Sense Oligonucleotides Research Articles

Insulin-like Growth Factor-1 Increases Skeletal Muscle Dihydropyridine Receptor α1S Transcriptional Activity by Acting on the cAMP-response Element-binding Protein Element of the Promoter Region

Previous work from our laboratory has shown that insulin-like growth factor 1 (IGF-1) increases the expression of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1) subunit by regulating DHPR alpha(1S) nuclear transcription. In this study, we investigated the mechanism by which IGF-1 enhances expression of the DHPR alpha(1S) gene. To this end, the promoter region of the mouse DHPR alpha(1S) gene was recently cloned and sequenced and various promoter deletion-luciferase reporter constructs were used. These constructs were transfected into C2C12 cells and IGF-1 effects were measured by recording luciferase activity. IGF-1 significantly enhanced DHPR alpha(1S) transcription in those constructs carrying cAMP-response element-binding protein (CREB) binding site but not in CREB core binding site mutants. Gel mobility shift assay using a double stranded oligonucleotide for the CREB site in the promoter region, and competition experiments with excess unlabeled or mutated promoter oligonucleotide, and unlabeled consensus CREB oligonucleotide demonstrated that IGF-1 induces CREB binding to the DHPR alpha(1S) promoter. IGF-1-mediated enhancement in charge movement was prevented by incubating the cells with antisense but not with sense oligonucleotides against CREB. These results support the conclusion that IGF-1 regulates DHPR alpha(1S) transcription in muscle cells by acting on the CREB element of the promoter.

The type 2 vascular endothelial growth factor receptor recruits insulin receptor substrate-1 in its signalling pathway.

Vascular endothelial growth factor (VEGF) isoforms exert their biological effects through receptors that possess intrinsic tyrosine kinase activity. Whether VEGF binding to its receptors recruits insulin receptor substrate (IRS) family of docking proteins to the receptor is not known. Following incubation of mouse kidney proximal tubular epithelial cells with VEGF, we observed an increase in tyrosine phosphorylation of several proteins, including one of approximately 200 kDa, suggesting possible regulation of phosphorylation of IRS proteins. VEGF augmented tyrosine phosphorylation of IRS-1 in kidney epithelial cells and rat heart endothelial cells in a time-dependent manner. In the epithelial cells, association of IRS-1 with type 2 VEGF receptor was promoted by VEGF. VEGF also increased association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase), and PI 3-kinase activity in IRS-1 immunoprecipitates was increased in VEGF-treated cells. Incubation of epithelial cells with antisense IRS-1 oligonucleotide, but not sense oligonucleotide, reduced expression of the protein and VEGF-induced PI 3-kinase activity in IRS-1 immunoprecipitates. Additionally, VEGF-induced protein synthesis was also impaired by antisense but not sense IRS-1 oligonucleotide. These data provide the first evidence that binding of VEGF to its type 2 receptor promotes association of IRS-1 with the receptor complex. This association may account for some of the increase in VEGF-induced PI 3-kinase activity, and the increase in de novo protein synthesis seen in renal epithelial cells.

Effects of l-arginine-derived nitric oxide synthesis on cardiovascular responses to stimulus-evoked somatosympathetic reflexes in the gracile nucleus

The purpose of these studies was to determine the role of gracile nucleus (Gr) and the effects of l-arginine-derived nitric oxide (NO) synthesis in the nucleus on the cardiovascular responses to somatosympathetic reflexes (SSR). Electrical stimulation of sural and tibial nerve to evoke excitatory and inhibitory SSR was carried out in anesthetized Sprague–Dawley rats. Arterial blood pressure and heart rate were monitored during stimulus-evoked SSR following microinjections of the agents into Gr. Cardiovascular responses to electrical stimulation of the sural and tibial nerves were blocked by microinjection of lidocaine into Gr. The hypertensive and tachycardiac responses to stimulation of the sural nerve were attenuated by bilateral microinjection of l-arginine into Gr, but enhanced by the presence of nNOS antisense oligodeoxynucleotides (oligos) in the area. Microinjection of l-arginine into Gr facilitated the hypotensive and bradycardic responses to stimulation of the tibial nerve while pretreatment with nNOS antisense oligos into Gr attenuated the tibial stimulation evoked inhibitory SSR. The stimulus-evoked responses were not altered by microinjection of nNOS sense oligos into Gr. The results show that the cardiovascular responses to stimulus-evoked SSR were inhibited by the presence of a blockade of neuronal conduction in the Gr. l-Arginine-derived NO synthesis in the Gr attenuates the cardiovascular responses to stimulus-evoked excitatory SSR and facilitates the responses to inhibitory SSR. We conclude that NO in the Gr plays an inhibitory role in the central cardiovascular control through SSR regulation.

Expression of matrix metalloproteinases in cyclosporin-treated gingival fibroblasts is regulated by transforming growth factor (TGF)-beta1 autocrine stimulation.

Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta1 (TGF-beta1). The aim of this study was to investigate the potential role of TGF-beta1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-beta1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-beta1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-beta1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or antisense oligonucleotides (AON). CsA simultaneously stimulated TGF-beta1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-beta1 production as demonstrated by ELISA, whereas TGF-beta1 mRNA expression levels were not significantly modified. The inhibition of TGF-beta1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-beta1 in CsA-treated human gingival fibroblasts. The data presented here suggest that TGF-beta1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extracellular matrix.

Phosphorylation of p85 βPIX, a Rac/Cdc42-specific Guanine Nucleotide Exchange Factor, via the Ras/ERK/PAK2 Pathway Is Required for Basic Fibroblast Growth Factor-induced Neurite Outgrowth

Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.

Deficiency in ClC-3 chloride channels prevents rat aortic smooth muscle cell proliferation.

Recent growing evidence suggests that chloride (Cl-) channels are critical to the cell cycle. In cultured rat aortic vascular smooth muscle cells (VSMCs), we have previously found that Cl- channel blockers inhibit endothelin-1 (ET-1)-induced cell proliferation. The present study was designed to further identify the specific Cl- channels responsible for VSMC proliferation. Due to the lack of a specific blocker or opener of any known Cl- channels, we used the antisense strategy to investigate the potential role of ClC-3, a member of the voltage-gated Cl- channel gene family, in cell proliferation of cultured rat aortic VSMCs. With [3H]-thymidine incorporation and immunoblots, we found that ET-1-induced cell proliferation was parallel to a significant increase in the endogenous expression of ClC-3 protein. Transient transfection of rat aortic VSMCs with antisense oligonucleotide specific to ClC-3 caused an inhibition in ET-1-induced expression of ClC-3 protein and cell proliferation of VSMCs in the same concentration- and time-dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 protein expression and cell proliferation. These results strongly suggest that ClC-3 may be the Cl- channel involved in VSMC proliferation and thus provide compelling molecular evidence linking a specific Cl- channel to cell proliferation. The full text of this article is available at http://www.circresaha.org.

Effect of antisense oligonucleotide to annexin II on the t-PA-mediated plasminogen activation in vitro.

In order to study the role of annexin II, a recombinant expression vector, pZeoSV2(+) ANN II, containing the annexin II cDNA, was developed. The 1.1-kb-length annexin II cDNA was inserted into a expression vector, PZeoSV(+) and transfected into HL-60 cells which had low baseline expression of Ann- II. pZeoSV(+) ANN II was analyzed by restriction mapping and the Ann- II sequence identified. The ability of the transfected cells, non-transfected and mock-transfected cells to stimulate t-PA-depend plasminogen activation was compared. The results showed that HL-60 with pZeoSV(+) ANN II transfection could significantly increase the plasminogen activation (8.9 +/- 1.2 U) in vitro with the difference being significant as compared with non-transfected (1.5 +/- 0.4 U) and mock-transfected cells (4.2 +/- 0.9 U), respectively. Antiannexin II oligonucleotides significantly inhibited the binding ability of t-PA and plasminogen to annexin II, and obviously reduced the plasminogen activation in vitro. The above findings showed human umbilical vein endothelial cells (HUVECs) treated with sense or missense oligonucleotides indicated no significant change in binding of t-PA and PLG. Treatment of HUVECs with antiannexin II oligonucleotides could significantly reduce the plasminogen activation by 2.4 +/- 0.3 U as compared with sense oligonucleotide group in binding of t-PA and PLG. These results, therefore, suggest that Ann- II can bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen, and that interference with Ann-II mRNA by antisense oligonucleotide may be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Surgical injury of rat arteries: genetic control of the remodelling process.

Remodelling and restenosis are complex biological processes responsible for bypass and percutaneous transluminal coronary angioplasty failures which are likely to affect many hundreds of genes. We evaluated the effectiveness of topically applied antisense oligonucleotides in reducing the translation of the messenger RNA for the transcription factor c-myc and in reducing stenosis. Surgery was performed under sterile conditions; 60 Wistar-Kyoto male rats were anaesthetized by ketamine. The carotid arteries were isolated through a median incision in the anterior neck region. At the same point, 0.5 mm longitudinal incisions were performed. Haemostasis was obtained by an adventitial 8.0 stitch. Thirty animals were given 150 microg of c-myc antisense oligonucleotide (Group A) while the other 30 animals received 150 microg of c-myc control sense oligonucleotide (Group B). Oligo molecules were locally applied through 100 microl of 20% pluronic gel. Rats were sacrificed at 30 days; carotid arteries were explanted and stained. Qualitative histological analysis was performed in all cases; serial sections were made every 25 micro in seven consecutive rats for each group. Morphometric analysis was also performed, luminal and medial area values recorded and the ratio between the two areas calculated. Data from each animal were compared with the corresponding contralateral carotid artery and expressed as mean+/-standard deviation. Statistical comparison between the two groups was carried out by one-way ANOVA text. Qualitative histological analysis showed marked remodelling with complete disarray of vessel wall, neointima accumulation and evidence of elastic fibres in the adventitia of all animals of Group B versus Group A. Morphometric analysis showed a significant reduction in the lumen area in Group A animals together with increased values of the medial area versus Group B animals. In addition, the ratio between the lumen and medial area was significantly higher in Group A than in Group B (2.61+/-0.18 versus 1.14+/-0.33, P<0.0001). c-myc antisense oligonucleotides applied intraoperatively can reduce post-operative stenosis.

Coexpression of Glucose Transporter 1 and Matrix Metalloproteinase-2 in Human Cancers

Cancer cells express higher levels of glucose transporter proteins (Gluts) than do normal cells. Glut-1 overexpression is associated with invasiveness. Because matrix metalloproteinase-2 (MMP-2) is also overexpressed in cancer cells and is associated with invasiveness, we tested the hypothesis that Glut-1 may regulate MMP-2 expression. We transiently transfected Glut-1 complementary DNA (cDNA) or Glut-1 antisense oligonucleotides in the human rhabdomyosarcoma cell line RD and analyzed MMP-2 mRNA expression and cell invasiveness. Empty vector and sense oligonucleotides were used for controls. We analyzed MMP-2 promoter activity in transfectants with a luciferase reporter assay and with p53 and Ets-1 gel mobility shift assays. Eight human cancer cell lines and 80 human cancer specimens were analyzed for coexpression of Glut-1 and MMP-2 by western blot and immunohistochemical analyses, respectively. Overexpression of Glut-1 in RD cells increased MMP-2 expression 4.3-fold (95% confidence interval [CI] = 3.7-fold to 4.9-fold) and invasiveness 3.2-fold (95% CI = 2.6-fold to 3.8-fold) relative to control transfected cells. Conversely, suppression of Glut-1 expression by antisense oligonucleotides decreased MMP-2 expression by 71.5% (95% CI = 71.1% to 71.9%) and invasiveness by 53.0% (95% CI = 47.5% to 58.5%). Glut-1-mediated MMP-2 expression involved the binding of the transcription factor p53 but not Ets-1. All eight human cancer cell lines coexpressed Glut-1 and MMP-2 by western blotting, and 45 of 80 human tumor tissues coexpressed Glut-1 and MMP-2 by immunohistochemistry. MMP-2 expression and cell invasiveness are tightly associated with Glut-1 expression in human cancer cell lines. Because suppression of Glut-1 decreased MMP-2 expression and cancer cell invasion, Glut-1 could be a target for therapy of various cancers that overexpress Glut-1.

Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H1.

The structural requirements for DNA/RNA hybrids to be suitable substrates for RNase H1 are well described; however the tolerance level of this enzyme towards modifications that do not alter the duplex conformation is not clearly understood, especially with respect to the sense RNA strand. In order to investigate the molecular requirements of Escherichia coli RNase H1 (termed RNase H1 here) with respect to the sense RNA strand, we synthesized a series of oligonucleotides containing 2'-deoxy-2'-fluoro-beta-D-ribose (2'F-RNA) as a substitute for the natural beta-D-ribose sugars found in RNA. Our results from a series of RNase H1 binding and cleavage studies indicated that 2'F-RNA/DNA hybrids are not substrates of RNase H1 and ultimately led to the conclusion that the 2'-hydroxyl moiety of the RNA strand in a DNA/RNA hybrid is required for both binding and hydrolysis by RNase H1. Through the synthesis of a series of chimeric sense oligonucleotides of mixed RNA and 2'F-RNA composition, the gap requirements of RNase H1 within the sense strand were examined. Results from these studies showed that RNase H1 requires at least five or six natural RNA residues within the sense RNA strand of a hybrid substrate for both binding and hydrolysis. The RNase H1-mediated degradation patterns of these hybrids agree with previous suggestions on the processivity of RNase H1, mainly that the binding site is located 5' to the catalytic site with respect to the sense strand. They also suggest, however, that the binding and catalytic domains of RNase H1 might be closer than has been previously suggested. In addition to the above, physicochemical studies have revealed the thermal stabilities and relative conformations of these modified heteroduplexes under physiological conditions. These findings offer further insights into the physical binding and catalytic properties of the RNase H1-substrate interaction, and have been incorporated into a general model summarizing the mechanism of action of this unique enzyme.

EGFR antisense treatment of human HNSCC cell lines down-regulates VEGF expression and endothelial cell migration

Overexpression of the epidermal growth factor receptor (EGFR) is thought to play a key role in the development of head and neck squamous cell carcinoma (HNSCC) primarily through its effect on promoting uncontrolled cell proliferation. Blocking EGFR ligand binding might also inhibit angiogenesis and down-regulate the production of angiogenic factors. Angiogenesis is increased in various human tumors, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. The vascular endothelial growth factor (VEGF) is thought to be the most important angiogenic factor. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human EGFR mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell migration was measured using a modified Boyden chamber. EGFR antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense oligonucleotide control. Addition of conditioned medium from EGFR antisense-treated tumor cells resulted in decreased endothelial cell migration. In conclusion, therapeutic strategies targeting EGFR signaling in head and neck cancer might have an antitumor effect mediated in part by inhibition of tumor angiogenesis.

Functions of epidermal growth factor-like growth factors during human urothelial reepithelialization in vitro and the role of erbB2.

Transitional epithelium of the urinary bladder can be damaged during, for example, catheterization, overstretching due to obstructed voiding, or partial resection. The subsequent repair process can be stimulated by specific proteins such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha). However, little is known about the role of EGF-like growth factors and their respective receptors in human urothelial repair. In this study, we examined the effects of EGF, TGFalpha, amphiregulin and heregulin-alpha (HRGalpha) on proliferation, wound closure, and the expression of their receptors c-erbB1-c-erbB4 in primary cultures of human urothelial cells in vitro. Under conditions representing intact urothelium, all EGF-like growth factors except HRGalpha induced proliferation. TGFalpha induced proliferation up to four times. Amphiregulin increased expression of c-erbB1. Treatment with either TGFalpha or amphiregulin resulted in higher c-erbB1 activation and c-erbB3 levels. None of the growth factors affected the constitutive expression of c-erbB2 and c-erbB4. In the repair model, both EGF and TGFalpha stimulated the wound closure most strongly. This was mainly achieved by increased cellular migration. Receptor expression was not affected by the addition of exogenous growth factor. The role of c-erbB2 in wound healing was further investigated with the use of antisense DNA. Wound closure could be delayed up to 50% by antisense c-erbB2 but not by mismatched or sense oligonucleotides. Excessive production (e.g. in bladder tumors) or application of EGF, TGFalpha or amphiregulin, but not HRGalpha may lead to either hyperplasia or a faster repair of damaged urothelium in vivo. These effects seem to be mediated not only via c-erbB1 but also via c-erbB2. Our results suggest that modified members of the EGF-EGFR family are potential targets for future therapies for bladder wound healing and malignancy.

Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product.

Malignant pleural mesothelioma is resistant to conventional therapies and to apoptosis. The bcl-2 family genes are major determinants of apoptotic homeostasis. Malignant pleural mesothelioma lines and tumors rarely express the antiapoptotic Bcl-2 protein but routinely express the antiapoptotic protein Bcl-xl and the proapoptotic proteins Bax and Bak. We have previously shown pharmacologic inhibition of bcl-xl expression in malignant pleural mesothelioma can lead to apoptosis, so we sought to determine whether antisense oligonucleotides directed at bcl-xl messenger RNA would engender apoptosis, possibly through a "forced imbalance" of bcl-2 family proteins. Malignant pleural mesothelioma lines REN (epithelial) and I-45 (sarcomatous) were exposed to modified bcl-xl antissense oligonecleotides directed near the messenger RNA initiation sequence with and without a liposomal delivery system. Untreated cells and bcl-xl sense oligonucleotides were controls. Cell viability was measured by colorimetric assay, and apoptosis was evaluated with Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis. Bcl-xl protein expression after antisense oligonucleotides was downwardly regulated in both cell lines relative to sense oligonucleotides (>65%). Significant cellular killing in both the I-45 and REN cell lines was achieved with antisense oligonucleotides (compared with sense oligonucleotides) without (P =.003 and.006, respectively) and with (P =.006 and.0005, respectively) liposomal delivery. Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis demonstrated apoptosis to be the mechanism of cellular death. Use of a liposomal delivery system increased therapeutic effect and allowed lower doses of antisense oligonucleotides. Antisense oligonucleotides directed at the bcl-xl gene product engender apoptosis in mesothelioma cell lines. The therapeutic potential of inhibiting expression of this protein in mesothelioma should be evaluated.

An NAD(P)H oxidase regulates growth and transcription in melanoma cells.

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappaB activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22(phox) and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappaB and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth.

Glucose transporter 8 expression and translocation are critical for murine blastocyst survival.

Glucose transporter (GLUT) 8 is an insulin-responsive facilitative glucose transporter expressed predominantly in the murine blastocyst. To determine the physiologic role of GLUT8, two-cell embryos were cultured to a blastocyst stage in antisense or sense oligonucleotides to GLUT8. Apoptosis was assessed using the TUNEL techniques and recorded as the percentage of TUNEL-positive nuclei/total nuclei. Embryos cultured in GLUT8 antisense experienced increased TUNEL-positive nuclei, whereas sense embryos did not. Embryos cultured in a control AS oligonucleotide, specific for heat shock protein 70-2, showed a rate of apoptosis similar to sense. To determine the outcome of these apoptotic embryos, blastocysts exposed to sense vs. antisense were transferred back into foster mice and the pregnancy continued until Day 14.5, at which time the uteri were examined for normal gestational sacs and resorptions. Embryos exposed to GLUT8 antisense experienced higher rates of resorptions and lower normal pregnancy rates compared to embryos cultured in GLUT8 sense. To examine the insulin growth factor (IGF)-1/insulin intracellular signaling pathways involved in GLUT8 translocation, IGF-1 receptor (IGF-1R) expression was decreased in the blastocysts with antisense oligonucleotides. Using confocal immunofluorescent microscopy, GLUT8 translocation in response to insulin was observed. Exposure to insulin in the embryos exposed to IGF-1R sense induced translocation of GLUT8 from intracellular compartments to the plasma membrane. Blastocysts exposed to IGF-1R antisense, however, failed to demonstrate any change in the intracellular location of GLUT8 with insulin treatment. The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared to sense embryos. These data suggest that GLUT8 expression and translocation in response to insulin are critical for blastocyst survival.

Differentiation of functional dendritic cells and macrophages from human peripheral blood monocyte precursors is dependent on expression of p21 (WAF1/CIP1) and requires iron.

Iron is required for monocyte/macrophage differentiation of HL-60 leukaemia cells. Differentiation requires induction of the cyclin-dependent kinase inhibitor p21 (WAF1/CIP1), and cell cycle arrest at the G1/S checkpoint. With iron depletion, p21 induction and differentiation are blocked. To establish the roles of iron and p21 in normal monocyte/macrophage differentiation, we examined generation of dendritic cells (DCs) and macrophages from peripheral monocytes. Monocytes were cultured with interleukin 4 and granulocyte-macrophage colony-stimulating factor (GM-CSF), then treated with lipopolysaccharide to produce DCs or with M-CSF to produce macrophages. Iron deprivation was induced by desferrioxamine (DF). Monocyte-derived DCs had characteristic phenotype and morphology, and stimulated proliferation of naïve allogeneic T lymphocytes. In contrast, DCs generated under iron deprivation were phenotypically undifferentiated and did not stimulate T cells. Similarly, macrophages expressed a characteristic phenotype and morphology, and phagocytosed latex beads, but macrophages generated under iron deprivation failed to develop a mature phenotype and had impaired phagocytosis. Iron deprivation blocked induction of p21 (WAF1/CIP1) expression in both DC and macrophage cultures. Furthermore, p21 antisense oligonucleotides, but not sense oligonucleotides, inhibited both DC and macrophage differentiation. These data indicate that a key role of iron in haematopoiesis is to support induction of p21 which, in turn, is required for DC and macrophage differentiation.

P2X4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X(4) receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X(4)R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X(4)R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5'-(alpha,beta-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5' region of cP2X(4)R abrogated the P2 agonist-stimulated (45)Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X(4) receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X(4) receptor, antibody against cP2X(4)R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X(4) receptor. The nonglycosylated 44-kDa P2X(4) receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X(4)R showed that only the glycosylated 58-kDa P2X(4) receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X(4) receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.

Is GHRH receptor essential to GHRP-2-induced GH secretion in primary cultured rat pituitary cells?

It is still controversial in rat whether the stimulation of GH secretion by GH-releasing peptides (GHRP) requires both GHRP receptor (GHRP-R) and GH-releasing hormone receptor (GHRH-R). To clarify this issue, we have postulated that inhibition of GHS-R or GHRH-R gene transcription should block GHRP-2-induced GH secretion. Rat pituitary cells were incubated for 3 days in the presence or absence of antisense 18-mer phosphorothiate oligonucleotides (ONs) complementary to the codon region of GHS-R or GHRH-R mRNAs. A significant decrease in GHRH-R and GHS-R mRNA levels was found in corresponding antisense-treated cells compared with the control cells treated with sense ON. Treatment with antisense GHS-R ON reduced (but not abolished) GHRP-2-induced GH secretion although GHRH-induced GH secretion was not altered. GHRH-stimulated GH secretion was totally abolished by the treatment with antisense GHRH-R ON, whereas GHRP-2 induced GH secretion was not affected. Treatment of cells with both GHS-R and GHRH-2 ONs however completely inhibited GHRH and GHRP-2-stimulated GH secretion. These results suggest that GHRH-R is vital for GHRH-induced GH secretion but only partially involved in GHRP-2-stimulated GH secretion under the condition of down-regulation of GHS-R gene transcription.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.