Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.
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PMID:Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli. 881 45

Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identify transcripts encoding proteins that bind Msx2. A lambdagt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. In toto, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter, nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix.
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PMID:The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter. 1045 62

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.
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PMID:Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity. 1083 28

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), exerts its effects through regulation of target gene transcription. Configuration at C-20 of 1,25(OH)(2)D(3) is important in determining potency, as shown by the high potency of analogs with inverted configuration at C-20 (20-epi compounds). Gemini analogs of 1,25(OH)(2)D(3) contain two side chains, combining a C-20-normal with a C20-epi side chain. We studied the potency of analogs combining double (Gemini) side chains with a 23-triple bond and a C-26,27-hexafluoro substitution in either the 20-epi (analog 20R) or 20-normal (analog 20S) side chain. These novel Gemini analogs were 8-50-fold more potent than 1,25(OH)(2)D(3) in inducing U937, HL-60G, and THP-1 differentiation and 5-50-fold more potent in inducing transcription from the osteocalcin vitamin D response element or the 25-hydroxyvitamin D(3)-24-hydroxylase (24OHase) promoter. In vivo, following i.p. injection in vitamin D-deficient mice, the 20S analog induced significantly higher levels of calbindin-D(9K) mRNA in intestine, and 24OHase and calbindin-D(28K) in kidney than 1,25(OH)(2)D(3) or analog 20R. Increased potency did not correlate with ligand-receptor binding affinity. In GST-pull down assays using in vitro translated VDR, Gemini analogs showed equivalent (or even attenuated) potency to 1,25(OH)(2)D(3) in recruiting cofactors DRIP205 and GRIP-1 to VDR. However, Gemini analogs were up to 15-fold more potent than 1,25(OH)(2)D(3) in recruiting the same cofactors to VDR in GST-pull down assays using equal amounts of VDR from nuclear extracts of VDR transfected and hormone treated (24 hr) COS-7 cells. Deletion of C-19 in either 20S or 20R Gemini analogs resulted overall in slightly less potent analogs compared to Gemini itself. We conclude that enhanced potency of the novel Gemini analogs is at least partly due to increased metabolic stability of the analogs, resulting in more cofactor binding and elevated levels of transcription.
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PMID:Novel Gemini analogs of 1alpha,25-dihydroxyvitamin D(3) with enhanced transcriptional activity. 1501 48

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor-alpha (TNF-alpha), an effect mediated by nuclear factor kappa B (NfkappaB). To determine the mechanism of NfkappaB inhibition of D-stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF-alpha or transfected with the p65 subunit of NfkappaB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N-terminus and a midmolecular region were both required for the inhibitory action of p65. Pull-down assays were done using an immobilized glutathione S-transferase (GST)-VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator-1 (SRC-1), a major D-dependent coactivator. p65 inhibited VDR-SRC-1 binding in a dose-dependent manner. Mutations of p65 that abrogated the inhibitory effect on D-stimulated transcription also failed to inhibit VDR-SRC-1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC-1 or the general coactivators, Creb binding protein or SRC-3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF-alpha treatment prevented D recruitment of SRC-1 to the transcription complex. These results show that TNF-alpha inhibition of vitamin D-action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC-1, thus decreasing the efficiency of D-stimulated gene transcription.
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PMID:Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription. 1521 79

Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1alpha,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1alpha,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1alpha,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a "master" transcriptional regulator of osteoblast differentiation, and 1alpha,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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PMID:Bone-specific transcription factor Runx2 interacts with the 1alpha,25-dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteoblastic cells. 1545 60

The role of ATF4 (activating transcription factor 4) in osteoblast differentiation and bone formation was recently described using ATF4-deficient mice (Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., and Karsenty, G. (2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.
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PMID:Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression. 1600 Mar 5

Parathyroid hormone (PTH) potently activates cAMP-protein kinase A (PKA)-driven molecular cascades in osteoblasts. The NR4A/NGFI-B orphan nuclear receptor (NR) Nurr1 is a PTH-induced, cAMP-responsive primary response gene (PRG) that transactivates osteocalcin (Ocn) expression through a putative NGFI-B response element (NBRE) in the proximal promoter. As a true orphan NR, Nurr1's expression level and coactivator recruitment regulate its transactivation capacity. We postulated that Nurr1's induction through cAMP-PKA signaling might favor a coactivator that is likewise cAMP-dependent. A possible candidate is the cAMP-inducible coactivator PPARgamma coactivator-1alpha (PGC-1alpha). We hypothesize that PGC-1alpha is a PTH-induced PRG that synergizes with Nurr1 to induce target gene transcription in osteoblasts. We show that 10 nM PTH for 2 h maximally induced PGC-1alpha mRNA in primary mouse osteoblasts (MOBs) and calvariae. Selective signaling agonists and antagonists demonstrated that PTH induced PGC-1alpha mRNA primarily through the cAMP-PKA pathway. Protein synthesis inhibition sustained PTH-induced PGC-1alpha expression. PGC-1alpha enhanced Nurr1-induced transactivation of a consensus 3xNBRE-luciferase construct and the rat (-1050)Ocn promoter-luciferase construct from 3.7- to 9.6- and 10.1-fold, respectively. This synergy required Nurr1-DNA binding, since a mutation of the Ocn promoter NBRE abolished both Nurr1- and Nurr1-PGC-1alpha-induced transactivation. Using GST pull-down assays, PGC-1alpha directly interacted with in vitro-generated and nuclear Nurr1. We conclude that PGC-1alpha is a PTH-induced, cAMP-dependent PRG that directly synergizes with Nurr1 to transactivate target genes in osteoblasts. Taken together with published data, our findings suggest that Nurr1 and PGC-1alpha may be pivotal mediators of cAMP-induced osteoblast gene expression and osteoblast function.
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PMID:PGC-1alpha is induced by parathyroid hormone and coactivates Nurr1-mediated promoter activity in osteoblasts. 1676 61

Nephroblastoma overexpressed (Nov), a member of the CCN family of proteins, is expressed by osteoblasts and its transcription is regulated by transforming growth factor (TGF)-beta and bone morphogenetic proteins (BMP). CCN proteins can interact with TGF-beta, BMPs, and Wnt. We explored the function of Nov in skeletal cells, in vitro and in vivo. Constitutive overexpression of Nov in cells of the osteoblastic lineage impaired osteoblastic differentiation, opposed the biological effects of BMP-2 and Wnt 3 and impaired BMP-2 and Wnt signaling, indicating that Nov has BMP-2 antagonistic activity. Transgenic mice overexpressing Nov under the control of the osteocalcin promoter exhibited osteopenia secondary to decreased bone formation, confirming the effects in vitro. GST pulldown experiments demonstrated direct Nov-BMP interactions. In conclusion, Nov has BMP antagonistic properties and inhibits osteoblastogenesis and osteoblastic function.
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PMID:Nephroblastoma overexpressed (Nov) is a novel bone morphogenetic protein antagonist. 1808 20

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA gamma is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.
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PMID:General transcription factor IIA-gamma increases osteoblast-specific osteocalcin gene expression via activating transcription factor 4 and runt-related transcription factor 2. 1817 74


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