Levels In Untreated Cells Research Articles

Gamma-amino Butyric Acid (GABA) Prevents the Induction of Nicotinic Receptor–Regulated Signaling by Chronic Ethanol in Pancreatic Cancer Cells and Normal Duct Epithelia

Pancreatic cancer has a high mortality rate and alcoholism is a risk factor independent of smoking. We have shown that nicotinic acetylcholine receptors (nAChR) regulate pancreatic ductal epithelia and pancreatic ductal adenocarcinoma (PDAC) cells in an autocrine fashion by stimulating their production of the stress neurotransmitters noradrenaline and adrenaline that signal through β-adrenergic receptors (β-AR). Our current study has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by γ-amino butyric acid (GABA). Using MTT assays, cell migration assays, Western blotting, immunoassays, and gene knockdown of individual nAChRs in two PDAC cell lines and in immortalized human pancreatic duct epithelial cells, our data show that treatment for seven days with ethanol induced the protein expression and sensitivity of nAChRs α3, α5, and α7 resulting in increased production of noradrenaline and adrenaline, which drive proliferation and migration via cyclic AMP (cAMP)-dependent signaling downstream of β-ARs. Treatment with GABA prevented all of these responses to chronic ethanol, reducing cell proliferation and migration below base levels in untreated cells. Our findings suggest that alcoholism induces multiple cAMP-dependent PDAC stimulating signaling pathways by upregulating the protein expression and sensitivity of nAChRs that regulate stress neurotransmitter production. Moreover, our data identify GABA as a promising agent for the prevention of PDAC in individuals at risk due to chronic alcohol consumption.

Effect of Cyclosporine A on the expression of GSTO2 metabolizing enzyme in Jurkat cell line

A B S T R A C T Cyclosporine A (CsA), a cyclic polypeptide metabolite extracted from the fungus, is used clinically to combat organ graft rejection in transplant subjects. Previous studies have shown that CsA exposure enhances the production of reactive oxygen species (ROS) and lipid peroxidation, which are directly involved in CsA toxicity. To protect cells and organs against ROS, the human body has evolved a highly antioxidant protection system to neutralize free radicals. Th e aim of this study was to investigate the effect of CsA on mRNA expression of anti -oxidant GSTO2. To do this, Jurkat cells were incubated for 24 h with different doses of CsA , ranging from 1-80 µg/ml, and the IC50 of CsA was calculated to be 40 µg/ml. Subsequently, Jurkat cells were treated with 3 µg/ml CsA for 24 h and the gene expression of GSTO2 was quantified by quantitative Real-time PCR. Results showed that the mean (SD) expression of the GSTO2 gene in CsA treated cells was 1.10 (0.07) (when assuming an expression level in untreated cells of 1.0). However, statistical analyses showed that the alterations were not significant (t=2.29 , df=2, P=0.149). These findings suggest that at this concentration of CsA, other antioxidant enzymes are up-regulated in Jurkat cell lines to detoxify free radical s induced by CsA.

Different effects of dibutyryl cAMP on monolayer permeability in human aortic and coronary arterial endothelial cells

Recent studies have demonstrated species- and/or organ-specific differences in the effects of dibutyryl cAMP (DBcAMP) on vascular permeability. In this study, we investigated the permeability of monolayers of human coronary arterial endothelial cells (HCAECs) and human aortic endothelial cells (HAECs) in the presence of DBcAMP using monolayer permeability models of endothelial cells and a double incubation chamber for measurement of FITC-labeled albumin or FITC-dextran (4 kDa) transfer through the monolayer. DBcAMP significantly lowered monolayer permeability in the HCAECs but had no effect in the HAECs when compared with untreated cells (P < 0.01). A cAMP-dependent protein kinase (PKA) inhibitor, Rp-8-Br-cAMPS (10-5 M), significantly reversed the DBcAMP induced decease in permeability of the HCAEC monolayer to the level of untreated cells. Rp-8-Br-cAMPS (10-5 M) and Sp-cAMPS (a PKA activator) had nosignificant effect on the permeability of the HAEC monolayer after treatment with DBcAMP. These results indicate that DBcAMP diminishes the permeability of an HCAEC monolayer via a cAMP-mediated PKA pathway but does not do so in HAECs, suggesting that the effects of cAMP on permeability of endothelial cell monolayers are dependent on the origin of the cells. Key words: Dibutyryl cAMP, endothelial cells, permeability.

Resveratrol Alters microRNA Expression Profiles in A549 Human Non-Small Cell Lung Cancer Cells

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.

The Sterol-sensing Domain (SSD) Directly Mediates Signal-regulated Endoplasmic Reticulum-associated Degradation (ERAD) of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Isozyme Hmg2

The sterol-sensing domain (SSD) is a conserved motif in membrane proteins responsible for sterol regulation. Mammalian proteins SREBP cleavage-activating protein (SCAP) and HMG-CoA reductase (HMGR) both possess SSDs required for feedback regulation of sterol-related genes and sterol synthetic rate. Although these two SSD proteins clearly sense sterols, the range of signals detected by this eukaryotic motif is not clear. The yeast HMG-CoA reductase isozyme Hmg2, like its mammalian counterpart, undergoes endoplasmic reticulum (ER)-associated degradation that is subject to feedback control by the sterol pathway. The primary degradation signal for yeast Hmg2 degradation is the 20-carbon isoprene geranylgeranyl pyrophosphate, rather than a sterol. Nevertheless, the Hmg2 protein possesses an SSD, leading us to test its role in feedback control of Hmg2 stability. We mutated highly conserved SSD residues of Hmg2 and evaluated regulated degradation. Our results indicated that the SSD was required for sterol pathway signals to stimulate Hmg2 ER-associated degradation and was employed for detection of both geranylgeranyl pyrophosphate and a secondary oxysterol signal. Our data further indicate that the SSD allows a signal-dependent structural change in Hmg2 that promotes entry into the ER degradation pathway. Thus, the eukaryotic SSD is capable of significant plasticity in signal recognition or response. We propose that the harnessing of cellular quality control pathways to bring about feedback regulation of normal proteins is a unifying theme for the action of all SSDs.

Cysteinyl leukotriene receptor expression pattern affects migration of breast cancer cells and survival of breast cancer patients

The fact that breast cancer patients with local or distal dissemination exhibit decreased survival, promotes a search for novel mechanisms to suppress such tumor progression. Here, we have determined the expression of proinflammatory cysteinyl leukotriene receptors (CysLTRs) in breast tumor tissue and their signaling effect on breast cancer cell functions related to tumor progression. Patients with breast tumors characterized by high CysLT(1)R and low CysLT(2)R expression levels exhibited increased risk of cancer-induced death in univariate analysis for both the total patient group (hazard ratio [HR] = 2.88, 95% confidence interval [CI] = 1.11-7.41), as well as patients with large (>20 mm) tumors (HR = 5.08, 95% CI = 1.39-18.5). Multivariate analysis revealed that patients with large tumors exhibiting high CysLT(1)R and low CysLT(2)R expression levels had a significantly reduced survival, also when adjusted for established prognostic parameters (HR = 7.51, 95% CI = 1.83-30.8). In patients with large (>20 mm) tumors, elevated CysLT(2)R expression predicted an improved 5-year survival (log-rank test p = 0.04). Surprisingly, for longer time periods, this prognostic value was lost. This disappearance coincided with the termination of hormonal treatment. Tamoxifen preserved and even induced transcription of CysLT(2)R, but not CysLT(1)R, in estrogene receptor-positive MCF-7 breast cancer cells. This elevated CysLT(2)R expression decreased, even below the level of untreated cells, when tamoxifen was withdrawn. CysLT(2)R signaling reduced MCF-7 cell migration, but had no effect on either proliferation or apoptosis. Our data indicate that low CysLT(1)R together with high CysLT(2)R expression levels might be useful parameters in prognostication and treatment stratification of breast cancer patients.

Melatonin suppresses cyclosporine A‐induced autophagy in rat pituitary GH3 cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0-100 microm) decreased cell survival of rat pituitary GH3 cells in a dose-dependent manner. At concentrations ranging from 1.0 to 10 microm, CsA induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-I and LC3-II. Cells treated with 2.5 microm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0-10 microm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol-requiring enzyme 1 alpha (IRE1alpha), increased as compared those levels in untreated cells. These results suggested that CsA-induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA-induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 microm) suppressed autophagy induced by 2.5 and 5 microm CsA. Furthermore, co-treatment with 100 microm melatonin inhibited LC3-II expression, and increased catalase and phosphorylated p-ERK levels in the presence of 2.5 and 5 microm CsA. BiP and IRE1alpha expression in melatonin-co-treated cells was superior to that in cells treated with 2.5 and 5 microm CsA alone. Thus, melatonin suppresses CsA-mediated autophagy in rat pituitary GH3 cells.

Topoisomerase I levels in the NCI-60 cancer cell line panel determined by validated ELISA and microarray analysis and correlation with indenoisoquinoline sensitivity

Topoisomerase I (Top1) is a proven target for cancer therapeutics, and the level of Top1 in tumors has been used as a biomarker for chemotherapeutic efficacy. In this study, we report the development and validation of a two-site enzyme chemiluminescent immunoassay for Top1, which we used to measure Top1 levels in the NCI-60 cancer cell line panel. Top1 levels ranged from 0.9 to 9.8 ng/mL/microg protein extract in these cell lines. Levels varied both within and between cancer types but were generally highest in colon cancer and leukemia cell lines and lowest in central nervous system and renal cancer cell lines. Top1 mRNA levels in the NCI-60 cell lines were also measured by microarray; mRNA values generally showed a good correlation with protein levels (Pearson correlation = 0.8). When these expression levels were compared with the activity of the indenoisoquinoline class of Top1 inhibitors across the NCI-60 cell panel, low levels of Top1 were associated with increased resistance to these drugs. The results of our studies indicate that our Top1 assay can be used to quantify Top1 levels in untreated cells as well as cells treated with Top1 inhibitors and that the assay has the potential to be adapted for use in predicting clinical response to Top1-active antineoplastic agents.

Hemin inhibits hypertensive rat vascular smooth muscle cell proliferation through regulation of cyclin D and p21

We tested the hypothesis that HO-1 (heme oxygenase-1) activity varied between vascular smooth muscle cells (VSMC) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. HO-1 levels were measured under baseline and hemin-stimulated conditions and cell proliferation was monitored. Basal HO-1 levels in untreated cells were lower in SHR compared to WKY rats. Treatment with hemin increased HO-1 mRNA and protein levels in the cells obtained from WKY rats compared to that of SHR rats. However, hemin-treatment showed a greater inhibitory effect on VSMC proliferation in SHR rats than in WKY rats. Tin protoporphyrin IX (SnPPIX) showed a greater reversal of the anti-proliferative effect of hemin on cells from SHR rats than WKY. Similarly, VSMC proliferation from SHR was significantly inhibited in VSMC transfected with the HO-1 gene. These inhibitory effects were associated with cell cycle arrest in the G1 phase. The level of cyclin D, and cyclin dependent kinase inhibitor p21 was higher in SHR cells progressing through the G1 phase. Treatment of the cells with hemin down-regulated the expression of cyclin D and up-regulated that of p21. These results indicate that hemin, an HO-1 inducer, may play a more critical role in VSMC proliferation in SHR than WKY.

Impact of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial DNA and RNA in Human Skeletal Muscle Cells

We previously reported that 2',3'-dideoxyinosine (didanosine, or ddI) significantly altered mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells in human immunodeficiency virus type 1 (HIV-1)-infected children who had undetectable plasma HIV-1 RNA for more than 2 years while receiving highly active antiretroviral therapy. This research examines the in vitro effects of nucleoside reverse transcriptase inhibitors (NRTIs) on mitochondria of human skeletal muscle cells (HSMCs), including myoblasts and differentiated myotubes. mtDNA, mitochondrial RNA (mtRNA), and mRNA levels for nuclear mitochondrial regulatory factors were quantified in vitro using HSMCs, including myoblasts and differentiated myotubes, treated with NRTIs singly and in combination. After 5 days of treatment, mtDNA was significantly decreased in myoblasts and myotubes treated with ddI (P < 0.001 and P = 0.01, respectively) and ddI-containing regimens (P < 0.001 and P < 0.001, respectively) compared to levels in untreated cells. mtRNA (MTCYB) was also significantly decreased in the myoblasts and myotubes treated with ddI (P = 0.004) and ddI-containing regimens (P < 0.001). Regardless of the NRTI regimens examined, NRTI combinations significantly decreased mtRNA (MTCO3) in myoblasts and myotubes (P = 0.02 and P = 0.01, respectively). No significant differences were observed for nuclear mitochondrial regulatory factor mRNA in myoblasts or myotubes when treated with NRTIs (P > 0.07). ddI and ddI-containing regimens significantly decrease mtDNA and mtRNA in HSMCs, most notably in myoblasts. These findings may be of particular importance in developing countries, where ddI is widely used for first-line treatment of HIV-infected children.

Antioxidants and doxorubicin supplementation to modulate CD14 expression and oxidative stress induced by vitamin D3 and seocalcitol in HL60 cells

1alpha,25-dihydroxyvitamin D3 (VD3) and the EB1089 analog are well known for their roles in the modulation of proliferation and the differentiation of several malignant cells. In addition, VD3 or EB1089 displayed a high disposal of oxidant features and the ability to cause release of reactive oxygen species (ROS). We attempted to enhance HL60 cell differentiation and to limit ROS generation, by the association of deltanoids with doxorubicin and the antioxidants catalase (CAT), superoxide dismutase (SOD) and N-acetyl cystein (NAC). Differentiation of HL60 cells into monocytic lineage was studied by expression of mRNA, protein CD14 and functional differentiation by the nitroblue tetrazolium assay. The 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) dye allowed to evaluate in situ ROS generation. When associated with 0.1 nM EB1089, 15 nM doxorubicin induced an increase of differentiated cell percentage from 29% to 87% and did not affect VD3-treated cells. The association with doxorubicin also induced a significant increase of ROS release (p<0.05) versus VD3 and EB1089-treated cells. These results correspond to additivity of individual effects of doxorubicin and deltanoids. Antioxidant agents (10 nM NAC, 50 U/ml SOD or 2000 U/ml CAT) were associated with 10 nM VD3 or 1 nM EB1089 for 72 h. Compared to VD3 and EB1089 treatments, associations with antioxidants induced a slight increase of differentiated cells and a significant increase of CD14 mRNA. The highest differentiation effect occurred in the case of the EB1089-NAC association. Antioxidants induced a decrease (p<0.05) in ROS release generated by VD3 or EB1089 near the level of untreated cells. Thus, antioxidant agents demonstrated a protective effect against VD3 and EB1089 oxidative cytotoxicity and an enhancement of the monocyte differentiation. Combinations of antioxidants with deltanoids could dissociate the oxidative stress and differentiation.

Effects of TNF-α on Leukocyte Adhesion Molecule Expressions in Cultured Human Lymphatic Endothelium

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.

Inflammatory response and genotoxicity of seven wood dusts in the human epithelial cell line A549

Exposure to wood dust is common in many workplaces. Epidemiological studies indicate that occupational exposure to hardwood dusts is more harmful than to softwood dusts. In this study, human epithelial cell line A549 was incubated with well-characterized dusts from six commonly used wood species and from medium density fibreboard (MDF), at concentrations between 10 and 300 μg/ml. After 3 and 6 h of incubation, genotoxicity was assessed by measurement of DNA damage with the single-cell gel electrophoresis (comet) assay and inflammation was measured by the expression of IL-6 and IL-8 mRNA and by the amount of IL-8 protein. There was a 1.2–1.4-fold increase in DNA strand breaks after incubation with beech, teak, pine and MDF dusts compared with the levels in untreated cells, but after 6 h only the increase induced by the MDF dust remained. Increased expression of cellular IL-6 and IL-8 mRNA was induced by all of the wood dusts at both times. Similar to IL-8 mRNA expression, the amounts of secreted IL-8 protein were elevated, except after incubation with oak dust, where a marginal reduction was seen. On the basis of the effects on IL-8 mRNA expression, the wood dusts could be divided into three groups, with teak dust being the most potent, MDF, birch, spruce and pine being intermediate, and beech and oak being the least potent. The induction of DNA strand breaks did not correlate well with the interleukin response. In conclusion, all wood dusts induced cytokine responses, and some dusts induced detectable DNA damage. The inflammatory potency seemed intermediate for dusts from the typical softwoods spruce and pine, whereas the dusts from species linked to cancer, beech and oak, were the least inflammatory. The variation of the effects induced by different wood dusts over time indicates that the DNA damage was not secondary to the cytokine response. Although hardwoods are often considered more harmful than softwoods by regulatory agencies, the current experiments do not provide evidence for a clear-cut distinction between toxicities of hardwood and softwood dust.

Telomerase activity in bleomycin-induced epithelial cell apoptosis and lung fibrosis

Epithelial cell injury and apoptosis are recognised as early features in idiopathic pulmonary fibrosis and bleomycin-induced fibrosis in mice. Telomerase is a known apoptosis-alleviating factor. The role of telomerase was studied during bleomycin-induced lung epithelial cell (LEC) apoptosis in vitro in a mouse LEC line, and in vivo in LECs isolated from bleomycin-treated mice. The current authors evaluated changes in murine telomerase reverse transcriptase (mTERT) mRNA levels and changes in telomerase activity with the TRAPeze Detection Kit, telomeric length with the TeloTTAGGG Telomere Length Kit, and LEC apoptosis with FACScan and 4,6-diamino-2-phenylindole dihydrochloride stain. There was a significant elevation in mTERT mRNA and a transient 41% increase in telomerase activity 24 h after in vitro bleomycin treatment. At 72 h, telomerase activity had fallen to 26% below levels in untreated cells. Reduction of telomerase activity over time, or by direct inhibition, significantly elevated LEC apoptosis. No change in average telomeric length was noted. In vivo, telomerase activity of LECs from bleomycin-treated mice increased at 7 and 14 days. In conclusion, telomerase activity may play a protective role against robust bleomycin-induced lung epithelial cell apoptosis. Moreover, stabilising telomerase activity may decrease epithelial cell apoptosis and the resulting lung fibrosis.

Effects of RNA Interference on CD80 and CD86 Expression in Bone Marrow‐derived Murine Dendritic Cells

To investigate whether RNA interference (RNAi) induced by small interfering RNA (siRNA) could suppress CD80 and CD86 expression in bone marrow-derived murine dendritic cells (DC). The bone marrow-derived DC of mice were separated and cultured in vitro, chemically synthesized siRNA were then transferred into the cells by LipofectAMINE 2000, and the siRNA transfection efficacy was assessed by both fluorescence microscope and flow cytometry. The mRNA expression and protein synthesis were analysed by real-time RT-PCR and flow cytometry. The cell viability of transfected DC was determined by annexin V and propidium iodine staining. Transfection of bone marrow-derived murine DC with a non-silencing FITC-labelled control siRNA demonstrated a high (71.86%) transfection efficiency without affecting cellular viability. CD80-1 siRNA was the most effective siRNA to block CD80 expression in three candidates. Similarly, CD86-3 siRNA was extraordinarily effective in repressing the expression of CD86. Cotransfection of siRNA specific to CD80 and CD86 can enhance gene silencing that is not affected by DC activation-inducing signals. CD80 and CD86 siRNA suppressed the expression of CD80 and CD86 to 31.05 +/- 2.41% and 25.43 +/- 0.85%, respectively, of the level in untreated cells (P < 0.05). siRNA is capable of triggering RNAi in bone marrow-derived DC; it can specifically and effectively knock down CD80 and CD86 gene expression. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for allograft rejection.

Distinct β-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

C5L2 Is a Functional Receptor for Acylation-stimulating Protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

Regulation of hyaluronan synthase-2 expression in human intestinal mesenchymal cells: mechanisms of interleukin-1β-mediated induction

Elevated levels of hyaluronan are associated with numerous inflammatory diseases including inflammatory bowel disease. The purpose of this study was to determine whether a cause and effect relationship might exist among proinflammatory cytokines, IL-1beta, TNF-alpha, IFN-gamma, or transforming growth factor-beta (TGF-beta) and hyaluronan expression in human JDMC and, if so, to identify possible mechanisms involved in the induction of hyaluronan expression. TGF-beta, TNF-alpha, and IFN-gamma had little or no effect on hyaluronan production by these cells. Treatment with IL-1beta induced an approximate 30-fold increase in the levels of hyaluronan in the medium of human jejunum-derived mesenchymal cells. Ribonuclease protection analysis revealed that steady-state transcript levels for hyaluronan synthase (HAS)2 were present at very low levels in untreated cells but increased as much as 18-fold in the presence of IL-1beta. HAS3 transcript levels were also increased slightly by exposure of these cells to IL-1beta. Expression of HAS1 transcripts was not detected under any condition in these cells. IL-1beta induction of hyaluronan expression was inhibited in cells transfected with short interfering RNA corresponding to HAS2 transcripts. Inhibitors of the p38 and ERK1/2 mitogen-activated pathways but not JNK/SAPK blocked the IL-1beta-mediated induction of hyaluronan expression and the increase in HAS2 transcript expression. These results suggest that IL-1beta induction of HAS2 expression involves multiple signaling pathways that act in concert, thus leading to an increase in expression of hyaluronan by jejunum-derived mesenchymal cells.

Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells.

Endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% +/- 4% or 1% +/- 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% +/- 12% or 5% +/- 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% +/- 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.