Calcium Phosphate Coprecipitation Method Research Articles

Coprecipitation technique as a new approach of using solid-state scintillation

In this paper, the coprecipitation methods of selected radionuclides in connection with solid-state scintillation counting were explored. The calcium phosphate co-precipitation method exhibited best results from all investigated methods reaching almost absolute detection efficiencies for selected actinides and more than 60% for Sr/Y-90. The linear response of the measurement was in the range of 0.4 to 411.5 Bq for alpha/beta separation mode, respective 0.4 to 2000 Bq nuclear counting (high activity mode). Computed and optimized MDA values exhibited better results in comparison with previous works.

Expression of Recombinant Factor IX Using the Transient Gene Expression Technique

Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes. Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique. Materials and Methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEKhFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test. Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX. Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and lowyield methods.

Facile Construction of Chloroquine Containing PLGA-Based pDNA Delivery System for Efficient Tumor and Pancreatitis Targeting in Vitro and in Vivo.

Chloroquine diphosphate (CQ) was ingeniously used to take place of phosphate salt in traditional calcium phosphate coprecipitation method for pDNA transfection. With multiple roles of CQ in the novel Ca-CQ-pDNA complex including pDNA compaction and assistance in lysosome escape, the transfection efficiency of the pDNA was significantly increased relative to the traditional method. CQ did not intercalate into the DNA double helix as free CQ did, which was probably ascribed to the prior mixing of the pDNA with high concentration of calcium chloride. In order to construct efficacious vector for in vivo gene delivery, Ca-CQ-pDNA-PLGA-NPs was designed and prepared. With entrapment efficiency, particle size and pDNA integrity as screening conditions, the optimal prescription was obtained and CaPi-pDNA-PLGA-NPs made with classic calcium phosphate coprecipitation method after optimization was also prepared as control to systematically study the role of CQ in the novel vector. Physical characters of the vectors were comprehensively studied using TEM, DSC, and XRD. The safety of the vector both in vitro and in vivo was evaluated using MTT, hemolysis test, and histological sections. The Ca-CQ-pDNA-PLGA-NPs dramatically enhanced the gene tranfection efficiency in Human Embryonic kidney HEK293 cells compared with the CaPi-pDNA-PLGA-NPs and presented an increasing gene transfection for up 144 h. The relative fast release of the CQ compared with pDNA from the nanoparticles was responsive for the increased transfection. The Did-labeled-Ca-CQ-pDNA-PLGA-NPs exhibited excellent tumor targeting efficiency and sustained circulation time in CT26 mouse model. The Ca-CQ-pDNA-PLGA-NP loaded with the plasmid pVITRO2 expressing mSurvivin-T34A protein gave 70% tumor inhibition rate, which was partially ascribed to CQ. The Ca-CQ-pDNA-PLGA-NPs showed high targeting efficiency in C57 acute pancreatitis model. In all, the Ca-CQ-pDNA-PLGA-NP was a promising candidate for targeted gene delivery to both tumor and pancreatitis.

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.

Rapid determination of 210Po in water samples

A new rapid method for the determination of 210Po in water samples has been developed at the Savannah River National Laboratory (SRNL) that can be used for emergency response or routine water analyses. If a radiological dispersive device event or a radiological attack associated with drinking water supplies occurs, there will be an urgent need for rapid analyses of water samples, including drinking water, ground water and other water effluents. Current analytical methods for the assay of 210Po in water samples have typically involved spontaneous auto-deposition of 210Po onto silver or other metal disks followed by counting by alpha spectrometry. The auto-deposition times range from 90 min to 24 h or more, at times with yields that may be less than desirable. If sample interferences are present, decreased yields and degraded alpha spectrums can occur due to unpredictable thickening in the deposited layer. Separation methods have focused on the use of Sr Resin™, often in combination with 210Pb analysis. A new rapid method for 210Po in water samples has been developed at the SRNL that utilizes a rapid calcium phosphate co-precipitation method, separation using DGA Resin® (N,N,N′,N′ tetraoctyldiglycolamide extractant-coated resin, Eichrom Technologies or Triskem-International), followed by rapid microprecipitation of 210Po using bismuth phosphate for counting by alpha spectrometry. This new method can be performed quickly with excellent removal of interferences, high chemical yields and very good alpha peak resolution, eliminating any potential problems with the alpha source preparation for emergency or routine samples. A rapid sequential separation method to separate 210Po and actinide isotopes was also developed. This new approach, rapid separation with DGA resin plus microprecipitation for alpha source preparation, is a significant advance in radiochemistry for the rapid determination of 210Po.

Tumor Necrosis Factor Receptor-associated Factor-6 and Ribosomal S6 Kinase Intracellular Pathways Link the Angiotensin II AT1 Receptor to the Phosphorylation and Activation of the IκB Kinase Complex in Vascular Smooth Muscle Cells

Activation of NF-κB transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-κB transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-κB transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKKβ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKCα, IKKβ subunit, tumor growth factor β-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKKβ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKβ requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKβ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKβ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKKβ, which is an essential step in the proinflammatory activity of Ang II.

Abstract 5261: Transfection of nerve growth factor cDNA into SK-N-BE(2)C neuroblastoma modulates TrkA and p75 receptor expression and activation

Abstract The diverse biological role of nerve growth factor (NGF) as a necessary modulator of neuroectodermal cell survival, proliferation and differentiation has been previously well described in both normal and neoplastic cell lines. However, recent studies demonstrate that neuroectodermal tumors, such as neuroblastoma, contain genetic alterations that result in limited secretion of active NGF, as well as expression abnormalities of its receptors TrkA and p75. To better define the differential effects of exogenous and endogenously synthesized NGF, the in vitro response of wild-type and genetically engineered neuroblastoma cells was investigated. Replication-defective NGF-expressing retroviral vectors were prepared using the calcium phosphate co-precipitation method and used to transfect SK-N-BE(2)C cells. Presence of the NGF cDNA was confirmed by RT-PCR, and secretion of NGF into the conditioned media by western immunoblotting. NGF transfection resulted in decreased cell proliferation as determined by nonradioactive cell proliferation assay. Wild type and NGF transfected (BE(2)C-NGF) cells were then incubated with exogenous NGF at 200ng/mL for varying time periods up to 24 hours. The expression of TrkA and p75, as well as proteins of their associated survival and apoptosis cascades were determined by Western immunoblotting of whole cell lysates. p75 expression in the NGF transfected cell line was equivalent to the parental wild type cell, but TrkA expression was significantly lower in BE(2)C-NGF. At baseline, BE(2)C-NGF cells demonstrate significant down-regulation of phospho-cRAF and its downstream effector phospho-p42/44 in comparison to the wild type cell line. Additionally, NGF stimulation further down-regulated phospho-c-RAF expression in the NGF transfected cell line, with maximal down-regulation occurring at 24 hours. BE(2)C-NGF cells also demonstrate significant baseline activation of phospho-p38 in comparison to the wild type cell line, with expression further up-regulated with NGF stimulation at 5 minutes and 1 hour. In conclusion, nerve growth factor cDNA gene transfer into wild type SK-N-BE(2)C neuroblastoma results in decreased activation of the p-42/44 survival pathway and concomitant up-regulation of the p-38 apoptotic pathway, leading to a overall decreased rate of proliferation. This effect is likely the result of a decreased TrkA to p75 receptor ratio, and further understanding of these pathways may facilitate the discovery of novel therapeutic targets for neuroblastoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5261.

TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1beta is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1beta is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1beta interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1beta enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.

The Proinflammatory Actions of Angiotensin II Are Dependent on p65 Phosphorylation by the IκB Kinase Complex

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.

Negative Regulation of the Retinoic Acid-inducible Gene I-induced Antiviral State by the Ubiquitin-editing Protein A20

Activation of the interferon regulatory factors (IRFs) 3 and 7 transcription factors is essential for the induction of type I interferon (IFN) and development of the innate antiviral response. Retinoic acid-inducible gene I has been shown to contribute to virus-induced IFN production independent of the Toll-like receptor pathways in response to a variety of RNA viruses and double-stranded RNA. In the present study, we demonstrate that the NF-kappaB-inducible, anti-apoptotic protein A20 efficiently blocks RIG-I-mediated activation of NF-kappaB-, IRF-3-, and IRF-7-dependent promoters but only weakly interferes with TRIF-TLR-3-mediated IFN activation. Expression of A20 completely blocked CARD domain containing DeltaRIG-I-induced IRF-3 Ser-396 phosphorylation, homodimerization, and DNA binding. The level of A20 inhibition was upstream of the TBK1/IKKepsilon kinases that phosphorylate IRF3 and IRF7 and paradoxically, A20 selectively degraded the TRIF protein but not RIG-I. A20 possesses two ubiquitin-editing domains, an N-terminal deubiquitination domain and a C-terminal ubiquitin ligase domain consisting of seven zinc finger domains. Deletion of the N-terminal de-ubiquitination domain had no significant effect on the inhibitory effect of A20, whereas deletion or mutation of zinc finger motif 7 ablated the inhibitory function of A20 on IRF- or NF-kappaB-mediated gene expression. Furthermore, cells stably expressing the active form of RIG-I induced an antiviral state that interfered with replication of vesicular stomatitis virus, an effect that was reversed by stable co-expression of A20. These results suggest that the virus-inducible, NF-kappaB-dependent activation of A20 functions as a negative regulator of RIG-I-mediated induction of the antiviral state.

P300 Modulates ATF4 Stability and Transcriptional Activity Independently of Its Acetyltransferase Domain

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.

DTL, the Drosophila Homolog of PIMT/Tgs1 Nuclear Receptor Coactivator-interacting Protein/RNA Methyltransferase, Has an Essential Role in Development

We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m3G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5' end of the message. This product of dtl is responsible for the formation of the m3G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m3G cap-containing RNAs and lethality in the early pupa stage. The 5' region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.

Bone Morphogenic Protein-7 Induces Mesenchymal to Epithelial Transition in Adult Renal Fibroblasts and Facilitates Regeneration of Injured Kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.

Notch Signals Inhibit the Development of Erythroid/Megakaryocytic Cells by Suppressing GATA-1 Activity through the Induction of HES1

The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.

Glucocorticoids Inhibit the Transcriptional Activity of LEF/TCF in Differentiating Osteoblasts in a Glycogen Synthase Kinase-3β-dependent and -independent Manner

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation-related cell cycle is associated with a decrease in beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser(473) within minutes, and this inhibition was also observed after 12 h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and Wnt signaling pathways. Consistent with a role for Wnt signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt signaling and the osteoblast differentiation-related cell cycle.

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

Casein Kinase Iϵ Plays a Functional Role in the Transforming Growth Factor-β Signaling Pathway

The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

Reciprocal Inhibition between MyoD and STAT3 in the Regulation of Growth and Differentiation of Myoblasts

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.