity, or cell growth. The results show that NaF stimulates osteoprogenitor cell number in vitro and that the maximal effect occurs at concentrations close to toxic levels.

β-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study

Abstract: Human adipose tissues surgically resected from the subcutaneous abdominal region were enzymatically processed to obtain Human Adipose Stem cells (fibroblast-like adipose tissue-derived stromal cells—ADSC-FL) that were immunophenotypically characterized using a panel of mesenchymal markers by flow cytometry. The formation of new hydroxyapatite crystals in culture dishes, by differentiating cells, further demonstrate the osteogenic potential of purified cells. The aim of this study was to evaluate the osteogenic differentiation potential of ADSC-FL seeded onto a porous β-tricalcium phosphate (β-TCP) matrix. ADSC-FL was cultured on the β-TCP matrix in medium with or without osteogenic differentiation additives. Time-dependent cell differentiation was monitored using osteogenic markers such as alkaline phosphatase (activity assay), osteocalcin and ostopontin (ELISA method) expression. Our results reveal that β-TCP triggers the differentiation of ADSC-FL toward an osteoblastic phenotype irrespective of whether the cells are grown in a proliferative or a differentiative medium. Hence, a β-TCP matrix is sufficient to promote osteoblastic differentiation of ADSC-FL. However, in proliferative medium, alkaline phosphatase activity was detected at lower level respect to differentiative medium and osteocalcin and osteopontin showed an expression delay in cells cultured in proliferative medium respect to differentiative one. Moreover, we observed an increase in FAK phosphorylation at level of tyrosine residue in position 397 (Western-blot) that indicates a good cell adhesion to β-TCP scaffold. In conclusion, our paper demonstrates that a three-dimensional β-TCP scaffold in vitro triggers on its own the differentiation of ADSC-FL toward an osteoblastic phenotype without the need to use differentiative media.

Probiotic bacteria Lactobacillus rhamnosus influences the blood profile in rainbow trout Oncorhynchus mykiss (Walbaum).

Abstract: This paper reports the effect of feeding probiotic diets on blood profiles in rainbow trout. Two experiments were performed: in the first, fish of average weight 75 g were offered either a commercial feed or the same incorporated with 109 CFU g−1 of lactic acid bacteria Lactobacillus rhamnosus for 30 days; in the second study performed for a similar duration, fish of average weight 126 g were offered formulated diets that either contained the same bacteria in heat-killed or freeze-dried form (nearly 1011 CFU g−1), or the basal diet without the bacteria. Blood samples were collected at different times after commencement of probiotic feeding to determine the total cholesterol, triglyceride contents, the plasma alkaline phosphatase activity, plasma protein and hematocrit value. The plasma cholesterol significantly increased upon probiotic feeding in the first experiment. A significant elevation (P < 0.05) of plasma cholesterol and triglyceride and alkaline phosphatase activity level was found in the freeze-dried probiotic fed groups at 20 and 30 days postfeeding. This was concomitant with the increased plasma protein and hematocrit values in FD group at 20 and 30 days. Likewise, the heat-killed probiotic fed group registered significantly high values of triglycerides, alkaline phosphatase activity, and plasma protein compared to the control diet fed groups after 20 days of feeding. Thus, alterations in the blood profiles could serve as supplementary information when examining the benefits of probiotics for fish.

Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cell.

Abstract: Dexamethasone (Dex) is used widely to induce differentiation in human mesenchymal stem cells (hMSCs); however, using a pharmaceutical agent to stimulate hMSC differentiation is not the best choice for engineered tissue transplantation due to potential side-effects. The goal of the present study was to investigate the effects of dynamic compressive loading on differentiation and mineralized matrix production of hMSCs in 3D polyurethane scaffolds, using a loading regimen previously shown to stimulate mineralised matrix production of mature bone cells (MLOA5). hMSCs were seeded in polyurethane scaffolds and cultured in standard culture media with or without Dex. Cell-seeded scaffolds were compressed at 5% global strain for 2 h on day 9 and then every 5 days in a media-filled sterile chamber. Samples were tested for mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), collagen type 1 (col 1) and runt-related transcription factor2 (RUNX-212 h) after the first loading, cell viability by MTS assay and alkaline phosphatase activity at day 12 of culture and cell viability, collagen content by Sirius red and calcium content by alizarin red at day 24 of culture. Neither Dex nor loading had significant effects on cell viability. Collagen content was significantly higher (p<0.01) in the loaded group compared with the non-loaded group in all conditions. There was no difference in ALP activity or the amount of collagen and calcium produced between the non-loaded group supplemented with Dex and the loaded group without Dex. We conclude that dynamic loading has the ability to stimulate osteogenic differentiation of hMSC in the absence of glucocorticoids.

Defects in mesenchymal stem cell self-renewal and cell fate determination lead to an osteopenic phenotype in Bmi-1 null mice.

Abstract: In parathyroid hormone-related protein 1-84 [PTHrP(1-84)] knockin mice, expression of the polycomb protein Bmi-1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi-1(-/-) mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM-MSCs) from Bmi-1(-/-) mice ex vivo in culture. Neonatal Bmi-1(-/-) mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar-gamma expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1-84) knockin mouse. The efficiency of colony-forming unit fibroblast (CFU-F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase-positive CFU-F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar-gamma expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM-MSCs were associated with upregulation of senescence-associated tumor-suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi-1(-/-) mice suggests that Bmi-1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi-1 maintains self-renewal of BM-MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM-MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi-1 therefore plays a critical role in promoting osteogenesis.

A Role for Intestinal Alkaline Phosphatase in the Maintenance of Local Gut Immunity

Abstract: Background and Aims Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains poorly defined. We investigated immune responses of wild-type (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Abstract: Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both αIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses. (J Bone Miner Res 2000;15:188–197)

Protective Role of Tinospora cordifolia against Lead-induced Hepatotoxicity.

Abstract: The importance of Tinospora cordifolia stem and leaves extract was investigated for its possible hepatoprotective effect in Swiss albino male mice against lead nitrate induced toxicity. Oral administration of plant extracts prevented the occurrence of lead nitrate induced liver damage. The decreased level of tissue enzymes, i.e., superoxide dismutase (SOD), catalase (CAT) and increased level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and acid phosphatase (ACP) were observed in mice treated with lead. Administration of aqueous stem extract (400 mg/kg body weight, orally) and aqueous leaves extract (400 mg/kg body weight, orally) along with the lead nitrate (5 mg/kg body weight, i.p. for 30 days) increased the activities of SOD and CAT and decreased the levels of AST, ALT, ALP, and ACP enzymes in mice. These biochemical observations were supplemented by histopathology/histological examinations of liver section. Results of this study revealed that plant extract could afford protection against lead-induced hepatic damage.

Increased serum alkaline phosphatase as a predictor of long-term mortality after stroke

Abstract: Background: Although the critical role of alkaline phosphatase in bone mineralization is clearly understood, the potentially adverse effect of high alkaline phosphatase levels on the cardiovascular system was only recently suggested. In this study, we hypothesized that increased levels of serum alkaline phosphatase may be associated with poor outcome after stroke in terms of mortality. Methods: We prospectively included patients with acute stroke admitted consecutively to our hospital, from October 2002 to September 2008. A total of 2,029 patients were selected for the analyses. In the analyses of mortality, the patients were divided by baseline measurements into quintiles of alkaline phosphatase levels ( 97 IU/L). Results: In the Cox proportional hazard models, compared with the first alkaline phosphatase quintile, adjusted hazard ratios of the third, fourth, and fifth quintiles for all-cause death were 1.67 (95% confidence interval 1.12–2.49), 1.79 (1.20–2.67), and 2.83 (1.95–4.10). When we divided the patients into ischemic and hemorrhagic stroke, the association was also significant for both subtypes of stroke. In terms of vascular death, compared to the first alkaline phosphatase quintile, the adjusted hazard ratios of the fourth and fifth quintiles of alkaline phosphatase were 1.81 (95% confidence interval 1.14–2.86) and 2.78 (1.87–4.15). Conclusion: Our study demonstrated that increased serum levels of alkaline phosphatase are an independent predictor of all-cause and vascular death after either ischemic or hemorrhagic stroke.

Perinatal hypophosphatasia presenting as neonatal epileptic encephalopathy with abnormal neurotransmitter metabolism secondary to reduced co-factor pyridoxal-5′-phosphate availability

Abstract: We describe two neonates presenting with perinatal hypophosphatasia and severe epileptic encephalopathy resulting in death. Both had increased levels of urinary vanillactate, indicating functional deficiency of aromatic amino acid decarboxylase, a pyridoxal-5-phosphate (PLP)-dependent enzyme required for dopamine and serotonin biosynthesis. Clinical findings and results of subsequent metabolic investigations were consistent with secondary pyridoxine-deficient encephalopathy. These patients highlight the importance of tissue non-specific alkaline phosphatase in the neuronal PLP-dependent metabolism of neurotransmitters. In addition, the disturbance of PLP metabolism appears to underlie the predominant neurological presentation in our patients. We recommend the measurement of serum alkaline phosphatase (ALP) during the assessment of perinatal seizures.