Alkaline Phosphatase Type Research Articles

Stimulation of intramembranous bone repair in rats by ghrelin

Researchers in our laboratory have previously shown that ghrelin, a gastric peptide hormone, may regulate mesenchymal cell differentiation into adipocytes and myocytes. Here we show that ghrelin promotes osteogenesis of intramembranous bone and improves the repair of calvarial bone defects in rats. Rats with a 9 mm full-thickness calvarial bone defect received either Bio-Oss (control group) or Bio-Oss mixed with 20 mug ghrelin (treatment group), followed by local administration of saline or ghrelin (10 microg), respectively, on days 5, 10 and 15. After 6 and 12 weeks, new bone formation was assessed. Animals treated with ghrelin showed a significant increase in new bone formation as demonstrated by an increment in bone mineral density and fluorescence labelling of tetracycline relative to the control group. At 6 weeks, bone mineral density increased from 54 +/- 7 (control group) to 78 +/- 9 mg cm(-2) in the treatment group, while the tetracycline fluorescence labelling increased by 61 +/- 15%. A similar increment was observed at 12 weeks. Quantitative reverse transcriptase-polymerase chain reaction showed that expression of alkaline phosphatase (ALP), osteocalcin and collagen type I was elevated. Relative to the control animals, mRNAs for ALP, osteocalcin and collagen type I increased 2.4 +/- 0.4-, 4.7 +/- 1.9- and 4.0 +/- 1.7-fold, respectively, in animals treated with ghrelin for 6 weeks (P < 0.05). At 12 weeks, mRNA levels of ALP, osteocalcin and collagen type I showed a decline relative to levels at 6 weeks but still remained significantly higher than in the control group, with fold changes of 2.4 +/- 0.8, 2.4 +/- 1.2 and 2.1 +/- 0.7, respectively (P < 0.05). This study demonstrated that ghrelin stimulates intramembranous osteogenesis.

日本臨床化学会(JSCC)勧告法による血中小腸型アルカリホスファターゼの臨床的評価

日本臨床化学会(JSCC)勧告法による血中小腸型アルカリホスファターゼの臨床的評価

Insulin-Like Effects of Visfatin on Human Osteoblasts

Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel adipocytokine that is highly expressed in visceral fat and upregulated in obesity and type 2 diabetes mellitus. Visfatin binds to and activates the insulin receptor (IR), thereby exerting insulin-mimetic effects in various cell lines. IR has been detected in osteoblasts, which is consistent with the role of insulin as an important osteotropic hormone. This study investigated the actions of visfatin on human primary osteoblasts. The expression and tyrosine phosphorylation of IR, IR substrate-1 (IRS-1), and IRS-2 were determined by immunoprecipitation and immunoblotting. Cell proliferation was determined by measuring [(3)H]thymidine incorporation and cell number. Glucose uptake was determined by measuring 2-[(3)H]deoxyglucose incorporation. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was used for determining alkaline phosphatase (ALP), osteocalcin, and type I collagen mRNA expression. Enzyme-linked immunosorbent assay and radioimmunoassay were used for measuring ALP activity, osteocalcin secretion, and type I collagen production. We found that visfatin induced tyrosine phosphorylation of IR, IRS-1, and IRS-2. Moreover, the effects of visfatin - glucose uptake, proliferation, and type I collagen enhancement of cultured human osteoblast-like cells - bore a close resemblance to those of insulin and were inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester, a specific inhibitor of IR tyrosine kinase activity. We also unexpectedly found that visfatin downregulated osteocalcin secretion from human osteoblast-like cells. These data indicate that the regulation of glucose uptake, proliferation, and type I collagen production by visfatin in human osteoblasts involves IR phosphorylation, the same signal-transduction pathway used by insulin.

Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Un adénome hépatique révélé par une élévation isolée des phosphatases alcalines

A 25-year-old woman had a high serum level of alkaline phosphatase activity (2571 UI/L). Serum levels of transaminases, gamma glutamyl transferase and bilirubin were normal. Abdominal ultrasonography revealed a tumor nodule in the right liver lobe. There was no evidence of biliary obstruction. The serum activity of alkaline phosphatase returned to normal after surgical removal of the liver tumor. Histologic examination showed that the tumor was a liver adenoma with no evidence of degeneration. The adenoma cells reacted strongly positive to alkaline phosphatase by histochemical staining. The production of hepatic and biliary type alkaline phosphatase by the tumor is the most likely mechanism for the high serum levels observed in this patient.

A method for measuring the catalytic activity of small intestine type alkaline phosphatase using L-phenylalanine as an inhibitor

A method for measuring the catalytic activity of small intestine type alkaline phosphatase using L-phenylalanine as an inhibitor

Dexamethasone suppresses Smad3 pathway in osteoblastic cells

Central in the pathogenesis of glucocorticoid (GC)-induced osteoporosis is the effects of GC on bone formation. However, the mechanism of GC-inhibited bone formation is not well known. Transforming growth factor (TGF)-beta is most abundant in bone matrix compared with other tissues, and we have recently proposed that Smad3, a TGF-beta signaling molecule, is important for promoting bone formation. However, no reports have been available about the effects of GC on Smad3 in osteoblasts. In the present study, we investigated whether dexamethasone (Dex), an active GC analog, would affect the expression and activity of Smad3 in mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Dex significantly suppressed Smad3-stimulated alkaline phosphatase (ALP) activity, although it did not affect TGF-beta-inhibited ALP activity in MC3T3-E1 cells. Moreover, pretreatment with Dex suppressed TGF-beta-enhanced expression of type I collagen in MC3T3-E1 and UMR-106 cells. In the luciferase assay using p3TP-Lux with a Smad3-specific response element, Dex significantly suppressed the transcriptional activity induced by TGF-beta as well as Smad3. However, Dex did not affect the expression of Smad3 in these cells at both mRNA and protein levels. In conclusion, the present study indicates that Dex inhibits ALP activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblastic cells.

Zinc Transporters, ZnT5 and ZnT7, Are Required for the Activation of Alkaline Phosphatases, Zinc-requiring Enzymes That Are Glycosylphosphatidylinositol-anchored to the Cytoplasmic Membrane

Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.

Role of Ascorbic Acid in Periodontal Ligament Cell Differentiation

Periodontal ligament (PDL) cells form mineralized nodules in vitro. Ascorbic acid is known to be required in this process, although its effect on osteoblastic differentiation of PDL cells remains unclear. The purpose of this study was to determine the role of ascorbic acid on the early osteoblastic differentiation of PDL cells, with regard to alkaline phosphatase (ALP) activity, type I collagen production and integrin expression. Cultured PDL cells were stimulated at confluence with ascorbic acid in the presence or absence of type I collagen inhibitor and blocking antibodies to integrins. After stimulation, the cells and culture supernatants were examined for ALP activity, type I collagen production, and integrin expression. The ALP activity was measured using a colorimetric assay with p-nitrophenyl phosphate and ALP staining. Enzyme-linked immunosorbent assay (ELISA) was used to determine type I collagen production, and ELISA and flow cytometric analysis were employed for assessment of integrin expression. Both ALP activity and type I collagen production were upregulated when PDL cells were cultured in the presence of ascorbic acid (200 microM). Inhibitor of the formation of collagen triple helices and blocking antibodies to alpha2beta1 integrin inhibited ALP activity by 50% in ascorbic acid-stimulated PDL cells. Furthermore, ascorbic acid increased the cell surface expression of alpha2beta1 integrin. Our findings indicated that ascorbic acid increases the ALP activity of PDL cells via type I collagen production and also enhances the expression of alpha2beta1 integrin, which is a major receptor of type I collagen. These results suggest that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating type I collagen-alpha2beta1 integrin interaction.

Non-cloning Synthesis of cRNA Probe and Its Application to In Situ Hybridization Analysis of Formalin-fixed Paraffin Embedded Tissue

Intestinal type alkaline phosphatase (IALP) mRNA-specific cRNA probe was synthesized by non-cloning method for in situ hybridization (ISH) analysis. Total RNA was extracted from small intestine of male F344 rat. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for IALP gene. T7 promoter adapter was directly added to IALP cDNA-specific PCR product by ligation reaction. IALP cDNA accompanied by T7 promoter adapter was then amplified by PCR using IALP gene-specific primer in combination with T7 promoter adapter primer sequence and used as a template of cRNA probe. Digoxigenin-labeled cRNA was synthesized by in vitro transcription method using RNA polimerase. ISH was performed using formalin-fixed, paraffin embedded small intestine tissue. As a result, the cytoplasm of mucosal epithelial cells was successfully stained with this cRNA probe. The present procedure is quite simple and a rapid way to prepare the cRNA probe and would be very useful for ISH analysis of formalin-fixed paraffin embedded tissue sample.

Involvement of phosphodiesterase isozymes in osteoblastic differentiation.

The cyclic monophosphate nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) are found ubiquitously in mammalian cells and act as second messenger transducers to effect the intracellular actions of a variety of hormones, cytokines, and neurotransmitters. In turn, these nucleotides also modulate the signal transduction processes regulated by a range of cytokines and growth factors. Previously, we have reported that pentoxifylline, a nonselective phosphodiesterase (PDE) inhibitor, can promote osteoblastic differentiation by elevating intracellular cAMP levels and, consequently, enhance bone formation in vivo and in vitro. In this study, reverse-transcription polymerase chain reaction (RT-PCR) analysis of the osteoblastic cell lines, MC3T3-E1 and ST2 revealed the presence of PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, and PDE9. We examined the effect of selective inhibitors for a respective PDE isozyme on the capacity of bone morphogenetic protein 4 (BMP-4)-induced alkaline phosphatase (ALP) activity, a cellular differentiation marker, in cells with osteogenetic potential. The results indicate that selective inhibitors for PDE2, PDE3, and PDE4 enhanced the BMP-4-induced ALP activity in a dose-dependent manner in ST2 cells but not in MC3T3-E1 cells. Northern blot analysis also revealed that the selective inhibitors for PDE2, PDE3, and PDE4 enhanced the levels of expression of messenger RNAs (mRNAs) of ALP, osteopontin (OP), and collagen type I in ST2 cells but not in MC3T3-E1 cells except for the treatment with PDE4 inhibitor. Given these data, we conclude that PDE isozymes are involved in the modulation of osteoblastic differentiation mainly at an early stage. Additionally, selective inhibitors for PDE2, PDE3, and PDE4 appear to promote the differentiation of osteogenic precursor cells toward an osteoblastic phenotype.

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion.

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein withamino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.

Col1a1-driven transgenic markers of osteoblast lineage progression.

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.

Temporal and Spatial Profiles of Alkaline Phosphatase Activity During Embryogenesis of Amphioxus Branchiostoma belcheri tsingtaunese

Temporal and spatial profiles of alkaline phosphatase (ALP) activity during the development of amphioxus have not been fully documented and thus the present study investigated this activity spectrophotometrically, electrophoretically and histochemically. The following results were observed: (1) spectrophotometrically, ALP activity increased markedly at the late gastrula stage and reached a plateau at 15 hr postfertilization; (2) the electrophoretic pattern of ALP isozymes changed dramatically during development; (3) ALP activity was initially localized in the posterior wall of the primitive gut and the anterior 5 to 6 somites at about the 15 hr larva stage, and then in the notochord and all the somites at about the 18 hr larva stage; (4) in 1-day larvae, ALP activity decreased in the posterior wall of the primitive gut and in the anterior 5 to 6 somites which had ALP activity at 15 hr, but it appeared in the newly formed somites, especially in the myosepta, the crevices cut in between adjoining somites; (5) in 2-day larvae, ALP activity was no longer visible in somites but became highly evident in most of the notochord except for its rostral region; and (6) when the lateral plate mesoderm pushed down ventrally on either side of the intestine and conjoined beneath the intestine, ALP activity was also detected in the conjoining lateral plate mesoderm. Apparently, two types of ALP exist in amphioxus larvae, the transient endodermal ALP and the constant mesodermal ALP, and the spatial and temporal correlation of ALP activity with the developing mesoderm, including the notochord, suggests that it plays a role in the differentiation of mesodermal structures during the development of amphioxus.

Association between two types of vitamin d receptor gene polymorphism and bone status in premenopausal Japanese women.

Polymorphisms in the vitamin D receptor (VDR) gene using ultrasound (US) bone mass and bone metabolic markers were investigated as potential genetic markers for osteoporosis in 126 premenopausal Japanese women aged 27.2 +/- 10.1 (mean +/- SD) years. The relationship between their VDR gene polymorphisms and bone states was determined. VDR genotypes were based on the absence (B) or presence (b) of the Bsm I restriction site (B polymorphism), and ATG (the M allele) and ACG (the m allele) sequences at the translation initiation site (M polymorphism). Genotype frequencies were 73.8%, bb; 24.6%, Bb; 1.6%, BB; 15.1%, MM; 51.6%, Mm and 33.3%, mm. The stiffness index of calcaneal bone minerals measured by an US bone densitometer was significantly higher in the mm types (P <0.05 versus MM) than in the Mm types (P <0.01 versus MM) and MM types. There was no significant difference between in B polymorphisms. Furthermore, bone mass was correlated with serum bone type alkaline phosphatase (ALP) activity and urinary deoxypyridinoline concentration in M polymorphisms. Because the distribution of B polymorphisms in each M polymorphism genotype did not differ, M polymorphisms were affected independently from B polymorphisms to bone mass or bone metabolic markers. No significant difference was observed in nutritional intake and food consumption among genotypes. In the MM and Mm types, the bone mass was closely related to the frequency of milk intake during the periods of elementary and junior high school. In contrast, bone mass was not associated with nutritional intake or the frequency of past milk intake in B polymorphisms. Therefore, the M polymorphism of the VDR gene is a stronger genetic indicator of osteoporosis than the B polymorphism in premenopausal Japanese woman.

Signal Peptide Determinants of SecA Binding and Stimulation of ATPase Activity

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.

Parathyroidectomy for primary hyperparathyroidism induces positive uncoupling and increases bone mineral density in cancellous bones.

Osteopenia is an important feature of primary hyperparathyroidism (PHP). However, little is known about the change of bone mineral density (BMD) in PHP after surgery. The aim was to investigate the mechanisms of increased BMD after parathyroidectomy in patients with PHP. Prospective observational study. Ten patients with PHP (7 women, 3 men; mean age 53.2+/-9.1 years). All patients underwent parathyroidectomy for excision of parathyroid adenoma. BMDs of two cancellous bone-rich sites (L2-L4 lumbar spine and ultra-distal end of the radius, RUD) and one cortical bone-rich site (distal third of the radius, R33%) were measured using dual energy X-ray absorptiometry, before, and 3, 6 and 12 months after surgery. Serum intact PTH, intact osteocalcin, bone type alkaline phosphatase (b-ALP), alkaline phosphatase, calcium, and urinary deoxypyridinoline (Dpd) were measured before, and 1 and 3 days, and 1, 2, 3, 4, 6, 8, 12, and 24 weeks after surgery. Parathyroidectomy resulted in a significant increase in BMDs of L2-L4 and RUD at 3 months postoperatively. Urinary Dpd levels decreased within a few days after surgery, while b-ALP and osteocalcin decreased more slowly throughout the first few months after surgery. The ratio of osteocalcin/Dpd at 1 week after surgery correlated significantly with the percentage change in BMD of L2-L4 at 3 and 6 months after surgery. The ratio of osteocalcin/Dpd at 2 weeks correlated significantly with the percentage change in BMD of L2-L4 at 3, 6 and 12 months after surgery. The preoperative values of osteocalcin, b-ALP, PTH and calcium were positively correlated with the change in BMD of RUD at 3 months and L2-L4 at 12 months, RUD at 6 months, RUD at 3 months and L2-L4 at 12 months, respectively. In primary hyperparathyroidism patients, the major increase in bone mineral density following parathyroidectomy occurs within 3 months. Parathyroidectomy resulted in a marked increase in bone mineral density of cancellous bones compared to that of cortical bones. The early increase in bone mineral density was due to a preferential activation of bone formation over bone resorption as evidenced by changes in bone metabolic markers. Our results also showed that the preoperative levels of bone metabolic markers may predict the gain in bone mineral density after parathyroidectomy.

Effects of retinoic acid on N-glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells.

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.

Establishing an immortalized human osteoprecursor cell line: OPC1.

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.

Effects of nonsteroidal anti-inflammatory drugs and prostaglandins on osteoblastic functions

It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress bone repair and bone remodeling but only mildly inhibit bone mineralization at the earlier stage of the repair process. We proposed that the proliferation and/or the earlier stage of differentiation of osteoblasts may be affected by NSAIDs. This study was designed to investigate whether NSAIDs affect the proliferation and/or differentiation of osteoblasts and whether these effects are prostaglandin (PG) mediated. The effects of PGE 1 and PGE 2, indomethacin, and ketorolac on thymidine incorporation, cell count, intracellular alkaline phosphatase (ALP) activity, and Type I collagen content in osteoblast-enriched cultures derived from fetal calvaria were evaluated. The results showed that both PGs and NSAIDs inhibited DNA synthesis and cell mitosis in a time- and concentration-dependent manner. However, intracellular ALP activity and Type I collagen content were stimulated at an earlier stage of differentiation in osteoblasts. These results suggested that (i) the inhibitory effect of ketorolac on osteoblastic proliferation contributes to its suppressive effects on bone repair and remodeling in vivo; (ii) PGEs and NSAIDs may be involved in matrix maturation and biologic bone mineralization in the earlier stage of osteoblast differentiation; and (iii) the effects of ketorolac and indomethacin on cell proliferation and differentiation may not be through the inhibition of the synthesis of PGE 1 or PGE 2.