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Express High Levels (express + high_level)
Selected AbstractsHuman cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2003Lubing Zhou Abstract We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E,+,6 molecules/,g total RNA) relative to that in the kidney (3E,+,4 molecules/,g total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E,+,5 molecules/,g total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E,+,4 molecules/,g total RNA (in skeletal muscle) to 3.2E,+,6 molecules/,g total RNA (in kidney), levels 10,100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart. J. Cell. Biochem. 90: 339,346, 2003. © 2003 Wiley-Liss, Inc. [source] Divergent roles of the DEAD-box protein BS-PL10, the urochordate homologue of human DDX3 and DDX3Y proteins, in colony astogeny and ontogenyDEVELOPMENTAL DYNAMICS, Issue 6 2006Amalia Rosner Abstract Proteins of the highly conserved PL-10 (Ded1P) subfamily of DEAD-box family, participate in a wide variety of biological functions. However, the entire spectrum of their functions in both vertebrates and invertebrates is still unknown. Here, we isolated the Botryllus schlosseri (Urochordata) homologue, BS-PL10, revealing its distributions and functions in ontogeny and colony astogeny. In botryllid ascidians, the colony grows by increasing the number of modular units (each called a zooid) through a whole colony synchronized and weekly cyclical astogenic budding process (blastogenesis). At the level of the colony, both BS-PL10 mRNA and its protein (78 kDa) fluctuate in a weekly pattern that corresponds with the animal's blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D. At the organ/module level, a sharp decline is revealed. Primary and secondary developing buds express high levels of BS-PL10 mRNA and protein at all blastogeneic stages. These levels are reduced four to nine times in the new set of functional zooids. This portrait of colony astogeny differed from its ontogeny. Oocytes and sperm cells express high levels of BS-PL10 protein only at early stages of development. Young embryos reveal background levels with increased expressions in some organs at more developed stages. Results reveal that higher levels of BS-PL10 mRNA and protein are characteristic to multipotent soma and germ cells, but patterns deviate between two populations of differentiating stem cells, the stem cells involved in weekly blastogenesis and stem cells involved in embryogenesis. Two types of experimental manipulations, zooidectomy and siRNA assays, have confirmed the importance of BS-PL10 for cell differentiation and organogenesis. BS-PL10 (phylogenetically matching the animal's position in the evolutionary tree), is the only member of this subfamily in B. schlosseri, featuring a wide range of biological activities, some of which represent pivotal roles. The surprising weekly cyclical expression and the participation in cell differentiation posit this molecule as a model system for studying PL10 protein subfamily. Developmental Dynamics 235:1508,1521, 2006. © 2006 Wiley-Liss, Inc. [source] Pulmonary stromal cells induce the generation of regulatory DC attenuating T-cell-mediated lung inflammationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2008Qian Li Abstract The tissue microenvironment may affect the development and function of immune cells such as DC. Whether and how the pulmonary stromal microenvironment can affect the development and function of lung DC need to be investigated. Regulatory DC (DCreg) can regulate T-cell response. We wondered whether such regulatory DC exist in the lung and what is the effect of the pulmonary stromal microenvironment on the generation of DCreg. Here we demonstrate that murine pulmonary stromal cells can drive immature DC, which are regarded as being widely distributed in the lung, to proliferate and differentiate into a distinct subset of DCreg, which express high levels of CD11b but low levels of MHC class II (I-A), CD11c, secrete high amounts of IL-10, NO and prostaglandin E2 (PGE2) and suppress T-cell proliferation. The natural counterpart of DCreg in the lung with similar phenotype and regulatory function has been identified. Pulmonary stroma-derived TGF-, is responsible for the differentiation of immature DC to DCreg, and DCreg-derived PGE2 contributes to their suppression of T-cell proliferation. Moreover, DCreg can induce the generation of CD4+CD25+Foxp3+ Treg. Importantly, infusion with DCreg attenuates T-cell-mediated eosinophilic airway inflammation in vivo. Therefore, the pulmonary microenvironment may drive the generation of DCreg, thus contributing to the maintenance of immune homoeostasis and the control of inflammation in the lung. [source] Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec-7-dependent and -independent mechanismsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2003Gavin Nicoll Abstract Siglec-7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for ,2,8-linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec-7-GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec-7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase-transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec-7-Fc protein. Surprisingly, GD3 synthase-transfected P815 cells were killed more effectively by untreated cells in a siglec-7-independent manner. However, following sialidase treatment of NK cells, a siglec-7-dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside. [source] Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studiesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2003Dan Chen Abstract Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DR,, DR,1, DR,3, DQ,, and DQ, regions. The transgenic mouse (4D1/C2D) in an I-A,o background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4+ T cells develop normally. Characterization of the transgene expression demonstrates that ,90% of B cells express high levels of DR3 and 50,70% of B cells express DQ2. CD11c+ dendritic cells express high levels of DR and DQ. Approximately12,18% of resting T cells are positive for DR expression, and further up-regulation to 40,50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions. [source] The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Tania Vitalis Abstract In the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB1Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB1R expression as well as effects of CB1R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB1Rs are strongly expressed in both reelin-expressing Cajal-Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB1R protein is localized first to somato-dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB1Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP-55940 results in the relocation of receptors from axons to somato-dendritic endosomes, indicating the functional competence of embryonic CB1Rs. The adult phenotype of CB1R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB1R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB1R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB1R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption. [source] Endothelial cell-derived bone morphogenetic proteins regulate glial differentiation of cortical progenitorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2008Tetsuya Imura Abstract Gliogenesis is an important component of cortical development during the postnatal period. Two macroglial cells are generated in a particular order, i.e. astrocytes first and oligodendrocytes later. The mechanisms underlying this sequence of glial differentiation are unknown but interactions with blood vessels are postulated to play a role. We show, using a mouse in-vitro coculture system, that endothelial cells promote astrocyte differentiation but inhibit oligodendrocyte differentiation of postnatal cortical progenitors. Endothelial cells produce bone morphogenetic proteins (BMPs) to activate Sma- and Mad-related protein (Smad) signalling in progenitors and the effects of endothelial cells on glial differentiation are blocked by the BMP antagonist Noggin. Differentiation of progenitors into astrocytes results in the inhibition of endothelial cell growth, accompanied by changes in gene expression of angiogenic factors, indicating bidirectional interactions between progenitors and endothelial cells. In vivo, Smad signalling is activated in various types of cortical cells including progenitors in association with astrogenesis but is inactivated before the peak of oligodendrogenesis. Capillary vessels isolated from the developing cortex express high levels of BMPs. Together, these results demonstrate that endothelial cells regulate glial differentiation by secreting BMPs in vitro and suggest a similar role in cortical gliogenesis in vivo. [source] Plakoglobin-dependent disruption of the desmosomal plaque in pemphigus vulgarisEXPERIMENTAL DERMATOLOGY, Issue 6 2007Alain De Bruin Abstract:, We recently reported that the pathogenesis of pemphigus vulgaris (PV), an autoimmune blistering skin disorder, is driven by the accumulation of c-Myc secondary to abrogation of plakoglobin (PG)-mediated transcriptional c-Myc suppression. PG knock-out mouse keratinocytes express high levels of c-Myc and resemble PVIgG-treated wild-type keratinocytes in most respects. However, they fail to accumulate nuclear c-Myc and loose intercellular adhesion in response to PVIgG-treatment like wild-type keratinocytes. This suggested that PG is also required for propagation of the PVIgG-induced events between augmented c-Myc expression and acantholysis. Here, we addressed this possibility by comparing PVIgG-induced changes in the desmosomal organization between wild-type and PG knock-out keratinocytes. We found that either bivalent PVIgG or monovalent PV-Fab (known to trigger blister formation in vivo) disrupt the linear organization of all major desmosomal components along cell borders in wild-type keratinocytes, simultaneously with a reduction in intercellular adhesive strength. In contrast, PV-Fab failed to affect PG knock-out keratinocytes while PVIgG cross-linked their desmosomal cadherins without significantly affecting desmoplakin. These results identify PG as a principle effector of the PVIgG-induced signals downstream of c-Myc that disrupt the desmosomal plaque at the plasma membrane. [source] 3T3-L1 adipocyte apoptosis induced by thiazolidinediones is peroxisome proliferator-activated receptor-,-dependent and mediated by the caspase-3-dependent apoptotic pathwayFEBS JOURNAL, Issue 3 2010Yuanyuan Xiao Although thiazolidinediones (TZDs) are potent promoters of adipogenesis in the preadipocyte, they induce apoptosis in several other cell types, such as cancer cells, endothelial cells and T-lymphocytes. In this study, we investigated the proapoptotic effect of TZDs in mature 3T3-L1 adipocytes, which express high levels of the peroxisome proliferator-activated receptor-, (PPAR,) protein. Apoptosis was induced in mature 3T3-L1 adipocytes by treatment with troglitazone, pioglitazone or prostaglandin J2, and could be blocked by the PPAR, antagonist GW9662. Treatment with PPAR, agonists also decreased Akt-1 protein and phosphorylation levels without affecting phosphoinositide 3-kinase and PTEN. Further analysis indicated that in troglitazone-treated 3T3-L1 adipocytes, Bad phosphorylation and Bcl-2 protein levels were reduced, and Bax translocation to the mitochondria was increased. Subsequently, cytochrome c release and caspase-3 cleavage were observed. TZD-induced adipocyte apoptosis could be blocked by the caspase-3 inhibitor Ac-DEVD-CHO or by overexpression of Bcl2. In cultured rat primary adipocytes, similar apoptosis-inducing effects of troglitazone were also observed. Thus, TZDs promote apoptosis in adipocytes through a PPAR,-dependent pathway. This apoptosis is mediated by the inhibition of Akt-1, which decreases Bad phosphorylation and activates the mitochondrial apoptotic pathway. [source] Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissuesFEBS JOURNAL, Issue 6 2001Sven Moosmang Hyperpolarization-activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1,4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1,4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1,4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue-specific expression of HCN channel genes. [source] Olfactory ensheathing cell membrane properties are shaped by connectivityGLIA, Issue 6 2010Lorena Rela Abstract Olfactory ensheathing cells (OECs) have been repeatedly implicated in mediating plasticity, particularly in situ in the olfactory nerve in which they support the extension of olfactory sensory neuron (OSN) axons from the olfactory epithelium to the olfactory bulb (OB). OECs are specialized glia whose processes surround OSN axon fascicles within the olfactory nerve and across the OB surface. Despite their purported importance in promoting axon extension, and following transplants, little is known about either morphology or biophysical properties of OECs in situ. In particular, cell,cell interactions that may influence OEC function are largely unexplored. Here, we studied OEC connectivity and morphology in slice preparations, preserving tissue structure and cell,cell interactions. Our analyses showed that OECs form a matrix of cellular projections surrounding axons, unique among glia, and express high levels of connexin-43. Lucifer Yellow injections revealed selective dye coupling among small subgroups of OECs. Two types of OECs were biophysically distinguished with whole-cell voltage-clamp recordings: (1) with low-input resistance (Ri), linear current profiles, and frequently dye coupled; and (2) with high Ri, nonlinear current profiles, and infrequent dye coupling. Pharmacological blockade of gap junctions changed OEC membrane properties such that linear OECs became nonlinear. Double recordings indicated that the appearance of the nonlinear current profile was associated with the loss of electrical coupling between OECs. We conclude that the diversity of OEC current profiles can be explained by differences in gap-junction connectivity and discuss implications of this diversity for OEC influences on axon growth and excitability. © 2009 Wiley-Liss, Inc. [source] Fas and TNFR1, but not cytolytic granule-dependent mechanisms, mediate clearance of murine liver adenoviral infection,HEPATOLOGY, Issue 1 2005Marwan S. Abougergi After intravenous injection of replication-deficient adenovirus, hepatocytes are transduced and express high levels of adenovirus-encoded genes. However, adenovirally encoded gene expression is ablated rapidly by CD8+ T-cell,dependent mechanisms. Thus, this model is suitable for examining intrahepatic cytotoxic T lymphocyte (CTL) effector mechanisms. In the present studies, recombinant adenoviruses encoding secreted (human apolipoprotein A-I) or intracellular (,-galactosidase) gene products were infused into mice with genetic deficiencies affecting the granule exocytosis-, Fas-, or tumor necrosis factor receptor 1 (TNFR1)-mediated pathways of CTL and natural killer cell effector function; the rates of clearance of adenovirus-encoded gene products were assessed. Clearance of secreted or intracellular adenoviral gene products was not delayed in perforin-deficient mice or dipeptidyl peptidase I-deficient mice, which fail to process and activate granzyme A or granzyme B. TNFR1-deficient mice also exhibited no delay in clearance of adenoviral gene products. However, adenoviral clearance from Fas-deficient mice was delayed, and such delays were much greater in mice deficient in both TNFR1 and Fas. In contrast, chimeric mice lacking both hepatic Fas and lymphocyte perforin function exhibited no greater delay in adenoviral clearance than chimeras deficient only in hepatic Fas expression. In conclusion, Fas-dependent mechanisms are required for efficient clearance of virally infected hepatocytes and, in Fas-deficient animals, TNFR1-dependent mechanisms provide an alternative mechanism for hepatic adenovirus clearance. In contrast, perforin- and granule protease,dependent cytotoxicity mechanisms play no apparent role in clearance of adenovirus from the liver. (HEPATOLOGY 2005;41:97,105.) [source] Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Devendra S. Dandekar Abstract Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE2 increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL- xL. Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer. © 2005 Wiley-Liss, Inc. [source] Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5 2010S. Kato Abstract Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies. [source] Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2003Lubing Zhou Abstract We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E,+,6 molecules/,g total RNA) relative to that in the kidney (3E,+,4 molecules/,g total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E,+,5 molecules/,g total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E,+,4 molecules/,g total RNA (in skeletal muscle) to 3.2E,+,6 molecules/,g total RNA (in kidney), levels 10,100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart. J. Cell. Biochem. 90: 339,346, 2003. © 2003 Wiley-Liss, Inc. [source] Characterization of the upstream mouse Cbfa1/Runx2 promoter,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Z. S. Xiao Abstract Cbfa1 (or Runx2/AML-3/PEPB2,) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5,-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located ,0.8 kb 5, upstream of the ATG encoding the MASN/p57 isoform and ,0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5, flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression. J. Cell. Biochem. 82: 647,659, 2001. © 2001 Wiley-Liss, Inc. [source] Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathwaysJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Margaret P. Quinlan Transformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion-dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F-actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell,cell, and not cell,substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase-, but not phosphatase-sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3-kinase-Akt-mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F-actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of epithelial cells. J. Cell. Physiol. 200: 277,290, 2004. © 2004 Wiley-Liss, Inc. [source] Symposium 1: Regulation of Neural Development by BMP and Activin Family MembersJOURNAL OF NEUROCHEMISTRY, Issue 2002J. A. Kessler The effects of BMP family members on stem cell lineage commitment depend upon the developmental age of the stem cell. BMP4 promotes apoptosis of early ventricular zone (VZ) stem cells, neuronal differentiation of later stage VZ cells, and astroglial differentiation of subventricular zone (SVZ) cells. BMP4 inhibits oligodendroglial lineage commitment at all stages of development. The effects of BMP4 in promoting commitment to a specific lineage reflect active suppression of alternate lineages by transcriptional inhibitors including ID and HEY family members and others. For example, BMP mediated increases in ID expression in SVZ stem cells suppress both oligodendroglial and neuronal differentiation. Similarly HEY 1 expression in SVZ cells suppresses neuronal differentiation, whereas HEYL expression by VZ cells inhibits glial differentiation and promotes neurogenesis. The differing effects of the BMPs on VZ and SVZ stem cells reflect also differences in the complement of transcription factors that are expressed. For example, VZ stem cells express high levels of neurogenin and HEY L whereas SVZ stem cells express lower levels of these factors but higher levels of HEY1. Thus lineage commitment by stem cells reflects interplay among stimulatory and inhibitory transcription factors, and responses to the BMPs depend upon the repertoire of transcription factors already expressed by the cell. [source] Patterns of laminins and integrins in the embryonic ventricular zone of the CNSTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Justin D. Lathia Abstract The extracellular matrix (ECM) provides both a physical framework and a microenvironment that supplies instructive signals from the earliest stages of multicellular development. As a first step toward understanding the role of the ECM in regulating the behavior of neural stem cells (NSCs), here we show the localization of laminins, a heterotrimeric family of ECM molecules expressed in many different stem cell microenvironments, and their corresponding receptors in the embryonic murine ventricular zone (VZ) within which the NSCs undergo symmetrical and asymmetrical divisions required for cortical development. In addition to the presence of laminins containing both the ,2 and ,4 chains, we find distinct patterns of ECM receptor expression in the VZ and in the overlying cortex. Neural stem cells derived from the VZ express high levels of the integrin laminin receptor ,6,1. At developmental stages at which NSCs undergo asymmetrical divisions, integrin ,1 was unevenly distributed in some mitotic pairs at the ventricular wall. These results suggest a significant role in the regulation of NSC fate for laminin/integrin signaling within the microenvironment of the VZ and provide a framework for future molecular and cellular analyses of the role of the ECM in neural development. J. Comp. Neurol. 505:630,643, 2007. © 2007 Wiley-Liss, Inc. [source] Elevated expression of interleukin-7 receptor in inflamed joints mediates interleukin-7,induced immune activation in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 9 2009Sarita A. Y. Hartgring Objective To evaluate the expression and functional ability of the high-affinity interleukin-7 receptor (IL-7R,) in patients with rheumatoid arthritis (RA). Methods Expression of IL-7R, and IL-7 was determined in synovial tissue from RA patients and was compared with that in synovial tissue from patients with undifferentiated arthritis (UA) and osteoarthritis (OA). IL-7R, expression on CD4 T cells, CD19 B cells, and CD14 monocyte/macrophages from RA synovial tissue, synovial fluid, and peripheral blood was also assessed. The proliferative capacity of IL-7R,bright and IL-7R,dim/, T cells was measured. In addition, we examined IL-7R blockade with soluble human IL-7R, (hIL-7R,) in the prevention of immune activation of peripheral blood mononuclear cells. Results We found significantly higher IL-7R, expression in RA and UA synovial tissue than in OA synovial tissue, and the level of IL-7R, expression correlated significantly with the levels of CD3 and IL-7 expression. CD4 T cells from RA synovial fluid and synovial tissue strongly expressed IL-7R,. A substantial percentage of B cells and macrophages from RA synovial fluid and synovial tissue also expressed IL-7R,, although less prominently than T cells. We found that peripheral blood IL-7R,bright T cells that did not express FoxP3 were highly proliferative as compared with IL-7R,dim/, T cells that did express high levels of FoxP3. Soluble hIL-7R, inhibited IL-7,induced proliferation and interferon-, production by mononuclear cells from RA patients. Conclusion Our data suggest that enhanced expression of IL-7R, and IL-7 in RA patients contributes significantly to the joint inflammation by activating T cells, B cells, and macrophages. The inhibition of IL-7R,mediated immune activation by soluble hIL-7R, further indicates an important role of IL-7R, in inflammatory responses in RA, suggesting IL-7R, as a therapeutic target for immunotherapy in RA. [source] | |||||||||||||||||||||||||