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Berberine is an essential phytochemical for the treatment of various diseases; however, when used to treat osteoporosis, it has minor effect as compared with that of the currently available drugs. This study aimed to find a new compound that would have a better anti-osteoporotic effect than that of berberine. Based on structure and activity relationship study, we identified compound 2c, a berberine derivative, to be the most potent compound to affect osteoblast differentiation. Compound 2c was more effective than berberine and exhibited no toxicity within its effective concentration. Compound 2c increased, in a dose-dependent manner, ALP activity during osteoblast differentiation and enhanced the mRNA expression of osteogenic factors including ALP, Runx2, and Osterix. Furthermore, compound 2c increased the transcriptional activity induced by BMP4 on the ALP and BSP promoter. Taken together, compound 2c shows promise as a therapeutic agent for osteoporosis by promoting osteoblast differentiation.

Retinoic acid (RA) plays a role in cancer therapy. However, its long-term treatment is hindered by the acquired resistance which is not fully understood. Our previous study indicated that the transcriptional activity of RA receptor (RAR) is enhanced by association of MED25 with CREB-binding protein (CBP) through the PTOV domain, which is also present in prostate tumor over-expressed protein 1 (PTOV1). Here, we show that MED25 and PTOV1 reciprocally regulate RAR transcriptional activity through competitive bindings to CBP and opposite regulation of CBP recruitment to the RA-responsive gene promoter. Finally, we demonstrate that MED25 and PTOV1 differentially modulate RA sensitivity in cancer cells depending on their expression levels, suggesting a potential molecular mechanism underlying RA resistance which frequently emerges during cancer treatments.

OCT4 plays a crucial role in pluripotency and self-renewal of embryonic stem cells. OCT4 is also expressed in testicular germ cell tumors (GCTs), suggesting the important function of OCT4 as an oncogenic factor in GCTs. To understand the molecular mechanism of human OCT4 (hOCT4) in tumorigenesis as well as stemness, we identified hOCT4 transactivation domains in human embryonic carcinoma cells. Context analyses of heterologous GAL4 and natural hOCT4 revealed that each N-terminal domain or C-terminal domain independently stimulated transcriptional activity, and that both domains are required for synergistic transactivation by deletion mapping analysis. Dose-dependent overexpression of exogenous hOCT4 significantly decreased the transcriptional activity of the hOCT4 promoter. This inhibition was reversed by the removal of one or both domains. These results suggest that the inhibitory effect of hOCT4 is mediated by transactivation domains, and that the self-regulation of hOCT4 may be mediated via a negative feedback loop in pluripotent cells.

We examined the effect of celecoxib on the expression of T-cell factors (TCFs) to clarify the mechanism by which celecoxib suppress beta-catenin/TCF-dependent transcriptional activity without reducing the level of beta-catenin protein, using HCT-116 cells. Celecoxib suppressed the expression of TCF-1 and TCF-4 in a time-dependent manner. Pretreatment of cells with the proteasome inhibitor MG132 inhibited the loss of TCF-1 and TCF-4 induced by celecoxib, suggesting that celecoxib induced the proteasome-dependent degradation of TCF-1 and TCF-4. Beta-catenin/TCF-dependent transcriptional activity was significantly decreased after the treatment with celecoxib for 6 h and the pretreatment of the cells with MG132 attenuated the effect of celecoxib. Further, celecoxib also suppressed the expression of TCF-1 and TCF-4 in another colon cancer cell line, DLD-1. Our results suggest that TCF-1 and TCF-4 degradation may involve the inhibition of the Wnt/beta-catenin signaling pathway induced by celecoxib.

As the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) is associated with resistance of tumor cells to current antitumor therapies, the spatiotemporal dynamics of HIF-1-active regions has been of great interest as a therapeutic target. In the present study, we established a unique cancer cell line, which changes color HIF-1-dependently, and monitored it during tumor progression. In imaging experiments, HIF-1-active cells appeared over the tumor xenograft, but dramatically decreased in number as blood vessels developed around the tumor. The remaining HIF-1-active cells at the center of the xenograft also disappeared after neovascularization. Thereafter, tumor growth was accelerated and HIF-1-active cells reappeared in different regions. The distance between HIF-1-active cells and the nearest vessels correlated to the diameter of the vessel ( r = 0.801). These results provide a basic knowledge of how to estimate the spatiotemporal dynamics of HIF-1-active cells using information about the image-guided architecture of tumor blood vessels.

Calcineurin is an important regulator of extracellular matrix (ECM) accumulation in the kidney but functions in a cell-specific manner. Previously, we identified a novel role for calcineurin in mesangial cells where calcineurin activity is required for TGFbeta-mediated induction of fibronectin expression. In this study, we examined the role of the calcineurin substrate NFATc in transcriptional regulation of fibronectin. First, inhibition of calcineurin blocks TGFbeta induction of the fibronectin promoter. Moreover, expression of constitutively active calcineurin in mesangial cells is sufficient to increase fibronectin transcription. Next, inhibition of the calcineurin substrate NFATc1 blocked TGFbeta-mediated activation of the fibronectin promoter. Finally, stable expression of a dominant-negative NFATc protein reduced transcriptional activation of the promoter and inhibited TGFbeta-mediated fibronectin expression. In conclusion, TGFbeta activation of calcineurin in mesangial cells results in regulation of ECM accumulation at least in part by direct transcriptional activity of NFATc on the fibronectin promoter.

Hypoxia-inducible factor-1alpha (HIF-1alpha) has a central role in neuroprotective responses to hypoxia in the brain. Hydroxylation of HIF-1alpha by prolyl-hydroxylase PHD and aspargynyl-hydroxylase FIH (factor inhibiting HIF-1alpha) causes proteasomal degradation and transcriptional inhibition of HIF-1alpha. Siah ubiquitin ligases regulate the abundance of PHD via targeting for proteasomal degradation. The present study identified Siah-1 as a binding partner for another hydroxylase FIH. Siah-1 and FIH coimmunoprecipitated each other in mammalian cells. Siah-1 was found both to interact with the JmjC domain of FIH through its substrate-binding domain and to specifically ubiquitinate FIH via its RING finger domain. Siah-1 facilitated FIH degradation via the ubiquitin-proteasome pathway under hypoxic conditions. Such findings suggest that Siah ubiquitin ligases might play a role as up-stream regulators of both hydroxylases for HIF-1alpha, i.e., PHD and FIH, by targeting them for proteasomal degradation, leading to increased HIF-1alpha abundance, and transcriptional activity in hypoxia.

International audience; O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified.

We recently showed that moderate hypoxia and hypoxia-mimetic agents CoCl(2) and desferrioxamine (DFO) induce differentiation of acute myeloid leukemic cells via hypoxia-inducible factor-1alpha (HIF-1alpha) that interacts with and increases the transcriptional activity of CCAAT/enhancer-binding protein alpha (C/EBPalpha), a critical factor for granulocytic differentiation. Here, we show that metavanadate antagonizes DFO-induced growth arrest and differentiation with the inhibition of HIF-1alpha protein accumulation in leukemic cells. Furthermore, DFO also increased C/EBPalpha expression rapidly but transiently, which was inhibited by metavanadate. Taken together, these findings provide further evidence for the role of HIF-1alpha and C/EBPalpha in DFO-induced leukemic cell differentiation.

One of the mechanisms underlying the induction of human inducible nitric oxide synthase (hiNOS) by cytokines is transcriptional regulation. However, previous reports suggest that in human cells, the cytokine activation of the hiNOS expression is accompanied by a drastic increase in cellular mRNA and protein content, but by little increment in transcriptional activity. We designed luciferase reporter constructs containing the hiNOS 5′-flanking region and 3′-untranslated region (UTR). The construct containing both 5′-flanking region and 3′-UTR plus its downstream canceled the constitutive activity of the reporter gene, and was more responsive to the cytokines. This is an initial attempt to highlight the important role played by the 3′-region of the hiNOS gene. Our studies provide the evidence that the cooperative interaction between the 5′-promoter and the 3′-region conveys marked induction of the gene in the presence of cytokines.

Our previous study has shown that PKCδ stimulates proteasome-dependent degradation of C/EBPα, which partially contributes to PKCδ-mediated apoptosis. However, the molecular interrelationship between these two important proteins is still unknown. In this study, we reported that C/EBPα was phosphorylated by activated PKCδ on three serines, two of which were reported for the first time. Phosphorylated C/EBPα underwent cytoplasmic translocation, which led to the inactivation of its transcriptional activity. Inactive cytoplasmic C/EBPα was finally subjected to proteasome degradation. This work reveals the exquisite molecular events linking activated PKCδ and C/EBPα degradation during cell apoptosis.

A human recombinant cDNA clone that encoded a zinc-finger protein ( M yc- a ssociated z inc-finger protein of human i slet; MAZi) was cloned by screening a cDNA library prepared from human pancreatic islet cells. The encoded protein showed a high degree of homology to theMyc-associated zinc-finger protein MAZ (ZF87 or Pur-1). However, differences between the cDNAs for MAZi and MAZ were found in the length of the encoded polyalanine stretch and in the sequence of the 5′-end leader. MAZi transcripts were significantly more abundant in rat pancreatic islet carcinoma tissue than in normal rat islet cells. Moreover, MAZi protein bound specifically to the pyrimidine-rich strand of the CT-element of thec-mycgenein vitroand strongly induced the expression of chloramphenicol acetyltransferase (CAT) from ac-mycpromoter/CAT reporter construct in human pancreatic cells. Our results suggest that a distinct member of the MAZ family is expressed in human islet cells and enhances the transcriptional activity of thec-mycgene.

The binding activity of the heat shock transcription factor (HSF) to the heat shock element (HSE) is observed in non-stressed liver and freshly isolated hepatocytes when the expression of hsp70 is undetectable. HSF binding activity in non-stressed liver/hepatocytes is specific for HSE and similar to the HSF binding activity observed in heat shocked hepatocytes that is associated with hsp70 transcription. However, the HSF binding activity in non-stressed and heat shock cells can be distinguished on the basis of the thermal stability in vitro. The HSE binding activity of cell extracts isolated from non-stressed liver/hepatocytes was lost rapidly when the extracts were incubated at 37 degrees C. In contrast, the HSF binding activity of cell extracts isolated from heat shocked hepatocytes was relatively stable at 37 degrees C. Based on our observations, we propose that the activation of HSF is a multistep process that involves a change in conformation after oligomerization and the acquisition of DNA binding to a form that is more thermostable and is associated with increased hsp70 transcription.

NS1 protein of influenza A virus is involved in regulating the apoptosis of infected cells. We found that exogenously expressed NS1 was able to associate with the tumor suppressor p53 that plays an essential role in regulating apoptosis of influenza A virus-infected cells. Exogenous expression of NS1 resulted in inhibition of p53-mediated transcriptional activity and apoptosis. The p53 inhibitory domain of NS1 was located between amino acids 144 and 188. This domain is necessary for NS1 to inhibit p53 activity, but it requires additional region(s) to cooperatively exert this inhibitory function.

The non-erythroid porphobilinogen deaminase (E.C. 4.3.1.8) promoter was investigated according to sequence changes and transcriptional activity. The minimal promoter sequence required for maximal transcription, was localized by deletion mapping to −243 to −115 relative to the translational start site. A negative transcriptional element was found between −55 and +1, indicating a repression mechanism. A new polymorphism was identified, at position −235 within the minimal promoter, both in acute intermittent porphyria patients and healthy control subjects. The polymorphic variants did not affect expression of cloned promoter fragments under the conditions used.

The Rev peptide that binds to nucleophosmin/B23 with the highest affinity exhibited the greatest cytotoxicity on Ras-3T3 cells and inhibited tumor growth most effectively in nude mice. The efficiency of colony formation in soft agar of Ras-3T3 cells was significantly inhibited by treatment with Rev peptide. In addition, Rev peptide could potentiate the doxorubicin-induced decrease of cellular viability in U1 bladder cancer cells and inhibition of tumor growth in nude mice. Treatment of Rev peptide increased protein expression and transcriptional activity of p53 and inhibited the nucleophosmin/B23-mediated PCNA promoter activation. Peptides having high affinity of binding to molecular targets such as nucleophosmin/B23 represent a potentially useful approach to anti-cancer biotherapeutics.

The glucocorticoid receptor (GR) is a ligand-induced transcription factor which modulates the transcriptional activity of target genes. Full transcriptional activity of GR is achieved with the help of accessory proteins that are able to interact with GR. We have identified a 95-kDa protein by a blotting technique which utilizes a radioactively labeled DNA-bound GR to detect proteins that bind to this complex. Biochemical purification of this protein followed by protein microsequencing resulted in the identification of human nucleolin. In addition we could show that a GR-deletion mutant localizes to the nucleolus, where nucleolin is one of the most abundant proteins. The binding of nucleolin to this deletion mutant was demonstrated by GST-pull-down experiments. We suggest a biological role of nucleolin in binding of GR in the nucleolus.

Numerous synthetic chemicals have estrogenic activity by interacting with the estrogen receptor. In this report, we test the hypothesis that some estrogenic chemicals may also modulate the human progesterone receptor (hPR) signaling pathway. This was evaluated by examining synthetic chemicals for their ability to modulate the activity of hPR expressed in yeast. The transcriptional activity of hPR was not increased in the presence of several synthetic chemicals. However, the estrogenic chemicals p-nonylphenol and 4-tert-octyphenol, and pentachlorophenol, effectively inhibited the activity of the hPR in yeast. Competition binding studies indicated these chemicals effectively competed with radiolabeled R5020, a synthetic progestin, for binding to the hPR in yeast. These results indicate that some synthetic chemicals directly inhibit the activity of hPR in yeast. The observations that some estrogenic chemicals can also inhibit hPR activity suggest a potential mechanism for the reported potent estrogenic activities of these chemicals.

Summary In this report, we show that androgen-dependent genes expressed in rat ventral prostate, Cl, C2 and C3, are enriched in residual DNA extracted from the nuclear matrix of ventral prostate. The enrichment is androgen-dependent and is likely to be related to their transcriptional activity. While this finding corroborates other data demonstrating that transcriptionally active genes can be protected from nuclease digestion by their association with the nuclear matrix, we have extended this analysis to show that nuclear matrix protection experiments can be used both to analyze tissue-specific expression among members of a highly related gene family and also to predict transcriptional activity of genes in particular tissues.

Thyroglobulin gene control by cAMP and thyrotropin has been compared in FRTL-5 thyroid cells maintained in the absence of serum, insulin, cortisol, and thyrotropin for 7 days. Readdition of thyrotropin induces an increase in the expression of the thyroglobulin gene with little effect on total RNA synthesis. In contrast, addition of 8-bromo cyclic AMP and cholera toxin stimulate total RNA synthesis as well as thyroglobulin gene expression. The same non-selective effect of cyclic AMP on the thyroglobulin gene was observed when transcriptional activity was measured in permeabilized FRTL-5 cells.