Cellular Alkaline Phosphatase Research Articles

Caffeine alters mitochondrial dehydrogenase and alkaline phosphatase activity of human gingival fibroblasts in vitro

Caffeine is one of the most widely consumed behaviorally active substances in the world. Although its effects on the central nervous system and bone metabolism have been documented, as yet there is no report on its effect on tissues in the oral cavity. In this study we analyzed the viability of human gingival fibroblasts (HGF) and alkaline phosphatase (ALP) enzyme activity after exposure to different concentrations of caffeine for different exposure time periods. The HGF were cultured with different concentrations of caffeine. Viability of cells exposed to caffeine was analyzed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay to assess mitochondrial dehydrogenase activity. The activity of ALP was analyzed at specific time intervals after caffeine addition. Our results showed that caffeine of concentrations <1mm did not affect the viability of HGF and the ALP enzyme activity. Nevertheless, caffeine at 5 and 10mm dramatically decreased the viability and ALP activity of the cells after 4days such that, by day 9, the viability of cells declined to near zero in the 10mm group. These results provided evidence that caffeine in high concentrations can decrease cellular viability and ALP activity in HGF.

The influence of prefreezing temperature on pore structure in freeze-dried β-TCP scaffolds

A combined method of tricalcium phosphate (TCP) scaffold production, which comprised negative mold and scaffold fabrication, was reported in this study. The negative mold structure was designed by computer and fabricated by fused deposition modeling (FDM) technology, while the TCP scaffold was produced by freeze-drying technology under different prefreezing temperatures of -10 degrees C, -30 degrees C, and -86 degrees C and thermal treatment to get beta-TCP. The scaffold structure was evaluated with X-ray, scanning electron microscopy (SEM), compressive mechanical testing, and micro-computerized tomography (micro-CT). The cell-scaffold interaction was studied by culturing dog bone marrow stromal cells (BMSCs) on the scaffolds and assessing differentiated BMSC function by measuring cellular alkaline phosphatase (ALP) activity. The results showed good interconnectivity and good pore distribution with the pore size ranging from 50 to 250 microm and compressive modulus of 1.18 MPa at a prefreezing temperatures of -10 degrees C. In vitro cell culture results indicated that the porous scaffolds were not toxic to bone cells. These results demonstrate that rapid prototyping and freeze-drying technologies for creating beta-TCP scaffolds are promising for bone tissue engineering.

Pulsed Low Intensity Electromagnetic Field (PEMF) Affects Cell Cycle of Human Osteoblast-like Cells &lt;i&gt;in &lt;/i&gt;&lt;i&gt;Vi&lt;/i&gt;&lt;i&gt;tro&lt;/i&gt;

High frequency electro magnetic field in the range of 4 - 33 kHz given in low frequency bursts of 7-15 Hz (PEMF) is efficient in stimulating osteoblast proliferation and differentiation in vitro. We hypothesize that osteoblast cell cycle changes following this stimulatory PEMF. In order to explore this effect we used primary explant cultures of human osteoblast-like cells. These cells were subjected to low frequency of 20-30 Hz, h igher frequency of 5-15 kHz electro magnetic fields and to PEM F (5-15 kHz basic frequency in pulses of 20-30 Hz) with maximal magnetic flu x of 10-12 Gauss. The maturation state of the cells was estimated by the measurement of cellular alkaline phosphatase activity and the overall cell death by the LDH activity in culture media. The cell cycle was analyzed by cytometry. The PEMF stimulation significantly increased the proportion of cells in the G1 phase (p<.001) and decreased the proportion of apoptotic and necrotic cells (p<.001), with parallel decrease in cellular alkaline phosphatase and media LDH act ivities. Pure 20-30 Hz or 5-15 kHz electro magnetic stimu lations had no effect on these parameters. Therefore we show that PEM F with a basic frequency in the kHz range provided in pulses in the infrasonic range alters human osteoblast cell cycle by reducing cell death and cellular maturation.

Bioactivity of nanostructure on titanium surface modified by chemical processing at room temperature

PurposeRecently, there has been considerable interest in finding novel applications and functions for existing dental materials. We found that, at room temperature and atmospheric pressure, titanium oxide spontaneously generates nanostructures very similar to the “nanotubes” created by TiO2 sputtering. The aim of this study was to evaluate the ability of this surface to affect the cellular osteogenic differentiation response. MethodsTitanium disks without and with a ‘nanosheet’ deposited on their surface were used as the control and test groups, respectively. Cell culture experiments were performed with SD rat bone marrow cells, which were seeded into microplate wells and cultured in media designed to induce osteogenic differentiation. We measured alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, calcium deposition and Runx2 gene expression to assess the levels of differentiation. ResultsAfter 14 and 21days, cellular ALP activity was significantly higher in the test group than in the control group. After 28days, cells in the test group also showed significantly more calcium deposition and OCN production than those in the control group. There was significantly different expression of Runx2 mRNA in the test group compared to the control group after 3days of culture. ConclusionIn conclusion, these data suggest that titanium implants modified by the application of nanostructures promote osteogenic differentiation, and may improve the biointegration of these implants into the alveolar bone.

Sanguis Draconis resin stimulates osteoblast alkaline phosphatase activity and mineralization in MC3T3-E1 cells

Ethnopharmacological relevanceSanguis Draconis (SD), “Dragon's Blood”, is a resin that is obtained from Daemonorops draco (Palmae). Used in traditional medicine, it has shown activity in the prevention of osteoporosis as well as promoting the healing of bone fractures. Materials and methodsIn this study, the effects of Sanguis Dranonis ethanol extract on β-glycerolphosphate and ascorbic acid induced differentiation using mouse calvaria origin MC3T3-E1 osteoblastic cells was examined. We looked at osteoblast differentiation, proliferation, and mineralization by measuring alkaline phosphatase (ALP) and specific bone marker activities. Osteoblast-like MC3T3-E1 cells were cultured in various concentrations of SD ethanol extract (0.005-1mg/mL) during the osteoblast differentiation period (1, 5, 15, and 25days). ResultsAs measured by 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay, SD extracts increased cell proliferation as compared to control. The most pronounced effect was observed at the concentration range between 0.01 and 0.1mg/mL (P<0.01). This SD stimulatory effect for cell proliferation was observed during the whole osteogenic period. Cellular (synthesized) ALP activity was increased during 15 days of culture, and was confirmed by the staining of ALP activity on cell matrix layers for matrix calcification. SD stimulatory effect for cell mineralization we observed in 14 and 21 days. Elevated mRNA or protein levels of bone morphogenetic protein-2(BMP 2), the differentiation marker osteocalcin, osteopontin, collgen I, and a master osteogenic transcription factor, Runx2, were observed in SD-treated cells. ConclusionsThese results suggest that SD may increase osteogenic effect by stimulating cell ALP activity and affect the BMP signaling pathway cascades in osteoblastic cells, then promotes osteoblast differentiation, mineralization, and bone formation.

Effects ofmyo-inositol on proliferation, differentiation, oxidative status and antioxidant capacity of carp enterocytes in primary culture

This study investigated the effects of myo-inositol (MI) on the growth and antioxidant capacity of carp enterocytes. The enterocytes were incubated in media containing 0, 15, 30, 45, 60 and 75 mg MI L−1 for 96 h. The results indicated that MI could increase cell viability. In addition, the activities of cellular alkaline phosphatase (AKP), gamma-glutamyl transpeptidase (γ-GT), Na+, K+-adenosine trisphosphatase (Na+, K+-ATPase) and creatinkinase (CK) increased with MI supplementation at levels ranging from 15 to 60 mg MI L−1 medium, indicating an improvement in cell differentiation and function. Further, enzymatic antioxidant ability, as measured by total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities, improved with MI supplementation. Finally, cell damage, as indicated by lactic acid dehydrogenase (LDH) activity, malondialdehyde (MDA) content of the medium and cellular protein carbonyls (PC), was all depressed by MI. Correlation analyses showed that cell viability (MTT) was positively related to the antioxidant enzyme activities, but negatively related to cell damage (LDH, MDA and PC). In summary, the data showed that MI could improve the growth of fish enterocytes. This result may be partly due to the enhanced antioxidant status and depressed oxidative damage.

In vitro performance of novel porous composites cultured with human mesenchymal stem cells

Novel porous BG–COL–HYA–PS bioactive composites composed of a sol–gel processed bioactive glass (BG), collagen (COL), hyaluronic acid (HYA) and phosphatidylserine (PS) have been developed for bone tissue engineering applications. In this study, the in vitro performance of the porous composites with or without bone morphogenetic protein (BMP) was evaluated after culturing with human mesenchymal stem cells (hMSCs) by: (1) phase contrast microscopy for cell morphology after culture with the composites; (2) MTT assay for cell viability and proliferation, and (3) alkaline phosphatase (ALP) and osteocalcin (OC) assays for cell differentiation after culture in extracts from the composites at 1, 3 and 7d. The results showed that cells attached and spread on and around the composites after 1d. The cells proliferated during the culture period. The composites are not cytotoxic and are compatible with hMSCs after 7d. Furthermore, the cellular ALP activity and OC concentration for the culture with BMP-combined composites are significantly higher than those for the composites alone and control. Human BMSCs were also labeled with Dil and seeded on the composite in order to investigate the effects of cell seeding density on the distribution and growth of human bone marrow stem cells (BMSCs) on the composite.

Anabolic effects and inhibition of interleukin 6 production induced by neridronate on human osteoblasts

Bisphosphonates (BPs) are pharmacological compounds widely used in the treatment of a variety of bone-related diseases, particularly where the bone-turnover is skewed in favour of osteolysis. The mechanisms by which BPs reduce bone-resorption directly acting on osteoclasts (OCs) are now largely clarified even at molecular level. The researches concerning the BPs effects on osteoblasts (OBs) have instead shown variable results. We have investigated the efficacy of neridronate (NER), an amino-BP, as anabolic agent on human OBs. Moreover, we have tried to verify if NER is able to negatively modulate the production of IL-6 on OBs stimulated or not by the pro-inflammatory cytokine IL-1beta. We have tested if different concentrations of NER (from 10-11 M to 10-3 M), added to primary human OB cultures, could affect the cells number, the endogenous cellular alkaline phosphatase (ALP) activity, the collagen I (COL-I) synthesis, the formation of mineralized nodules and the IL-6 production. Our experimental approach was performed testing a wide range of NER concentrations because, under physiological conditions, OBs seems to be exposed to variable and transient levels of the drug. Our results show that NER doesn't negatively affect in vitro the viability, proliferation and cellular activity of human OBs, even after 20 days of exposure to concentrations < or =10-5 M (therapeutic dose). In addition, NER seems to enhance the differentiation of cultured OBs in mature bone-forming cells. A maximum increase of COL-I synthesis (+25% after 4 days; p < 0.05), ALP activity (+50% after 10 days; p < 0.01) and mineralized nodules (+48% after 20 days; p < 0.05) was observed in cultures treated with NER 10-8 M. A maximal reduction of IL-6 secretion (-24% on IL-1beta stimulated cultures and -29% on unstimulated cultures) was observed for NER 10-9 M. These results encourage the use of neridronate in therapy of demineralizing metabolic bone disorders.

In vitro biocompatibility of magnesium-incorporated submicro-porous titanium oxide surface produced by hydrothermal treatment

This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) oxide surface incorporating magnesium ions (Mg), produced by hydrothermal treatment using an alkaline Mg-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and optical profilometry. Mouse calvaria-derived osteoblastic cell (MC3T3-E1) attachment, spreading, proliferation, alkaline phosphatase (ALP) activity, and osteoblastic gene expression on Mg-containing surfaces were compared with untreated Ti surfaces. Hydrothermal treatment resulted in Mg-incorporated Ti oxide layer with submicro-porous surface structures approximately 2 μm in thickness. ICP-AES analysis revealed Mg ions release from treated surfaces into the solution. The Mg-incorporated surface displayed significantly increased cellular attachment and ALP activity compared with untreated surface ( p < 0.05), and supported better cell spreading. Real-time polymerase chain reaction analysis showed notably higher mRNA expression of the osteoblast transcription factor genes (Dlx5, Runx2) and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on the Mg-incorporated surfaces than untreated surfaces. These results demonstrate that the Mg-incorporated submicro-porous Ti oxide surface produced by hydrothermal treatment may improve implant osseointegration by enhancing the attachment, spreading and differentiation of osteoblastic cells.

Red yeast rice stimulates osteoblast proliferation and increases alkaline phosphatase activity in MC3T3-E1 cells

Red yeast (Monascus purpureus) is used as a traditional hypocholesterolemic dietary food component in Asia due to its bioactive component, lovastatin. Recently, new evidence suggesting that the statins in red yeast enhance bone formation has been reported, but more research is still needed in order to support these claims of osteogenic effects. Therefore, in this study, we hypothesized that red yeast rice (in which red yeast is fermented) can improve osteogenic function through osteoblast cell proliferation and differentiation. We studied the effect of methanol extract of red yeast rice powder (RYRP) on osteoblast proliferation and differentiation by measuring mitochondrial enzyme activity and bone marker alkaline phosphatase (ALP) activity, respectively. Osteoblast-like MC3T3-E1 cells were cultured in various concentrations of RYRP methanol extract (0.001-1 mg/mL) during the osteoblast differentiation period (1, 5, 10, and 15 days). As measured by 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay, RYRP extracts stimulated cell proliferation during a 24-hour period, compared to cooked white rice powder extract. The most pronounced effect was observed at the concentration range between 0.075 and 0.1 mg/mL. This RYRP stimulatory effect for cell proliferation was observed during the whole osteogenic period. Cellular (synthesized) ALP activity was increased at a RYRP extract concentration of 0.075 mg/mL during 15 days of culture, but the medium (secreted) ALP activity did not show any significant change. This cellular ALP activity stimulation by RYRP extract was confirmed by the staining of ALP activity on cell matrix layers for matrix calcification. The results imply that RYRP extract may increase osteogenic effect by stimulating cell proliferation and ALP activity in osteoblastic cells.

The Role of Activin A in The Human Osteoblast Cell Cycle: A Preliminary Experimental in Vitro Study

Animal studies have previously shown that activin A enhances osteoblast proliferation IN VITRO and increases bone formation and bone mechanical strength IN VIVO. For the further understanding of its action in human osteoblast, we studied the pattern of a cell cycle response to the treatment with activin A. We hypothesize that activin A alters the cell cycle pattern of human osteoblast. Primary cultures of human osteoblast-like cells were treated by activin A in a biologically effective concentration (100 ng/mL). The cells in cultured samples were counted, assayed for cellular alkaline phosphatase activity and calcitonin expression, LDH activity in the medium, cellular BrdU incorporation, cell cycle cytometry and compared to untreated controls. The treated by activin A cells responded by a significant shift toward the G1 phase of the cell cycle with parallel decrease in cell death rate (lower LDH activity and less necrotic cells in cytometric analysis). The treated cells also showed a lower alkaline phosphatase activity and calcitonin expression, indicating their undifferentiated state, and didn't change their proliferation rate. The number of cells in culture increased following treatment with activin A. We show that activin A increases the net osteoblast number in culture by reducing the cell death rate without affecting the cell proliferation. These findings should be part of cellular pathways that are involved in the initial stages of bone tissue generation.

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

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.

Block Copolymer of Polyphosphoester and Poly(l-Lactic Acid) Modified Surface for Enhancing Osteoblast Adhesion, Proliferation, and Function

Surface modification is often needed in tissue engineering to enhance the interaction between cells and synthetic materials and improve the cytocompatibility and cellular functions. In this study, block copolymers of poly(L-lactic acid) and poly(ethyl ethylene phosphate) (PLLA-b-PEEP) were synthesized and used to modify the PLLA surface via a spin-coating process, to understand whether surface modification with polyphosphoester-based polymer will be osteoinductive for potential bone tissue engineering applications. X-ray photoelectron spectra measurements revealed that phosphorus atomic compositions after surface modification increased from 2.09% to 4.39% with increasing PEEP length of PLLA-b-PEEP from 58 to 224 units, which also led to a more hydrophilic surface property compared with unmodified PLLA. The initial osteoblast attachment and proliferation on the modified surfaces were significantly enhanced. Moreover, cellular alkaline phosphatase activity and mineral calcium depositions were also promoted by PEEP modification. The gene expression determined by reverse transcription polymerase chain reaction further revealed that type I collagen and osteocalcin expression were upregulated in osteoblasts cultured on the modified surfaces, indicating that PEEP modification might be potentially osteoinductive and favorable for further application in bone tissue engineering.

Biocompatibility of a new medical magnesium alloy in vitro

Objective To evaluate the in vitro biocompatibility of new biomaterials (AZ-1 and AZ-3) which result from film oxidation of AZ9ID by step anodization. Methods Osteoblastic cells were harvested by sequential enzyme digestion from calvaria of Sprague Dawhy rats and cultured in vitro onto the magnesium alloys AZ-1 and AZ-3 respectively. The morphology and function of the osteoblastic cells were observed by scanning electron microscopy, MTT assay and alkaline phosphatase activity assay. The bio-materials were also tested by in vitro hemolytic test with fresh cony blood. Results The osteoblastic cells adhered to, and proliferated and grew normally and perfectly on surface of the biomedical magne-sium alloys. Their cellular activity and alkaline phosphatase activity were not influenced by the new biomedical material. The in vitro hemolytic test showed that the hemolytic rates of all samples were lower than 5 %. Conclusion Since the new magnesium alloys seem to possess good biocompatibility in vitro, it may be a new type of material as bone implant. Key words: Magnesium; Alloys; Materials testing; Osteoblastic cells; Step anodization

Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway

To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free alpha-minimum essential media plus 10% fetal bovine serum (dextran-coated charcoal stripped) supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L beta-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 micromol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. The treatment of HBMSC with NTG (0.1-10 micromol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evidenced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 micromol/L) could not be abolished by either N(G ) -nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity. NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.

Osteogenese unter dem Einfluß ionisierender Bestrahlung in vitro

Irradiation is a well established therapeutical concept to prevent heterotopic ossification after joint replacement. The influence of irradiation on proliferation of mature osteoblasts and their potential osteoprogenitors, matrix formation and mineralization are not well known in this setting. We therefore studied the effect of different doses of ionizing irradiation on the several steps of osteogenesis in vitro, using cells isolated from the juvenile rat. A colony forming test, the MTT-viability assay, a cell count, measurement of the cellular protein content and alkaline phosphatase activity, as well as determination of in vitro mineralisation have been applied to calvarian osteoblasts, fibroblasts and stromal bone marrow cells. Irradiation results in a dose-dependent suppression of clonogenic activity in all mitotically active cells, but metabolic activity and matrix synthesis were not impaired. In dense cultures alkaline phosphatase expression and in vitro mineralisation were not significantly affected by irradiation. Our experimental in vitro data suggest that irradiation inhibits the initial phase of in vivo osteogenesis due to the cytostatic effect. Postoperative irradiation after THR must therefore take place as early as possible. The homoeostasis of normal, orthotopic bone does not seem to be severely affected by local low-dose irradiation.

Biodegradable polycation and plasmid DNA multilayer film for prolonged gene delivery to mouse osteoblasts

Sustained release of functional plasmid DNA from the surfaces of materials which support cell adhesion for tissue formation could have a significant impact on gene therapy and tissue engineering. We report here layer-by-layer assembled multilayer film from a degradable cationic poly(2-aminoethyl propylene phosphate) and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) for mouse osteoblast cell adhesion and prolonged gene delivery. Multilayer film growth was monitored by UV spectrophotometry and intensity of absorbance at 260 nm related to incorporated DNA increased in an exponential manner with increase the number of deposited polymer and plasmid layers. It degraded upon incubation in phosphate-buffered saline (PBS) at 37 °C and sustained the release of bioactive plasmid DNA up to 2 months. The multilayer film facilitated initial mouse osteoblast cell adhesion onto the surface and enhanced cellular alkaline phosphatase activity and calcium accumulation. It sustained delivering transcriptional active DNA to mouse osteoblast cells cultured on the film, and directly prolonged gene expression in the presence of serum without any exogenous transfection agent. This biodegradable multilayer assembly is promising for the local and sustained delivery of plasmid DNA and such a layer-by-layer system suggests an alternative method for plasmid DNA incorporation which may be useful for surface modification of implanted materials or scaffold for gene therapy and tissue regeneration.

A NOVEL ALKALINE PHOSPHATASE IN THE COCCOLITHOPHOREEMILIANIA HUXLEYI(PRYMNESIOPHYCEAE) AND ITS REGULATION BY PHOSPHORUS1

Alkaline phosphatase (AP) plays an important role in the regeneration of bioavailable phosphate from organic compounds and allows phytoplankton growing in low inorganic phosphate environments to acquire phosphorus. We report the isolation, cloning, and initial characterization of the first AP (ehap1) in the coccolithophoreEmiliania huxleyi(Lohm.) Hay and Mohler. This novel AP is a major form of AP released from the cell surface into the medium at late exponential and stationary phase of P‐limited batch cultures but has no significant sequence similarity to other known APs. The cDNA sequence encodes a protein of roughly 94 kDa, while the processed active EHAP1 released from the cell surface is roughly 75 kDa. This difference is due to the cleavage of the signal sequence at its N‐terminus and perhaps some truncation at its C‐terminus. In response to variations in phosphate levels, the expression ofehap1was found to correlate well with cellular AP activity. Theehap1transcript was induced 4 h after phosphate depletion, increasing 1000‐fold within a day, and was repressed rapidly upon phosphate addition. These results provide the basis for developing specific probes to study the expression of AP, and thus phosphate stress, of field populations.

An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats

Human cathepsin K, a cysteine proteinase of the papain family, has been recognized as a potential drug target for the treatment of osteoporosis. The predominant expression of cathepsin K in osteoclasts has rendered the enzyme into a major target for the development of novel antiresorptive drugs. Now, we report the pharmacological properties of OST-4077 [furan-2-carboxylic acid (1-{1-[4-fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide] as a novel selective cathepsin K inhibitor. Human and rat cathepsin K were inhibited in vitro by OST-4077 with the IC50 values of 11 and 427 nM, respectively. OST-4077 suppressed bone resorption induced by rabbit osteoclasts (IC50, 37 nM) but did not affect bone mineralization or cellular alkaline phosphatase activity in MC3T3-E1 cells. Parathyroid hormone-induced bone resorption was inhibited in a dose-dependent manner in thyroparathyroidectomized rats gavaged with a single dose of OST-4077 (ED50, 69 mg/kg). When given orally twice daily for 4 weeks to 3-month-old ovariectomized (OVX) rats, OST-4077 dose-dependently prevented bone loss, as monitored by bone densitometry, ash content, and urinary excretion of deoxypyridinoline. No change in serum osteocalcin in the OVX rats by OST-4077 suggested that bone formation might not be affected by the agent. In summary, OST-4077 selectively inhibited bone resorbing activities of osteoclasts and prevented bone loss induced by estrogen deficiency but did not affect bone formation. OST-4077, an orally active selective human cathepsin K inhibitor, may have the therapeutic potential for the treatment of diseases characterized by excessive bone loss including osteoporosis.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue

A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue. Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation. After differentiation, AP activity increased 5.1 +/- 1.3-fold in the absence and 8.9 +/- 2.8-fold (P < 0.05) in the presence of levamisole. However, adipogenesis increased 1.95 +/- 0.11-fold in the absence but only 1.36 +/- 0.06-fold (P < 0.001) in the presence of levamisole. There was a 4.2 +/- 2.2-fold increase in AP activity in the absence and a 0.51 +/- 0.46-fold (P < 0.05) decrease in the presence of histidine. Adipogenesis increased 2.09 +/- 0.35-fold in the absence of histidine but only 1.22 +/- 0.30-fold (P < 0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell. This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.