Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intermittent doses of parathyroid hormone (PTH) stimulate bone formation in animals and humans, but the molecular mechanisms underlying this phenomenon are not understood. Bone formation culminates with the expression of type I collagen, osteocalcin, and alkaline phosphatase, but genes that initiate and support the anabolic response are not known. To identify novel PTH-regulated genes in bone during the anabolic response, we used differential display-polymerase chain reaction (DDRT-PCR) to analyze RNA from young male rats injected with either human PTH (1-34) or vehicle control, once daily for 5 days. Total RNA was isolated from the distal femur metaphysis at 1, 6, and 48 h after the final injection and subjected to DDRT-PCR. We identified three PTH-responsive transcripts as matrix metalloproteinase-9 (MMP-9), creatine kinase, and the alpha1 (I) polypeptide chain (COL1A1) of type I collagen. The concomitant upregulation of MMP-9 and COL1A1 during bone formation was particularly intriguing. Further characterization of MMP-9 expression revealed that it was localized to osteoblasts, osteocytes, megakaryocytes, and cells of the bone marrow in the rat distal femur metaphysis. Northern analysis for MMP-9 expression in other tissues indicated that this transcript was present in the kidney and brain. In vitro, PTH regulated the protein synthesis of MMP-9 by osteoblasts of the primary spongiosa. We propose that PTH may promote bone formation by mediating the subtle variation in MMP activities, thus preparing the extracellular matrix for the subsequent bone cell migration and deposition of new osteoid.
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PMID:Intermittent administration of parathyroid hormone (1-34) stimulates matrix metalloproteinase-9 (MMP-9) expression in rat long bone. 970 76

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to </=28% of preimplantation values. In contrast, OPN mRNA levels increased up to 7-fold by day 10 and thereafter, returned to 1.7-fold above control values. COL1A1 mRNA decreased 2-fold at day 3 and increased to 3.5-, 1.6-, and 2.8-fold above control at days 10, 28, and 56, respectively. MMP-9 levels increased 5- to 10-fold by days 3-10, but fell to undetectable levels by 28-56 days. These results indicate that the formation of mineralized matrix (bone nodules) seen in the 56-day DC of ROS 17/2.8 cells was preceded by coordinate temporal expression of IEGs, matrix proteins, and matrix-modifying enzymes. Additionally, these results substantiate that measurement of molecular parameters in tissues formed by cells incubated in DC in vivo may be a useful predictor of the osteogenic process.
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PMID:Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo. 1043 Jun 46

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.
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PMID:Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone. 1268 18

Genetic variants that affect collagen Ialpha1 metabolism may be important in the development of osteoporosis or osteoporotic fractures. A -1997G-->T polymorphism in the promoter of the collagen Ialpha1 gene (COL1A1) was shown to be associated with bone mineral density (BMD) for the lumbar spine in postmenopausal Spanish women. The relation of this polymorphism to BMD in Japanese women or men has now been examined in a population-based study. The subjects (1,110 women, 1,126 men) were 40 to 79 years of age and were randomly recruited for a population-based prospective cohort study of aging and age-related diseases. BMD for the lumbar spine, right femoral neck, right trochanter, and right Ward's triangle was measured using dual-energy x-ray absorptiometry. Genotypes for the -1997G-->T polymorphism of COL1A1 were determined with a fluorescence-based allele-specific DNA primer assay system. When all women were analyzed together, BMD for the lumbar spine and trochanter was significantly lower in subjects with the COL1A1 *G/*G genotype than in those in the combined group of COL1A1 *G/*T and COL1A1 *T/*T genotypes. When postmenopausal women were analyzed separately, BMD for the femoral neck and trochanter was also significantly lower in those with the COL1A1 *G/*G genotype than in those with the COL1A1 *G/*T genotype or those in the combined group of COL1A1*G/*T and COL1A1 *T/*T genotypes. BMD was not associated with -1997G-->T genotype in premenopausal women or in men. Multivariate regression analysis revealed that -1997G-->T genotype affected BMD at various sites with a variance of 0.46-0.62% for all women and 0.61-1.01% for postmenopausal women. The -1997G-->T genotype was not related to the serum concentration of osteocalcin, the serum activity of bone-specific alkaline phosphatase, or the urinary excretion of deoxypyridinoline or cross-linked N-telopeptides of type I collagen in men or in premenopausal or postmenopausal women. These results suggest that COL1A1 is a susceptibility locus for reduced BMD in postmenopausal Japanese women.
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PMID:Association of a -1997G-->T polymorphism of the collagen Ialpha1 gene with bone mineral density in postmenopausal Japanese women. 1611 14

In order to investigate the mechanisms by which 1alpha,25(OH)2 vitamin D3 (VD3) stimulates the differentiation of human osteoblasts, we cultured MG-63, which is a human osteoblastic cell line, in the presence or absence of VD3 and/or L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. The cell growth rate was decreased by the presence of VD3 in the culture medium. Type I collagen synthesis and alkaline phosphatase (ALP) activity, which are markers of early stage osteoblast differentiation, were stimulated by the presence of VD3 as well as by that of Asc 2-P. The co-presence of Asc 2-P and VD3 had a synergistic effect on the collagen synthesis and ALP activity of the cells. Inhibition of collagen synthesis by the addition of inhibitors of collagen synthesis to the medium attenuated the stimulative effect of VD3 and Asc 2-P on the ALP activity. Transfection of the cells with siRNA-expressing vectors for COL1A1 decreased the expression level of ALP mRNA in addition to that of COL1A1. On the other hand, ALP activity was significantly increased, and the growth rate was decreased, when the cells were cultured on type I collagen-coated dishes. These effects were not seen when the cells were cultured on dishes coated with heat-denatured collagen. VD3 also increased the mRNA levels for Runx2 and osterix, which are transcription factors critical for osteoblast differentiation, as well as those of differentiation markers such as bone/liver/kidney type ALP, COL1A1, (the gene for the alpha1 chain of type I collagen), and osteocalcin, in the cells. Normal human osteoblasts and human bone marrow-derived mesenchymal stem cells (hBMSC) showed quite similar responses to VD3. These results indicate that VD3-stimulated gene expression of type I collagen and that mature type I collagen produced in the presence of Asc 2-P mediates at least a part of the stimulative effects of Asc 2-P and VD3 on the differentiation of these human osteoblastic cells. Levels of mRNAs for ALP and COL1A1 were increased, but the level of Runx2 was decreased, by the expression of osterix in MG-63 cells. These results also suggest that VD3 controls the growth and differentiation of human osteoblastic cells by regulating the gene expression of osteoblast-related transcription factors as well as that of type I collagen, and that the co-presence of both signals is essential for VD3 to express full activity toward the differentiation of human osteoblasts.
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PMID:Both direct and collagen-mediated signals are required for active vitamin D3-elicited differentiation of human osteoblastic cells: roles of osterix, an osteoblast-related transcription factor. 1626 99

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.
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PMID:Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts. 1674 51

Previous studies have shown a generalised increase in bone mass in patients with osteoarthritis (OA). Using molecular histomorphometry, this study examined the in vivo expression of mRNA encoding bone anabolic factors and collagen type I genes (COL1A1, COL1A2) in human OA and non-OA bone. Bone samples were obtained from the intertrochanteric (IT) region of the proximal femur, a skeletal site distal to the active site of disease, from individuals with hip OA at joint replacement surgery and from autopsy controls. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed elevated mRNA expression levels of alkaline phosphatase (p < 0.002), osteocalcin (OCN) (p < 0.0001), osteopontin (p < 0.05), COL1A1 (p < 0.0001), and COL1A2 (p < 0.002) in OA bone compared to control, suggesting possible increases in osteoblastic biosynthetic activity and/or bone turnover at the IT region in OA. Interestingly, the ratio of COL1A1/COL1A2 mRNA was almost twofold greater in OA bone compared to control (p < 0.001), suggesting the potential presence of collagen type I homotrimer at the distal site. Insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor-beta1 mRNA levels were similar between OA and control bone. Bone histomorphometric analysis indicated that OA IT bone had increased surface density of bone (p < 0.0003), increased trabecular number (Tb.N) (p < 0.0003), and decreased trabecular separation (Tb.Sp) (p < 0.0001) compared to control bone. When the molecular and histomorphometric data were plotted, positive associations were observed in the controls for OCN/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) versus bone tissue volume (r = 0.82, p < 0.0007) and OCN/GAPDH versus Tb.N (r = 0.56, p < 0.05) and a negative association was observed for OCN/GAPDH versus Tb.Sp (r = -0.64, p < 0.02). These relationships were not evident in trabecular bone from patients with OA, suggesting that bone regulatory processes leading to particular trabecular structures may be altered in this disease. The finding of differential gene expression, as well as architectural changes and differences in molecular histomorphometric associations between OA and controls, at a skeletal site distal to the active site of joint degeneration supports the concept of generalised involvement of bone in the pathogenesis of OA.
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PMID:Differential gene expression of bone anabolic factors and trabecular bone architectural changes in the proximal femoral shaft of primary hip osteoarthritis patients. 1718 61

Distraction osteogenesis is a special form of bone healing in which well-controlled distraction stresses and consequent tensile strains within callus tissue induce very efficient new bone formation. Proinflammatory cytokines are involved during the early phase of fracture healing and callus remodeling. Temporal expression patterns of proinflammatory cytokines were assessed in Sprague-Dawley rat tibial models of distraction osteogenesis and acute lengthening, and only interleukin-6 (IL-6) was found to be specifically induced during the distraction phase. IL-6 immunoreactivity was detected not only in hemopoietic cells and osteoblasts but also in the spindle-shaped cells of the fibrous interzone, where most of the tensile strains are concentrated. In vitro study revealed that IL-6 did not affect the proliferation of C3H10T1/2 cells, mouse bone marrow stromal cells (MSCs), or MC3T3-E1 cells; but its blocking antibody reduced the proliferation of C3H10T1/2 cells and MSCs. The mRNA expression of COL1A1 and osteopontin were not changed by IL-6 or its blocking antibody, but the alkaline phosphatase activities of MC3T3-E1 cells were increased by IL-6 and decreased by its blocking antibody. These findings indicate that IL-6 is a proinflammatory cytokine that responds to tensile strain during distraction osteogenesis. IL-6 negatively affects the proliferation of primitive mesenchymal cells, whereas the differentiation of more mature osteoblastic lineage cells is enhanced by IL-6 in vitro. IL-6 appears to be one of the cytokines involved in the complex network of signal cascades evoked during distraction osteogenesis and may differentially affect immature and mature osteoblastic lineage cells.
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PMID:Expression and role of interleukin-6 in distraction osteogenesis. 1734 Feb 23

To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.
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PMID:Small interfering RNA of alkaline phosphatase inhibits matrix mineralization. 1831 13

We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications.
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PMID:cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models. 1923 69


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