Murine mRNA Research Articles

Haploinsufficiency of SNEV Causes Defects of Hematopoietic Stem Cells Functions

The quality of hematopoietic stem cells (HSCs) is essentially defined by two characteristics, i.e., multilineage differentiation and self-renewal capacity. Thus, it is of high priority to clarify mechanisms that regulate these functions and to understand them at the molecular level. In the present study, we investigated the role of senescence evasion factor (synonymously hPrp19,hPSO4,hNMP200: SNEV), a multifunctional protein involved in pre-mRNA splicing, regulation of replicative life span, and DNA repair. Here we report that murine SNEV mRNA expression is high in lineage-depleted (Lin(-)) precursor cells of the bone marrow immediately after isolation as compared to fully differentiated peripheral blood lymphocytes (PBLs). Furthermore, the progenitor cell subset with highest colony-forming ability and self-renewal capacity (Lin(-), Sca-1(+)) showed also the highest SNEV expression. To test if the observed differences in SNEV mRNA levels cause stem cell defects, Lin(-) cells derived from heterozygous SNEV knockout mice were tested for primary as well as secondary colony-forming potential as a measure of self-renewal capacity. Interestingly, both, primary and secondary colonies were significantly less formed from SNEV(+/-) cells, a defect that was rescued by ectopic SNEV expression. Similarly, bone marrow cells derived from the short-lived Senescence-Accelerated-Mouse-Prone (SAMP8) model showed similar differences in comparison to the aging-resistant (SAMR1) control strain. These data suggest that the expression of SNEV is closely associated with the growth of murine HSCs and determines the proliferative and repopulating capacity of phenotypically defined HSC subsets.

Ucma, a Novel Secreted Cartilage-specific Protein with Implications in Osteogenesis

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.

CBP/p300-Interacting Protein CITED1 Modulates Parathyroid Hormone Regulation of Osteoblastic Differentiation

PTH regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo, depending on the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of CITED1 transcriptional coactivator were strongly up-regulated by human (h) PTH(1-34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester, and the cAMP-selective PTH analog [G(1),R(19)] hPTH (1-28) and inhibited completely by the protein kinase A inhibitor, H89 and partially by phorbol ester-induced protein kinase C depletion. Increased CITED1 expression was not maintained during persistent (24 h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison with wild type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1-34) (10 nm, for 4 h every 48 h) for 3-6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial cell cultures. Whereas the cAMP-selective [G(1),R(19)]hPTH(1-28) analog [at 100 nm, equivalent to 10 nm hPTH(1-34)] did not stimulate mineralization in WT cultures, it was twice as effective as hPTH(1-34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence.

Expression of High Levels of Human γ-Globin in Adult Mice Carrying a Transgene of the Brazilian Type of Hereditary Persistence of Fetal Hemoglobin.

The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.

The Anti-inflammatory Effects of Selenium Are Mediated through 15-Deoxy-Δ12,14-prostaglandin J2 in Macrophages

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-kappaB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-kappaB cascade, IkappaB-kinase beta (IKKbeta) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKbeta in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKbeta activity. Further analysis revealed that inhibition of IKKbeta activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKbeta, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-gamma in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium.

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-β peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.

Murine CXCR1 Is a Functional Receptor for GCP-2/CXCL6 and Interleukin-8/CXCL8

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

27 EFFECT OF ACTIVATION METHODS AND BIOPSY SAMPLING ON NUCLEAR TRANSFER BLASTOCYST PRODUCTION AND CLONING EFFICIENCY IN A HORSE

In the horse, rates of blastocyst production after nuclear transfer are low. This study was conducted to examine the effect of activation via injection of different volumes of sperm extract or via injection of murine mRNA for PLC-ζ, a sperm-specific protein which induces Ca2+ oscillations in all species thus far studied, on blastocyst development after nuclear transfer. Two biopsy samples from the same horse were also examined for cloning efficiency. Donor fibroblasts cultured from a skin biopsy taken from a 19-year-old mare were treated with 15 µm R-roscovitine for 18 to 24 h before direct injection into enucleated ooplasts. Ionomycin treatment, 5 µm for 4 min, was used in all activation treatments, as the combination of ionomycin with sperm extract provided the highest blastocyst development and foaling rates in our previous report (Hinrichs et al. 2007 Reproduction 134, 319–325). All treatments were followed by incubation in 2 mm 6-dimethylaminopurine for 4 h. Differences in blastocyst development between treatments were analyzed using Fisher's exact test. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 s at a fixed pipette diameter and injection pressure, and then treated with ionomycin. The blastocyst rate (9.8%) for 0.1 s was significantly higher than that for 0.2 s (0%) or 0.8 s (1.4%). In Experiment 2, murine PLC-ζ mRNA (0.25 µg µL–1) was injected into reconstructed oocytes 20 to 30 min before or after ionomycin treatment and compared with a control treatment (injection of 2 to 4 pL sperm extract 20 to 50 min after ionomycin exposure). There were no differences in blastocyst development among treatments (0, 4.5, and 5.6%, respectively). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in 5 pregnancies: however, all were lost before 70 days of gestation. In Experiment 3, a second skin biopsy was obtained from the same mare and cells from this tissue sample were used for nuclear transfer concurrently with cells from the first sample, using the control method above. Blastocyst production was higher using cells from the second biopsy sample (4/23 v. 0/23; P = 0.05). Transfer of these four blastocysts yielded four pregnancies, two of which continued to term and produced viable foals. These results indicate that blastocyst development after injection of sperm extract is dependent upon the volume injected, that injection of murine PLC-ζ mRNA does not improve blastocyst formation under the given conditions, and that the efficiency of cloning may vary with biopsy sample even from the same animal.

Reduction of plasma triglycerides in apolipoprotein C-II transgenic mice overexpressing lipoprotein lipase in muscle

LPL and its specific physiological activator, apolipoprotein C-II (apoC-II), regulate the hydrolysis of triglycerides (TGs) from circulating TG-rich lipoproteins. Previously, we developed a skeletal muscle-specific LPL transgenic mouse that had lower plasma TG levels. ApoC-II transgenic mice develop hypertriglyceridemia attributed to delayed clearance. To investigate whether overexpression of LPL could correct this apoC-II-induced hypertriglyceridemia, mice with overexpression of human apoC-II (CII) were cross-bred with mice with two levels of muscle-specific human LPL overexpression (LPL-L or LPL-H). Plasma TG levels were 319 +/- 39 mg/dl in CII mice and 39 +/- 5 mg/dl in wild-type mice. Compared with CII mice, apoC-II transgenic mice with the higher level of LPL overexpression (CIILPL-H) had a 50% reduction in plasma TG levels (P = 0.013). Heart LPL activity was reduced by approximately 30% in mice with the human apoC-II transgene, which accompanied a more modest 10% decrease in total LPL protein. Overexpression of human LPL in skeletal muscle resulted in dose-dependent reduction of plasma TGs in apoC-II transgenic mice. Along with plasma apoC-II concentrations, heart and skeletal muscle LPL activities were predictors of plasma TGs. These data suggest that mice with the human apoC-II transgene may have alterations in the expression/activity of endogenous LPL in the heart. Furthermore, the decrease of LPL activity in the heart, along with the inhibitory effects of excess apoC-II, may contribute to the hypertriglyceridemia observed in apoC-II transgenic mice.

Penumbra Encodes a New Tetraspanin Critical to Effective Erythropoiesis.

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra−/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra−/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra−/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias.

Characterisation Erythroid-Specific Cis-Elements Regulating the Key Transcription Factor GATA1.

The critical myeloid transcription factor GATA1 can specify erythroid cells at expense of granulocytes/macrophages (GM) from the common myeloid progenitor (CMP). In red cells sustained GATA1 expression is required for terminal maturation. Conversely, GATA1 expression has to be extinguished to allow GM differentiation. Therefore, one component in dissecting myeloid specification will be to define how GATA1 expression is regulated. The level of GATA1 is mainly controlled transcriptionally. Here, we show that murine(m) GATA1 mRNA rises 6-fold as CMPs differentiate to MEPs (megakaryocyte-erythroid progenitor) and another 4-fold as MEPs differentiate to Ter119+ erythroid cells. As a step towards understanding the molecular basis of erythroid-specific GATA1 expression, we have been characterising GATA1 cis-elements. Previously, we and others showed that an upstream enhancer (mHS−3.5) is required to direct erythroid-specific GATA1 expression in cooperation with sequences near the mGata1 gene (IE promoter and intron element mHS+3.5) in transgenic mice. Though a mGata1 transgene regulated by mHS−3.5-IE-mHS+3.5 grossly rescues erythropoiesis in GATA1 knock out mice, germ line deletion of mHS−3.5 leaves red cell GATA1 expression unaffected. This suggests other cis-elements in the mGata1 locus can substitute for mHS−3.5 in red cells. Recently, we identified an erythroid-specific DNase I hypersensitive site (DHS), mHS-25, with enhancer activity in erythroid cell lines and where the chromatin associated with it is hyperacetylated at histone H3/H4. We now demonstrate by fine DHS mapping that mHS-25 is more complex, being composed of two adjacent DHSs ~500 bp apart (mHS-25 and mHS-26). These DHSs are present only in primary red cells and not other primary cells. Chromatin immunoprecipitation (ChiP) shows that H3 and H4 associated with both sites is hyperacetylated only in Ter119+ cells. We tested mHS25/6 function in mice transgenic for a mGata1-LacZ reporter construct regulated by mHS-25/6-IE-mHS+3.5. β-galactosidase expression was quantitated in myeloid lineages by FACS analysis using lineage-specific antibodies and the β-galactosidase substrate FDG. 6 out of 7 F0 transgenic embryos expressed β-galactosidase in 5–23% of fetal liver Ter119+ cells. FDG staining was not detected in CD61+Mac1- megakaryocytes or Mac-1+ macrophages. <0.2% of Ter119+ cells stained with FDG when mHS-25/6 was not present. Erythroid-specific reporter gene expression was confirmed in bone marrow samples in 2 independent lines of mice transgenic for this reporter construct. ChiP analysis demonstrated that GATA1, SCL, E2A, the SCL-interacting protein Ldb1 and LMO2 bind in vivo to mHS-25/6 only in Ter119+ cells (and not primary megakaryocytes or neutrophils). Previously, we and others have shown that these transcription factors exist in a multi-protein complex to activate gene expression. Taken together, these findings suggest mHS-25/6 is an erythroid-specific GATA1 enhancer in primary murine cells. Finally, to begin to understand the relative roles of mHS−3.5 and mHS-25/6 during erythroid differentiation, we show by DHS mapping and ChiP analysis that in the multi-potential myeloid cell line FDCP-mix, only mHS−3.5 is present (but not mHS-25/6) and that it binds GATA1/SCL/E2A/Ldb1/LMO2 in vivo. Later in Ter119+ cells both mHS−3.5 and mHS-25/6 are seen with GATA1/SCL/E2A/Ldb1/LMO2 detected at these sites. This suggests a hierarchical utilisation of these two mGata1 cis-elements during erythroid differentiation.

Daxx Represses Expression of a Subset of Antiapoptotic Genes Regulated by Nuclear Factor-κB

Daxx is a nuclear protein that localizes to PML oncogenic domains, sensitizes cells to apoptosis, and functions as a transcriptional repressor. We found that Daxx represses the expression of several antiapoptotic genes regulated by nuclear factor-kappaB, including cIAP2, in human tumor cell lines. Daxx interacts with RelB and inhibits RelB-mediated transcriptional activation of the human cIAP2 gene promoter. Daxx also forms complexes with RelB while bound to its target sites in the cIAP2 promoter, as shown by electrophoretic mobility shift assays and chromatin immunoprecipitation experiments. Using cells from daxx-/- mouse embryos, we observed that levels of the corresponding murine c-IAP mRNA and protein are increased in cells lacking Daxx. Conversely, c-IAP mRNA and protein levels were reduced in relB-/- cells. Taken together, these observations provide a mechanism that links two previously ascribed functions of Daxx: transcriptional repression and sensitization to apoptosis.

Phenytoin Induction of the Cyp2c37 Gene Is Mediated by the Constitutive Androstane Receptor

The CYP2C subfamily of cytochrome P450 monooxygenases is responsible for the metabolism of approximately 20% of therapeutic drugs and many endogenous compounds in humans. These enzymes can be induced by prior treatment with drugs, resulting in changes in drug efficacy. Induction of human CYP2C enzymes by xenobiotics occurs at the transcriptional level and is reported to involve the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). In the present study, we report that murine CYP2C37 mRNA is induced by phenobarbital and phenytoin. In contrast, the mouse PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile did not induce CYP2C37 mRNA, suggesting that PXR does not regulate this gene. The induction of CYP2C37 mRNA by phenobarbital and phenytoin is essentially abolished in CAR-null mice; thus, induction of Cyp2c37 by these xenobiotics is CAR-dependent. A functional CAR response element (CAR-RE) was identified at -2791 base pairs from the translation start site of the Cyp2c37 gene. Mutation of this CAR-RE abolished mouse CAR transactivation of a Cyp2c37 -2.9-kilobase pair luciferase reporter construct in HepG2 cells.

Continued Administration of Ciliary Neurotrophic Factor Protects Mice from Inflammatory Pathology in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. We show that daily administration (days 8 to 24) of murine ciliary neurotrophic factor (CNTF), a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocytes, significantly ameliorates the clinical course of a mouse model of multiple sclerosis. In the acute phase of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide 35-55, treatment with CNTF did not change the peripheral immune response but did reduce the number of perivascular infiltrates and T cells and the level of diffuse microglial activation in spinal cord. Blood brain barrier permeability was significantly reduced in CNTF-treated animals. Beneficial effects of CNTF did not persist after it was withdrawn. After cessation of CNTF treatment, inflammation and symptoms returned to control levels. However, slight but significantly higher numbers of oligodendrocytes, NG2-positive cells, axons, and neurons were observed in mice that had been treated with high concentrations of CNTF. Our results show that CNTF inhibits inflammation in the spinal cord, resulting in amelioration of the clinical course of experimental autoimmune encephalomyelitis during time of treatment.

S100A7 (Psoriasin): a Story of Mice and Men

S100 proteins are calcium-regulated proteins that regulate fundamental biological processes. S100A7 (psoriasin), functions as a transglutaminase substrate/cornified envelope precursor, signal transduction protein, chemokine, and antibacterial protein in normal epidermis. S100A7 is markedly increased in epidermal hyperproliferative disorders. The murine homolog of S100A7 and S10015 has been identified, providing a valuable tool for studying the regulation and function of this protein in epidermis.

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.

The Ubiquitin-like Protein MNSFβ Regulates ERK-MAPK Cascade

MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.

Focus on Molecules: Rod photoreceptor cGMP-gated cation channel

The rod photoreceptor cGMP-gated cation (rod CNG) channel is an essential component of phototransduction and also may play a critical role in maintenance of photoreceptor outer segment structural integrity. It is a non-selective cation channel that belongs to the superfamily of voltage-gated channels, and the subfamily of cyclic nucleotide gated channels (Kaupp and Seifert, 2002). This channel is composed of a heterotetramer consisting of three α-subunits (encoded by the CNGA1 locus; also called A subunit) and one β-subunit (encoded by the CNGB1 locus; also called the B subunit; Genbank IDs for Murine and Human mRNA: α-subunit 34328042, 71143140; β-subunit 28386104, 4502918; uniprot IDs for Murine and Human proteins are: α-subunit P29974, P29973; β-subunit Q9UMG2, Q80XL8) arranged around a central pore. The αand β-subunits are the products of separate genes whose loci are designated CNGA1 and CNGB1. The CNGB1 locus is complex encoding multiple alternatively spliced transcripts that are expressed in several diverse tissues (Ardell et al., 2000). For example, an olfactory tissue specific transcript encodes a βsubunit variant that is required for normal channel function of the olfactory cyclic nucleotide gated channel.

A novel mammalian bubblegum-related acyl-CoA synthetase restricted to testes and possibly involved in spermatogenesis

We have characterized a new, membrane-associated acyl-CoA synthetase (ACS), termed bubblegum-related protein (BGR), which upon functional analysis demonstrated ACS activity capable of activating long- and very long-chain fatty acids. By multiple tissue RNA array and Northern blot analyses, human BGR mRNA was exclusively detected in testes. Murine Bgr mRNA was specifically expressed in pubertal and adult testes and was further demonstrated to be enriched in germ cells and Sertoli cells while present at a lower level in Leydig cells both by in situ hybridization and cell type fractionation. The complex 5′-end of the BGR mRNA appears to underlie translational control leading to differential utilization of alternative translation start sites. Thus, the BGR gene expands the bubblegum ACS family with a testes-specific, developmentally regulated member that may play a role in spermatogenesis.

Characterization of Gpr101 expression and G-protein coupling selectivity

This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Gαs protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.