Significant Increase In mRNA Research Articles

Homeobox gene Distal-less 3 (DLX3) is a regulator of villous cytotrophoblast differentiation

Dlx3, a member of the large homeobox gene family of transcription factors, is important for murine placental development. Targeted deletion of Dlx3 in the mouse results in embryonic death due to placental failure. This study investigated the role of human DLX3 in villous cytotrophoblast (VCT) differentiation in the placenta. Primary VCT from human term placentae, which spontaneously differentiate when maintained in culture over 72 h, showed a significant increase in mRNA and protein expression of DLX3 and 3βHSD. The functional role of DLX3 was determined using trophoblast derived-cell line, BeWo. Forskolin treated BeWo cells showed significantly increased DLX3 mRNA and protein expression. Forskolin stimulation also showed a significant increase in syncytin and 3βHSD mRNA expression, and increased release of βhCG into the cell culture supernatant. To determine whether DLX3 had a direct or indirect effect on VCT differentiation, mRNA and protein expression of DLX3 was increased using a plasmid DLX3 over-expression construct. Over-expression of DLX3 resulted in increased mRNA expression of 3βHSD and syncytin, as well as increased secretion of β-hCG protein in the cell culture medium. In conclusion, we provide evidence that DLX3 acts upstream of syncytin, 3βHSD and βhCG and that DLX3 has a regulatory role in VCT differentiation.

Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine β-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Physiological and molecular endocrine changes in maturing wild sockeye salmon, Oncorhynchus nerka, during ocean and river migration

Maturing adult sockeye salmon Oncorhynchus nerka were intercepted while migrating in the ocean and upstream in freshwater over a combined distance of more than 1,300 km to determine physiological and endocrine changes associated with ionoregulation. Sockeye migrating through seawater and freshwater showed consistent declines in gill Na+/K+ -ATPase (NKA) activity, plasma osmolality and plasma chloride concentration. In contrast, plasma sodium concentration became elevated in seawater as fish approached the river mouth and was then restored after sockeye entered the river. Accompanying the movement from seawater to freshwater was a significant increase in mRNA for the NKA α1a subunit in the gill, with little change in the α1b subunit. Potential endocrine signals stimulating the physiological changes during migration were assessed by measuring plasma cortisol and prolactin (Prl) concentrations and quantifying mRNA extracted from the gill for glucocorticoid receptors 1 and 2 (GR1 and GR2), mineralocorticoid receptor (MR), growth hormone 1 receptor (GH1R), and prolactin receptor (PrlR). Plasma cortisol and prolactin concentrations were high in seawater suggesting a preparatory endocrine signal before freshwater entry. Generally, the mRNA expression for GR1, GR2 and MR declined during migration, most notably after fish entered freshwater. In contrast, PrlR mRNA increased throughout migration, particularly as sockeye approached the spawning grounds. A highly significant association existed between gill PrlR mRNA and gill NKA α1a mRNA. GH1R mRNA also increased significantly, but only after sockeye had migrated beyond tidal influence in the river and then again just before the fish reached the spawning grounds. These findings suggest that cortisol and prolactin stimulate ionoregulation in the gill as sockeye salmon adapt to freshwater.

Ginkgolide B attenuates ethanol-induced neurotoxicity through regulating NADPH oxidases

Ethanol has long been demonstrated to trigger cell apoptosis in the central nervous system. The over-production of reactive oxygen species (ROS) is considered as one of the most important mechanisms involving in the apoptosis caused by ethanol. Ginkgolide B (GB), which was widely used as a monomer of traditional Chinese medicine, was reported to scavenge free radicals in endothelial cells and smooth muscle cells. But whether GB can prevent ethanol-induced neurotoxicity is still unknown. The aim of this study was to investigate effects of GB on ethanol-induced cytotoxicity, oxidative stress and apoptosis and explore potential protective molecular mechanism of GB. It was found that GB inhibited cell injury and apoptosis in a dose-dependent manner in ethanol-treated PC12 cells by MTT and LDH assays. It was also found that activities of caspase-3 increased by ethanol were mostly abrogated by GB. Further, GB decreased the production of ROS and subsequent over-production of lipid peroxides. A significant increase of alcohol dehydrogenase (ADH) and CYP2E1 enzyme activity was found in the ethanol-exposed PC12 cells as compared to controls. However, GB pretreatment did not significantly affect ethanol-induced ADH and CYP2E1 activities. Quantitative real-time PCR and Western blot analysis demonstrated that ethanol treatment resulted in a significant increase in mRNA and protein expression of NADPH oxidases, which are main oxidases producing ROS in neurons. Moreover, expression and activities of NADPH oxidases were down-regulated by GB. These results indicate that ethanol-induced neurotoxicity is ameliorated by GB mainly through regulating expression and activity of NADPH oxidases.

Infection with Influenza Virus Induces IL-33 in Murine Lungs

IL-33, a novel IL-1 family member, is crucially expressed and involved in pulmonary diseases, but its regulation in viral diseases such as influenza A virus (IAV) remains unclear. This study aimed to characterize the expression and release of IL-33 in lungs of IAV-infected mice in vivo and in murine respiratory epithelial cells (MLE-15) in vitro. Our results provide evidence of up-regulation of IL-33 mRNA in IAV-infected murine lungs, compared with noninfected control mice. The overexpression of IL-33 was positively correlated with a significant increase in mRNA encoding the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6, and was also associated with an increase in IFN-β mRNA. A profound overexpression of IL-33 protein was evident in IAV-infected murine lungs and bronchoalveolar lavages of influenza-infected mice, compared with low concentrations in naive lungs in vivo. Immunolocalization highlighted the cellular expression of IL-33 in alveolar epithelial and endothelial cells, along with increased infiltrate cells in virus-infected lungs. Further in vitro experiments showed an induction of IL-33 transcript-in MLE-15 cells and human epithelial cells (A549) infected with different strains of IAV in comparison with noninfected cells. In conclusion, our findings evidenced a profound expression of IL-33 in lungs during both in vivo and in vitro IAV infections, suggesting a role for IL-33 in virus-induced lung infections.

Toll‐like receptors expressed by dermal fibroblasts contribute to hypertrophic scarring

Hypertrophic scar (HTS), a fibroproliferative disorder (FPD), complicates burn wound healing. Although the pathogenesis is not understood, prolonged inflammation is a known contributing factor. Emerging evidence suggests that fibroblasts regulate immune/inflammatory responses through toll-like receptor 4 (TLR4) activated by lipopolysaccharide (LPS) through adaptor molecules, leading to nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases activation, cytokine gene transcription and co-stimulatory molecule expression resulting in inflammation. This study explored the possible role of TLR4 in HTS formation. Paired normal and HTS tissue from burn patients was collected and dermal fibroblasts isolated and cultured. Immunohistochemical analysis of tissues demonstrated increased TLR4 staining in HTS tissue. Quantitative RT-PCR of three pairs of fibroblasts demonstrated mRNA levels for TLR4 and its legend myeloid differentiation factor 88 (MyD88) in HTS fibroblasts were increased significantly compared with normal fibroblasts. Flow cytometry showed increased TLR4 expression in HTS fibroblasts compared with normal. ELISA demonstrated protein levels for prostaglandin E2, interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in HTS fibroblasts compared to normal. When paired normal and HTS fibroblasts were stimulated with LPS, significant increases in mRNA and protein levels for MyD88, IL-6, IL-8, and MCP-1 were detected. However, when transfected with MyD88 small interfering RNA (siRNA), then stimulated with LPS, a significant decrease in mRNA and protein levels for these molecules compared to only LPS-stimulated fibroblasts was detected. In comparison, a scramble siRNA transfection did not affect mRNA or protein levels for these molecules. Results demonstrate LPS stimulates proinflammatory cytokine expression in dermal fibroblasts and MyD88 siRNA eliminates the expression. Therefore, controlling inflammation and manipulating TLR signaling in skin cells may result in novel treatment strategies for HTS and other FPD.

IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT

Event Abstract Back to Event IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT Catherine Propper1*, Brian Searcy1, Angela Schwendiman1, Claire Ramirez1, Michael Owen1, Nik Veldhoen2 and Caren Helbing2 1 Northern Arizona University, Biological Sciences, United States 2 University of Victoria, Biochemistry and Microbiology, Canada Anthropogenic chemicals impact development, reproduction and behavior in wildlife and laboratory animals. Organisms living downstream from wastewater effluent (WWE) outlets are exposed to complex mixes of such compounds, and some exposed organisms show disrupted developmental processes. Using Xenopus laevis as a model, we tested the hypothesis that exposure to WWE decreased time to metamorphosis. Tadpoles were exposed from NF-stage 47 through the completion of metamorphosis to either artificial pond water, RO water balanced to equal salts in local WWE, or WWE. Animals exposed to WWE underwent metamorphosis sooner than either control group. Since thyroid hormone directly controls metamorphosis, our results suggest the WWE had thyroid hormone-like activity. To test this hypothesis, we used X. laevis whole tail cultures to determine whether WWE was impacting expression of four genes sensitive to thyroid hormone signaling: thyroid hormone receptor beta (TRbeta), deiodinase II (D2), corticotropin releasing hormone binding protein (CRHBP), and fibroblast activation protein alpha (FAPalpha). Tails were incubated for 48 hr to 0 or 10nM thyroid hormone (T3) and either culture media alone or extracts of WWE reconstituted in culture media to 1X or 5X the original WWE concentration. T3 induced a significant increase in mRNA in TRbeta, CRHBP, and FAPalpha, but not D2. Wastewater effluent alone induced a significant increase in FAPalpha (at both 1X and 5X) and CRHBP (at 1X only) expression, but not expression of TRbeta or D2. When tails were exposed to both T3 and WWE, all genes showed large, significant synergistic increases in expression compared to tails exposed to T3 or WWE alone. Our results suggest the decreased time to metamorphosis found in vivo is not solely a direct result of WWE compounds binding to TH receptor, but that some of the compounds have the potential to affect thyroid hormone signaling pathways upstream and/or downstream of receptor activation. Acknowledgements Acknowledgement: This work was supported by Arizona State TRIF funds to CRP. Keywords: Effluent, endocrine disruption, Gene Expression, Thyroid hormone, wastewater, Xenopus Conference: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology, Ann Arbor, United States, 11 Jul - 13 Jul, 2011. Presentation Type: Invited Symposium Topic: Endocrine disruption Citation: Propper C, Searcy B, Schwendiman A, Ramirez C, Owen M, Veldhoen N and Helbing C (2011). IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT. Front. Endocrinol. Conference Abstract: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology. doi: 10.3389/conf.fendo.2011.03.00038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Catherine Propper, Northern Arizona University, Biological Sciences, Flagstaff, AZ, 86011, United States, Catherine.Propper@nau.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Google Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Google Scholar Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing PubMed Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

SSRI augmentation of antipsychotic alters expression of GABAA receptor and related genes in PMC of schizophrenia patients

Clinical studies have shown that negative symptoms of schizophrenia unresponsive to antipsychotic given alone can improve after augmentation with SSRI antidepressant. Laboratory investigations into the mechanism of this synergism showed that co-administration of SSRI and antipsychotic produces changes in GABA(A) receptor and related systems, which differ from the effects of each drug alone. To examine the clinical relevance of these findings, the current study examined the effects of SSRI augmentation treatment on GABA(A) receptor and related systems in schizophrenia patients. Schizophrenia patients with high levels of negative symptoms unresponsive to antipsychotic treatment received add-on fluvoxamine (100 mg/d). Blood was taken before and 1, 3 and 6 wk after adding fluvoxamine and peripheral mononuclear cells (PMC) isolated. RNA encoding for GABA(A)β3, 5-HT2A, and 5-HT7 receptors, PKCβ2, and brain-derived neurotrophic factor (BDNF) was assayed with real-time RT-PCR. Plasma BDNF protein was assayed using ELISA. Clinical symptoms were assessed with validated rating scales. We found significant increase in mRNA encoding for GABA(A)β3 and 5-HT2A, 5-HT7 receptors and BDNF and a reduction in PKCβ2 mRNA. Plasma BDNF protein concentrations were increased. There were significant correlations among the genes. Clinical symptoms improved significantly. mRNA expression of PKCβ2, 5-HT2A and 5-HT7 showed significant associations with clinical symptoms. Combined SSRI+antipsychotic treatment is associated with changes in GABA(A) receptor and in related signalling systems in patients. These changes may be part of the mechanism of clinically effective drug action and may prove to be biomarkers of pharmacological response.

Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion

Studies have shown that the superoxide mechanism is involved in angiotensin II (ANG II) signaling in the central nervous system. We hypothesized that ANG II activates sympathetic outflow by stimulation of superoxide anion in the paraventricular nucleus (PVN) of streptozotocin (STZ)-induced diabetic rats. In α-chloralose- and urethane-anesthetized rats, microinjection of ANG II into the PVN (50, 100, and 200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP), and heart rate (HR) in control and STZ-induced diabetic rats. There was a potentiation of the increase in RSNA (35.0 ± 5.0 vs. 23.0 ± 4.3%, P < 0.05), AP, and HR due to ANG II type I (AT(1)) receptor activation in diabetic rats compared with control rats. Blocking endogenous AT(1) receptors within the PVN with AT(1) receptor antagonist losartan produced significantly greater decreases in RSNA, AP, and HR in diabetic rats compared with control rats. Concomitantly, there were significant increases in mRNA and protein expression of AT(1) receptor with increased superoxide levels and expression of NAD(P)H oxidase subunits p22(phox), p47(phox), and p67(phox) in the PVN of rats with diabetes. Pretreatment with losartan (10 mg·kg(-1)·day(-1) in drinking water for 3 wk) significantly reduced protein expression of NAD(P)H oxidase subunits (p22(phox) and p47(phox)) in the PVN of diabetic rats. Pretreatment with adenoviral vector-mediated overexpression of human cytoplasmic superoxide dismutase (AdCuZnSOD) within the PVN attenuated the increased central responses to ANG II in diabetes (RSNA: 20.4 ± 0.7 vs. 27.7 ± 2.1%, n = 6, P < 0.05). These data support the concept that superoxide anion contributes to an enhanced ANG II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.

PAW19 Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

BackgroundIncrease in intraneuronal iron is thought to be relevant to the pathogenesis of Parkinson's disease (PD), although the mechanism remains elusive. Here we investigate expression of the iron importers DMT1...

PRECLINICAL STUDY: BRIEF REPORT: Epigenetic modulation at the CCR2 gene correlates with the maintenance of behavioral sensitization to methamphetamine

The intermittent administration of methamphetamine produces behavioral sensitization to methamphetamine. In the limbic forebrain, mainly including the nucleus accumbens, of mice that had been intermittently treated with methamphetamine, we found a significant increase in mRNA of a chemokine, CCR2. This increase was accompanied by a significant increase in histone H3 lysine 4 (H3K4) trimethylation at its promoter. Interestingly, the maintenance of sensitization to methamphetamine-induced hyperlocomotion was significantly decreased in CCR2 knockout mice. These findings suggest that increased CCR2 associated with epigenetic modification after the intermittent administration of methamphetamine may be associated with the maintenance of sensitization to methamphetamine-induced hyperlocomotion.

Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 mug/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli(2), VEGF-A, Notch 1, and Hrts. Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

The Mandibular Cartilage Metabolism is Altered by Damaged Subchondral Bone from Traumatic Impact Loading

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease caused by excessive external loading. Recently, it was reported that the damage in the mineralized subchondral bone caused by traumatic impact-loading is responsible for the initiation and progression of cartilage degeneration. Thus far, we have hypothesized that cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1beta immunohistochemically and prominently up-regulated IL-1alpha and IL-1beta mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1beta in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1beta, -6, and TNF-alpha. Among them, osteoblasts pre-treated by IL-1beta affected chondrocytes most strongly. It was also shown that IL-1beta-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1beta synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA.

Selective Sensitization of Transduction pathways in Carotid Body Glomus Cells of Spontaneously Hypertensive Rats (SHR)

We have reported that the non‐voltage gated Acid‐Sensing Ion (Na+) Channels (ASICs) and the Tandem pore‐domain Acid‐Sensitive K+ channels (TASK) are low pH sensors in isolated rat glomus cells causing an initial rapid depolarization, and a sustained (Sust.) depolarization, respectively (Circ. Res. 2007; 101:1009‐19). TASK also mediate glomus cells' responses to hypoxia and to the mitochondrial inhibitor cyanide (Cn) (J Physiol. 2004; 556:175‐91).In the present study we tested whether hypersensitivity to low pH (6.0), which we had noted in glomus cells of young (4‐6 weeks) prehypertensive SHR (Hypertension, 2008; see table below), would also be evident with Cn‐induced mitochondrial inhibition. Our hypothesis was that the responses would be equally enhanced with Cn since both low pH and Cn inhibit TASK channels.Using real‐time PCR and western blot analyses we found significant increases in mRNA and protein expression of both ASIC3 and TASK1 in carotid bodies of SHR compared to WKY. However, the enhanced depolarizations (ΔmV) seen at low pH (6.0) in SHR compared to WKY (*P&lt;0.05) were not seen with Cn (2 mM).We conclude that the non‐voltage gated Na+ (ASICs) and K+ (TASK) channels are significantly more sensitive to low pH than to inhibition of mitochondrial electron transport in SHR. The possibility of alternative transduction pathways for low pH vs. hypoxia or Cn remains to be determined.

Effect of 7-nitroindazole on the expression of intracellular calcium channels in the kidney of spontaneously hypertensive rats

The spontaneously hypertensive rats (SHR) were fed with nitric oxide synthase (NOS) blocker 7-nitroindazole (7-NI, 10 mg/kg/day) for 6 weeks and an expression of intracellular calcium channels, SERCA and proapoptotic agents was evaluated in kidney. Treatment of rats with 7-NI resulted in a significant increase in mRNA and protein levels of the IP3 receptors type 1 and type 2, while mRNA levels of the IP3 receptor type 3 remained unchanged. The mRNA of other intracellular calcium channels, ryanodine receptors type 1 and type 2 was also upregulated by 7-NI treatment. Gene expression of the SERCA2a, calcium pump responsible for loading intracellular stores with calcium, revealed increased gene expression due to 7-NI as well. Interestingly, proapoptotic agents caspase 3 and Bax were also upregulated by the 7-NI treatment. These results may indicate that nNOS blocker 7-NI modifies intracellular calcium transport system, which may have impact on altered calcium handling and regulation of various metabolic pathways.

Involvement of Hypoxia-inducing Factor-1α-dependent Plasminogen Activator Inhibitor-1 Up-regulation in Cyr61/CCN1-induced Gastric Cancer Cell Invasion

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.

Lentiviral Transduction of ApoAI Into Hematopoietic Progenitor Cells and Macrophages

We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was >25% for HPCs and >70% for macrophages. ApoAI was found in the macrophage culture media, mostly associated with the HDL fraction. Interestingly, a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls, without influencing plasma HDL-C levels. Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression, exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque.

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after subarachnoid hemorrhage (SAH) had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross-sections of basilar arteries were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) and 4′,6-diamidino-2-phenylindole (DAPI). Positively stained cells were counted and numbers obtained were normalized to the cross-sectional area. The results were compared to normal dog basilar arteries contracted pharmacologically in vitro. SAH resulted in significant vasospasm ( P < 0.001 for each, paired t-tests). There were significant increases in mRNA for β-actin (441%, P = 0.01), glyceraldehyde-3-phosphate dehydrogenase (566%, P = 0.007) and 18S ribosomal RNA (320%, P = 0.025) 7 days after SAH. Total mRNA was increased 7 days after SAH relative to genomic DNA (157%, P = 0.009). There were significant increases in the number of cells in the tunica media and adventitia of arteries after SAH and a significant decrease in the media after contraction in vitro. Cells in the tunica media and adventitia labeled with PCNA were significantly increased at both times after SAH. Transcripts for housekeeping genes are increased after SAH, making standardization to them potentially invalid. The increase is due to proliferation of cells in the adventitia and increased total mRNA in the media and adventitia.

Intracellular localization and subsequent redistribution of metal transporters in a rat choroid plexus model following exposure to manganese or iron

Confocal microscopy was used to investigate the effects of manganese (Mn) and iron (Fe) exposure on the subcellular distribution of metal transporting proteins, i.e., divalent metal transporter 1 (DMT1), metal transporter protein 1 (MTP1), and transferrin receptor (TfR), in the rat intact choroid plexus which comprises the blood–cerebrospinal fluid barrier. In control tissue, DMT1 was concentrated below the apical epithelial membrane, MTP1 was diffuse within the cytosol, and TfR was distributed in vesicles around nuclei. Following Mn or Fe treatment (1 and 10 μM), the distribution of DMT1 was not affected. However, MTP1 and TfR moved markedly toward the apical pole of the cells. These shifts were abolished when microtubules were disrupted. Quantitative RT-PCR and Western blot analyses revealed a significant increase in mRNA and protein levels of TfR but not DMT1 and MTP1 after Mn exposure. These results suggest that early events in the tissue response to Mn or Fe exposure involve microtubule-dependent, intracellular trafficking of MTP1 and TfR. The intracellular trafficking of metal transporters in the choroid plexus following Mn exposure may partially contribute to Mn-induced disruption in Fe homeostasis in the cerebrospinal fluid (CSF) following Mn exposure.

Inflammation-induced up-regulation of ionotropic glutamate receptor expression in human skin

N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid (AMPA), and kainate (KA) receptors are members of the ionotropic glutamate receptor (iGluR) family and are increased in inflamed rat skin. These receptors contribute to inflammatory pain. In this study, we have examined whether there is a similar increase in iGluRs in inflamed human skin in the presence of inflammatory pain. Normal and inflamed-skin biopsies were obtained from eight patients undergoing elective wound-debridement surgery. Real-time-polymerase chain reaction (PCR) and western blot analysis were used for quantitation of iGluR mRNA and protein in normal and inflamed human skin. A significant increase in mRNA and protein for NMDA, AMPA, and KA receptor subunits was detected in inflamed compared with normal skin. The amounts of NMDA (NR1 subunit), AMPA (GluR2 subunit), and KA (GluR6 subunit) mRNA in inflamed skin were mean 6 (SD 1.6-fold), 2.5 (0.6-fold), and 3.8 (0.9-fold) (P<0.05), respectively, greater than that measured in normal skin. The ratio of NR1, GluR2, and GluR6 protein in inflamed compared with normal skin was 5.7 (1.2), 2.4 (0.5), and 3.6 (0.9) (P<0.05), respectively. These results, in human tissue, demonstrate that iGluR mRNA and protein expression are increased during persistent inflammation and that this increased activity may be involved in mediating clinical inflammatory pain in human skin.