Concentration-dependent Increase In Expression Research Articles

A novel concept of Pyridoxal 5'-phosphate permeability in E.coli for modulating the heterologous expression of PLP dependent proteins

PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies.

Bisphenol-A induced antioxidants imbalance and cytokines alteration leading to immune suppression during larval development of Labeo rohita.

Recently, the oxidative stress and immunotoxicity biomarkers have been extensively used in embryotoxicity using fish embryos as promising models especially after exposure to chemical-like environmental estrogens. Bisphenol-A (BPA) is an estrogenic endocrine disruptor and is ubiquitous in the aquatic environment. Larvae of Labeo rohita were exposed to low concentrations of BPA (10, 100, 1000μg/l) for 21days. Innate immune system, antioxidants parameters, and developmental alterations were used as biomarkers. Exposure to BPA caused developmental abnormalities including un-inflated swim bladder, delayed yolk sac absorption, spinal curvature, and edema of pericardium. Lipid peroxidation increased and activity of catalase (p < 0.05), superoxide dismutase (p < 0.05), and glutathione peroxidase (p < 0.01) decreased after exposure to BPA. Level of reduced glutathione also decreased (p < 0.05) in BPA-exposed group. Lower expression of tumor necrosis factor-α (p < 0.05) and interferon-γ (p < 0.001) was observed in BPA-exposed groups while expression of interleukin-10 increased (p < 0.05) in larvae exposed to 10μg/l BPA. Moreover, exposure of BPA caused a concentration-dependent increase in expression of heat shock protein 70 (p < 0.05). The present study showed that the exposure to BPA in early life stages of Labeo rohita caused oxidative stress and suppress NF-κB signaling pathway leading to immunosuppression. The results presented here demonstrate the cross talk between heat shock protein 70 and cytokines expression.

Sulfur Mustard Analog Mechlorethamine (Bis(2-chloroethyl)methylamine) Modulates Cell Cycle Progression via the DNA Damage Response in Human Lung Epithelial A549 Cells.

Nitrogen mustard, mechlorethamine (bis(2-chloroethyl)methylamine; HN2), and sulfur mustard are potent vesicants that modify and disrupt cellular macromolecules including DNA leading to cytotoxicity and tissue injury. In many cell types, HN2 upregulates DNA damage signaling pathways including ataxia telangiectasia mutated (ATM), ataxia telangiectasia mutated- and Rad3-related (ATR) as well as DNA-dependent protein kinase (DNA-PK). In the present studies, we investigated crosstalk between the HN2-induced DNA damage response and cell cycle progression using human A549 lung epithelial cells. HN2 (1-20 μM; 24 h) caused a concentration-dependent arrest of cells in the S and G2/M phases of the cell cycle. This was associated with inhibition of DNA synthesis, as measured by incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into S phase cells. Cell cycle arrest was correlated with activation of DNA damage and cell cycle checkpoint signaling. Thus, HN2 treatment resulted in time- and concentration-dependent increases in expression of phosphorylated ATM (Ser1981), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15). Activation of DNA damage signaling was most pronounced in S-phase cells followed by G2/M-phase cells. HN2-induced cell cycle arrest was suppressed by the ATM and DNA-PK inhibitors, KU55933 and NU7441, respectively, and to a lesser extent by VE821, an ATR inhibitor. This was correlated with abrogation of DNA damage checkpoints signaling. These data indicate that activation of ATM, ATR, and DNA-PK signaling pathways by HN2 are important in the mechanism of vesicant-induced cell cycle arrest and cytotoxicity. Drugs that inhibit activation of DNA damage signaling may be effective countermeasures for vesicant-induced tissue injury.

Gene structure and comparative and phylogenetic analyses of Catla catla CYP1A full-length cDNA and its responsiveness to benzo(a)pyrene and copper sulphate at early developmental stages.

In the present study, full-length CYP1A cDNA from Catla catla (Catla) has been identified, and its real-time quantitative reverse transcription PCR (qRT-PCR) expression has been evaluated in different tissues, developmental stages (0, 3, 6, 12 and 24h and 5, 7 and 9days post-fertilization) and copper sulphate and benzo(a)pyrene (BaP)-treated 5-day post-fertilization (dpf) larvae (6 to 6.5mm). Various structural, comparative and phylogenetic analyses of the deduced amino acid sequence revealed that the identified gene of Catla belongs to the CYP1A1 subfamily. Among different tissues of Catla, the highest CYP1A expression was observed in the kidney followed by the liver, muscle, gill, intestine and brain. CYP1A mRNA expression was detected during all the larval developmental stages, including the unfertilized egg with the highest expression on 9dpf. BaP (3.5ppb) and copper sulphate (sublethal dose 0.516ppm) challenge test for 96h to Catla larvae revealed the highest CYP1A1 expression at 48h post-challenge. CYP1A1 transcript also showed a concentration-dependent increase in expression following exposure at 1.75 and 3.5ppb of BaP for 48h. Its expression profiling indicates that it is functional at early developmental stages. It can also be used to develop a specific biomarker tool for monitoring environmental pollution.

Abstract 4021: The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations

Abstract Immunotherapy has led to major treatment breakthroughs for a number of cancers including non-small cell lung cancer (NSCLC). Although initial responses to immune checkpoint inhibitors are promising, a significant percentage of patients do not respond or rapidly acquire resistance. Although the mechanisms underlying intrinsic and acquired resistance remain largely unexplained; the expression of programmed cell death-ligand 1 (PD-L1), lack of tumor cell capacity to effectively present neoantigens, and presence of immunosuppressive cellular subsets have been implicated as potential mechanisms. Histone deacetylase (HDAC) inhibitors have emerged as a class of agents that may combat checkpoint inhibitor resistance by reversing immune evasion and eliciting an anti-tumor activity through a multi-faceted immuno-stimulatory mechanism of action. Mocetinostat is a spectrum-selective Class I/IV HDAC inhibitor specifically targeting HDAC-1, -2, -3 and -11. The present studies were designed to explore mocetinostat's effect as an immune-enhancer and ultimately, to evaluate its potential to be used in combination with immune checkpoint inhibitors (e.g., PD-1/PD-L1 antagonists). Specifically, we assessed mocetinostat's effect on the expression of various immunomodulatory factors by tumor cells as well as its effect on immune cell sub-populations in the tumor microenvironment in vivo. Mocetinostat elicited a concentration-dependent increase in PD-L1 mRNA expression which translated into increased PD-L1 surface protein expression in a panel of NSCLC cell lines. In addition, mocetinostat elicited a concentration-dependent increase in expression of MHC-class I related polypeptide-related sequence A (MIC-A) and MIC-B, and cluster of differentiation 86 (CD86). Furthermore, mocetinostat induced expression of several human leukocyte antigen (HLA) gene complex family members including HLA-A, -B, -DRA, and -DPA among others. To determine the effect of mocetinostat on systemic and tumor immune cell subpopulations we treated CT26 tumor-bearing mice. Mocetinostat increased splenic CD4-positive T effector cells and tumor mature cytolytic CD8-postive T cells and at the same time decreased tumor FoxP3-positive T regulatory cells and CD11b/Gr1-positive myeloid-derived suppressor cells (MDSC). These data provide evidence that mocetinostat modulates key immune regulators both in tumor cells as well as in relevant immune cell types in the tumor microenvironment and provides strong rationale for combination with immune checkpoint inhibitors. Citation Format: David Briere, Niranjan Sudhakar, Lars Engstrom, Jill Hallin, Ruth Tang, Harrah Chiang, Maryland Rosenfeld-Franklin, Peter Olson, James Christensen. The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4021.

Calcium oxalate nephrolithiasis and expression of matrix GLA protein in the kidneys

Polymorphism of the gene for matrix GLA protein (MGP), a calcification inhibitor, is associated with nephrolithiasis. However, experimental investigations of MGP role in stone pathogenesis are limited. We determined the effect of renal epithelial exposure to oxalate (Ox), calcium oxalate (CaOx) monohydrate (COM) or hydroxyapatite (HA) crystal on the expression of MGP. MDCK cells in culture were exposed to 0.3, 0.5 or 1 mM Ox and 33, 66 or 133-150 μg/cm(2) of COM/HA for 3-72 h. MGP expression and production were determined by Western blotting and densitometric analysis. Enzyme-linked immunosorbent assay was performed to determine MGP release into the medium. Hyperoxaluria was induced in male Sprague-Dawley rats by feeding hydroxyl-L-proline. Immunohistochemistry was performed to detect renal MGP expression. Exposure to Ox and crystals led to time- and concentration-dependent increase in expression of MGP in MDCK cells. Cellular response was quicker to crystal exposure than to the Ox, expression being significantly higher after 3-h exposure to COM or HA crystals and more than 6 h of exposure to Ox. MGP expression was increased in kidneys of hyperoxaluric rats particularly in renal peritubular vessels. We demonstrate increased expression of MGP in renal tubular epithelial cells exposed to Ox or CaOx crystals as well as the HA crystals. The most significant finding of this study is the increased staining seen in renal peritubular vessels of the hyperoxaluric rats, indicating involvement of renal endothelial cells in the synthesis of MGP.

Mir-21 Regulation of MARCKS Protein and Mucin Secretion in Airway Epithelial Cells

Hypersecretion of mucus characterizes many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Excess mucus causes airway obstruction, reduces pulmonary function, and can lead to increased morbidity and mortality. MicroRNAs are small non-coding pieces of RNA which regulate other genes by binding to a complementary sequence in the target mRNA. The microRNA miR-21 is upregulated in many inflammatory conditions and, interestingly, miR-21 has been shown to target the mRNA of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), a protein that is an important regulator of airway mucin (the solid component of mucus) secretion. In these studies, we determined that exposure of primary, well-differentiated, normal human bronchial epithelial (NHBE) cells to the pro-inflammatory stimulus lipopolysaccharide (LPS) increased expression of both miR-21 and MARCKS in a time-dependent manner. To investigate whether miR-21 regulation of MARCKS played a role in mucin secretion, two separate airway epithelial cell lines, HBE1 (papilloma virus transformed) and NCI-H292 (mucodepidermoid derived) were utilized, since manipulation of miR-21 is performed via transfection of commercially-available miR-21 inhibitors and mimics/activators. Treatment of HBE1 cells with LPS caused concentration-dependent increases in expression of both miR-21 and MARCKS mRNA and protein. The miR-21 inhibitor effectively reduced levels of miR-21 in the cells, coincident with an increase in MARCKS mRNA expression over time as well as enhanced mucin secretion, while the miR-21 mimic/activator increased levels of miR-21, which coincided with a decrease in expression of MARCKS and a decrease in mucin secretion. These results suggest that miR-21 is increased in airway epithelial cells following exposure to LPS, and that miR-21 downregulates expression of MARCKS, which may decrease mucin secretion by the cells. Thus, miR-21 may act as a negative feedback regulator of mucin secretion in airway epithelial cells, and may do so, at least in part, by downregulating expression of MARCKS.

CIMT: Refining CVD Risk Prediction and Evaluation Effects of Lipid Therapies †

s 227 3.9% for IFN-g alone, P 5 .0035. For ABCG1: 194.3 6 22.03% for resveratrol+IFN-g vs 64.1 6 3.5% for IFN-g alone, P 5 0.001. We found a concentration-dependent increase in expression of cholesterol efflux proteins upon resveratrol treatment. 10 mM resveratrol stimulated expression of ABCA1 and ABCG1 transporters up to 160.73 6 4.7% and 170.5 6 4.2% versus untreated cells, respectively (n 5 5, P , .01). 27-OHase message was elevated to 352.7 6 13.6% of untreated control by 25 mM resveratrol (n 5 5, P , 0.001). Expression of LXR-a was up-regulated by 72.6 6 7.5% with 25 mM resveratrol and by 94.52 6 4.5% with 50 mM resveratrol (n 5 5, P , .01). Macrophage foam cell formation was significantly reduced with resveratrol compared with control (22.2 6 5% vs. 35 6 5%, P 5 .03). Conclusions: This study shows that in a human cell culture model of atherosclerosis, resveratrol modulates expression of cholesterol efflux proteins in an atheroprotective manner and overcomes the atheroma-promoting properties of IFN-g. These findings imply a possible therapeutic role for resveratrol in the armamentarium against atherosclerotic cardiovascular disease. Since resveratrol has a strong safety profile and is well-tolerated, low cost, and can be used in combination with multiple other therapies without contraindication, further in vivo studies are indicated for this promising treatment option. Table Effect of Lipid therapy on CIMT in moderate/High CVD risk patients Study Patients Duration, year Treatments Baseline

The HIV Protease Inhibitor Nelfinavir: A Unique Oral Drug That Inhibits the Proteasome and AKT-Phosphorylation, Induces ER Stress and Sensitizes Bortezomib-Refractory Primary Myeloma Cells and Primary AML Cells towards Bortezomib

AbstractAbstract 4069 Background:HIV protease inhibitors (HIV-PI: Ritonavir, Lopinavir, Saquinavir, Nelfinavir, Amprenavir, Indinavir, Atazanavir, Tipranavir and Darunavir) are oral drugs approved for HIV treatment. Although designed to inhibit the HIV protease, HIV-PI likely have additional, yet unidentified, mammalian targets. Individual HIV-PI have been reported to inhibit the proteasome as well as AKT phosphorylation, induce ER stress and exert synergistic cytotoxicity with bortezomib in solid tumors, suggesting a therapeutic potential of HIV-PI especially in myeloma and AML. However, testing and comparing the effects of the different HIV-PI in AML or myloma cells, a prerequisite to identify the most promising HIV-PI to enter clinical testing, have not yet been performed. Methods:We systematically compared and analysed the effects of all currently approved HIV-PI on proteasome inhibition, cytotoxicity, induction or ER-stress, inhibition of p-AKT, and synergism with bortezomib in human myeloma and AML cell lines and primary cells in vitro. Results:HIV-PI can be divided into two subclasses, based on their activity on myeloma and AML cells: Lopinavir, Nelfinavir, Ritonavir and Saquinavir (LNRS-PI) showed biological and molecular activity at concentrations within or near to therapeutic drug levels (10-20 μ M). All remaining HIV-PI were inactive in this concentration range. The LNRS-PI induced ER stress in a very similar fashion (concentration-dependent increase in expression of the chaperones BIP and PDI at 10 μ M). This translated into a uniform pattern of ER stress-induced apoptosis, as deferred from increased expression of CHOP and cleaved PARP, leading to cell death. Likewise, the concentration-dependent inhibition of AKT-phosphorylation was similarly observed for all LNRS-PI, starting at 10 μ M. The LNRS-PI also showed a uniform synergistic cytotoxic activity with bortezomib at therapeutic drug levels, as observed with myeloma cell lines, as well as AML cell lines and primary cells in vitro. By contrast, all remaining HIV-PI did not show a synergistic effect with bortezomib. To assess the effects of the different HIV-PI on proteasome activity, we used activity-based proteasome-specific probes that visualize the activities of the proteasome β2 and β1/β5 active sites in intact, viable cells. Nelfinavir stood out as the only HIV-PI with proteasome-inhibiting activity at therapeutic drug levels. Nelfinavir led to a dose-dependent decrease in active proteasome β1/β5 as well as β2 species in the concentration range of 10–40 μ M. By contrast, low doses of Lopinavir, Ritonavir or Saquinavir induced proteasome β2 and β1/β5 activity, which then decreased to baseline activity levels upon higher drug concentrations (up to 80 μ M). All remaining HIV-PI had no effect on proteasome activity. When cells were pre-treated with bortezomib, which preferentially inhibits the proteasome β1/β5 subunits, and were then exposed to the different HIV-PI, only Nelfinavir led to an additional inhibition of the proteasomal β1/5 and also β2-activity, while all other HIV-PI did not have such an effect. Strikingly, primary myeloma cells from a patient who was refractory to bortezomib-lenalidomide combination therapy, and which were refractory to Bortezomib 10 nM also in vitro, showed robust (> 90%) cytotoxicity when bortezomib was combined with therapeutic drug levels of Nelfinavir (10 μ M). Conclusions:Nelfinavir is a unique drug in the class of HIV-PI, which leads not only to the induction of ER stress and inhibition of AKT-phosphorylation, but also to proteasome inhibition of all active subunits in intact cells at therapeutic drug levels. It re-sensitizes bortezomib-refractory myeloma cells towards bortezomib treatment. Nelfinavir may therefore be an active drug warranting clinical testing in hematologic malignancies, such as myeloma, mantle cell lymphoma or AML. Disclosures:No relevant conflicts of interest to declare.

P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress.

During ischaemia/reperfusion, cells of the blood-brain barrier are subjected to oxidative stress. This study uses primary cultured rat brain endothelial cells to examine the effect of such stresses on expression of multidrug transporters. H(2)O(2) up to 500 microm applied to cell monolayers caused a concentration-dependent increase in expression of P-glycoprotein (Pgp) but not of multidrug resistance-associated protein (Mrp1). Concentrations > 250 microm H(2)O(2) decreased cell viability. Application of 100 microm H(2)O(2) caused a significant increase after 48 h in Pgp functional activity, as assessed from [(3)H]vincristine accumulation experiments. At this concentration, H(2)O(2) produced a transient increase within 10 min followed by a sustained decrease in levels of intracellular reactive oxygen species (iROS), detectable by flow cytometry. Reoxygenation of cell monolayers after 6 h hypoxia gave rise to a similar transient increase in iROS and this also led to increased Pgp expression by 24 h. Increases were also observed within 4 h after both H(2)O(2) and hypoxia/reoxygenation treatments in mdr1a and mdr1b mRNA. Evidence suggests this was due to enhanced transcription rather than mRNA stabilization. Therefore, oxidative stress, by changing Pgp expression, may affect movement of Pgp substrates in and out of the brain.

Pharmacological characteristics of solid-phase von Willebrand factor in human platelets.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1.

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.

Promotion of the Development of Enteric Neurons and Glia by Neuropoietic Cytokines: Interactions with Neurotrophin-3

Neurotrophin-3 (NT-3) is known to promote enteric neuronal and glial development. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) were investigated to test the hypothesis that the development of subsets of enteric neurons and/or glia is also affected by a neuropoietic cytokine, by itself, or together with NT-3. Crest-derived cells, immunoselected from the fetal rat gut (E14) with antibodies to p75 NTR , were found by RT-PCR and immunocytochemistry (after culture) to express both α (CNTFRα) and β components (gp130 and LIFRβ) of the tripartite CNTF receptor. In situ , myenteric ganglia below the esophagus were CNTFRα-immunoreactive by E16–E18. In vitro , CNTF and LIF induced in crest-derived cells nuclear translocation of STAT3 (signal transducer and activator of transcription 3), a concentration-dependent increase in expression of neuronal or glial markers, and a decrease in expression of the precursor marker, nestin. LIFRβ was expressed by more cells than CNTFRα; therefore, although the factors were equipotent, the maximal effect of LIF > CNTF. The cytokines and NT-3 were additive in promoting neuronal but not glial development. Specifically, the development of neurons expressing NADPH-diaphorase activity (an early marker found in inhibitory motor neurons) was promoted by CNTF and NT-3. These observations support the idea that a ligand for the tripartite CNTF receptor complex plays a role in ENS development.

Promotion of the development of enteric neurons and glia by neuropoietic cytokines: Interactions with neurotrophin-3

Neurotrophin-3 (NT-3) is known to promote enteric neuronal and glial development. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) were investigated to test the hypothesis that the development of subsets of enteric neurons and/or glia is also affected by a neuropoietic cytokine, by itself, or together with NT-3. Crest-derived cells, immunoselected from the fetal rat gut (E14) with antibodies to p75 NTR, were found by RT-PCR and immunocytochemistry (after culture) to express both α (CNTFRα) and β components (gp130 and LIFRβ) of the tripartite CNTF receptor. In situ, myenteric ganglia below the esophagus were CNTFRα-immunoreactive by E16–E18. In vitro, CNTF and LIF induced in crest-derived cells nuclear translocation of STAT3 (signal transducer and activator of transcription 3), a concentration-dependent increase in expression of neuronal or glial markers, and a decrease in expression of the precursor marker, nestin. LIFRβ was expressed by more cells than CNTFRα; therefore, although the factors were equipotent, the maximal effect of LIF > CNTF. The cytokines and NT-3 were additive in promoting neuronal but not glial development. Specifically, the development of neurons expressing NADPH-diaphorase activity (an early marker found in inhibitory motor neurons) was promoted by CNTF and NT-3. These observations support the idea that a ligand for the tripartite CNTF receptor complex plays a role in ENS development.

Protein Kinase C Regulates Keratinocyte Transglutaminase (TGK) Gene Expression in Cultured Primary Mouse Epidermal Keratinocytes Induced to Terminally Differentiate by Calcium

During the final stage of epidermal differentiation, activation of keratinocyte transglutaminase results in covalent crosslinking of a variety of proteins to form highly protective cornified cell envelopes. We have studied the regulation of keratinocyte transglutaminase (TGK) gene expression in murine epidermal keratinocytes induced to terminally differentiate in vitro by increasing the level of extracellular Ca++ or treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Raising extracellular Ca++ induces squamous differentiation of cultured keratinocytes and elicits a concentration-dependent increase in expression of TGK mRNA; keratinocytes grown for 24 h in 0.12 mM Ca++ medium express approximately 12 times as much TGK mRNA as basal cells (grown in 0.05 mM Ca++ medium), whereas cultures exposed to 1.4 mM Ca++ express approximately 17 times as much. TPA induces squamous differentiation and TGK mRNA even in basal keratinocyte cultures grown in 0.05 mM Ca++ medium, suggesting that expression of this differentiation marker is regulated by the PKC signaling pathway. Induction of TGK mRNA in response to TPA treatment is transient, reaching a peak at 6-8 h and returning to baseline by 24 h. In contrast, elevation of TGK mRNA levels in response to Ca++ persists for at least 24 h. The increased abundance of TGK mRNA reflects increased transcription of the TGK gene, based on nuclear run-on analysis of Ca(++)- and TPA-treated keratinocytes. Induction of TGK mRNA by either TPA or Ca++ is blocked in the presence of cycloheximide, suggesting that a PKC-dependent protein factor is required for TGK gene expression in response to both stimuli. Furthermore, the accumulation of TGK mRNA in keratinocytes treated with TPA or Ca++ is blocked in cells treated with the PKC inhibitor GF 109203X or bryostatin. These results suggest that the induction of TGK gene expression by Ca++ is dependent on PKC, providing further support for the hypothesis that PKC plays a central role in regulating the late stages of epidermal differentiation.

TFIID can be rate limiting in vivo for TATA-containing, but not TATA-lacking, RNA polymerase II promoters.

We have studied the effect of exogenous expression of the basal transcription factor TFIID on the activities of several different TATA-containing and TATA-lacking promoters. Overexpression of TFIID from a transfected plasmid in Drosophila Schneider cells resulted in substantial concentration-dependent increases in expression from a cotransfected minimal TATA-containing promoter. Overexpression of TFIID activated expression from all TATA-containing promoters tested, with the maximum level of activation being inversely proportional to the strength of the promoter. In contrast, expression from TATA-less promoters was not enhanced, and could in fact be reduced, by increased expression of TFIID. Consistent with these findings overexpression of TFIID had opposite effects on Sp1-mediated activation observed from minimal synthetic promoters consisting of Sp1-binding sites and either a TATA box or initiator element. We discuss the significance of these results in terms of the role of TFIID in the initiation of transcription and as a possible regulatory target for expression from TATA-containing promoters, as well as the role TFIID may play in expression from TATA-less promoters.

Reversible abrogation of IL-3 dependence by an inducible H-ras oncogene.

Immortalized, interleukin-3 (IL-3)-dependent mouse mast cells (PB-3c) were transfected with a human activated c-H-ras gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat. Addition of increasing amounts of dexamethasone resulted in a concentration-dependent increase in expression of the H-ras oncogene. The elevation of p21 ras protein concentrations was paralleled by progressive growth of the transfectants in the absence of exogenous IL-3, leading to complete abrogation of growth-factor requirement at high p21ras levels. The maintenance of the IL-3-independent state required the continuous expression of the H-ras oncogene, since dexamethasone removal was followed by rapid cell death. Expression of the H-ras oncogene induced PB-3c cells to produce IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that their IL-3-independent proliferation may be due to an autocrine mechanism.