Cloning and characterizing of the ovine MX1 gene promoter/enhancer region

Ovine MX1 (MX1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. In this study we cloned the MX1 gene promoter/enhancer, and tested its response to interferon tau (IFN-tau). To address the role of IFN tau in regulating MX1 expression, serial deletion mutants were prepared along with a clone that contained a full-length promoter including the two proximal ISREs but lacking an intronic ISRE site. Promoter deletions showed the two proximal ISRE sites, but not the intronic ISRE site, were required for maximal response to IFN tau. Interestingly, MX1 promoter deletion mutants revealed the presence of distal positive (-920 to -715) and negative (-715 to -437) regulatory regions. Identifying positive and negative regulatory regions in MX1 promoter will help define the complex regulation of MX1 during early pregnancy in ruminants.     

ASRI2005-87 Generation of treg cells in the CBA/J × DBA/2J combination by vaccination with male BALB/c splenocytes rescues from abortion

Mx proteins are 70–80 kD intracellular antiviral proteins induced by viruses and type I interferons (IFNs). Mx proteins belong to the dynamin superfamily of large GTPases, thought to regulate endocytosis, intracellular vesicle trafficking and cytokinesis. Although antiviral Mx proteins are highly inducible during early pregnancy by embryo-derived IFN tau, cellular functions of Mx proteins in uninduced cells are poorly defined. To better understand the physiological role of Mx proteins during early pregnancy, expression and localization of ovine Mx1 (oMx1) protein were determined in a uterine derived cell line (oGE). oMx1 and beta tubulin were immunolabeled in cells at different stages of the cell cycle. oMx1 was uniformly distributed in the cytoplasm during interphase in the absence of roIFN tau, whereas oMx1 aggregated around vacuoles near the surface of the cells in the presence of roIFN. Interestingly, oMx1 co-localized with mitotic spindles during metaphase. During telophase, oMx1 co-localized with remnants of mitotic spindles at intercellular bridges between dividing cells. Our findings are consistent with a role for Mx1 in secretion by oGE cells and indicate that oMx1 may be involved in cell division. Supported in part by USDA NRICGP grant 2002-02398 and NIH NCRR grant P20-RR15587-01 to T.L.O.     

Cellular localization and function of the antiviral protein, ovine Mx1 (oMx1): II. The oMx1 protein is a regulator of secretion in an ovine glandular epithelial cell line

PROBLEM: Embryonic loss is a major contributor to infertility. Understanding factors affecting embryonic loss will help increase fertility. METHOD OF STUDY: We investigated if ovine Mx1 (oMx1) mediated secretion by ovine glandular epithelial (oGE) cells using small interfering RNA (siRNA). Effects on secretion were examined through the conventional endoplasmic reticulum-Golgi pathway using beta2- microglobulin (beta2MG) as a marker, and interferon-stimulated gene 15 (ISG15) as a marker for unconventional secretion. RESULTS: Mx1 siRNA reduced oMx1 mRNA levels at 12 and 24 hr after IFN-tau treatment (P < 0.05), without affecting levels of oMx2, ISG15, 2',5'-oligoadenylate synthetas or beta2MG. Mx1 siRNA reduced Mx1 protein levels at 48 and 120 hr after treatment (P < 0.05) and protein levels remained low at 120 hr. Transient oMx1 knock-down reduced secretion of oMx1 (P < 0.01). ISG15 protein in secretions was reduced without affecting intracellular levels (P < 0.05). Levels of beta2MG in secretions were not affected by Mx1 siRNA. CONCLUSION: We showed that oMx1 protein is secreted by oGE cells and that reduction in oMx1 protein levels by siRNA reduced secretion of ISG15, but not beta2MG. Results support the hypothesis that oMx1 is a regulator of secretion through unconventional secretory pathway(s).     

Cellular localization and function of the antiviral protein, ovine Mx1 (oMx1): I. Ovine Mx1 is secreted by endometrial epithelial cells via an 'unconventional' secretory pathway

PROBLEM: Embryonic loss is a major contributor to infertility. Understanding factors contributing to embryonic loss will aid in development of technologies to improve/regulate fertility in animals and humans. METHOD OF STUDY: We tested the hypothesis that the antiviral protein, ovine Mx1 (oMx1), is secreted by uterine epithelial cells. Uterine flushes were obtained from cyclic and early pregnant ewes and examined for levels of oMx1 protein. The pathway for ovine Mx1 secretion in ovine glandular epithelial (oGE) cells was determined using brefeldin A (BFA), an inhibitor of the conventional secretory pathway. Effects of BFA were determined using beta2-microglobulin (beta2MG) as a marker for the conventional secretory pathway, and interferon stimulated gene 15 (ISG15) and Galectin-1 (Gal-1) as markers for the unconventional secretory pathways. RESULTS: Ovine Mx1 protein levels were low in uterine flushes from cyclic ewes and levels increased in pregnant ewes after D 15. Ovine GE cells secreted oMx1 in response to interferon and secretion was not reduced by BFA, suggesting oMx1 was secreted via an unconventional secretory pathway. beta2MG secretion was reduced by BFA, whereas ISG15 and Gal-1 were not. CONCLUSION: This is the first report that the antiviral protein, oMx1, is secreted and provides evidence that secretion occurs via unconventional secretory pathway(s).     

An Mx1 promoter-reporter system to study interferon pathways in rainbow trout

A rainbow trout interferon (IFN) reporter system has been established by selection of a stable cell line, RTG-P1, transfected with a plasmid expressing the firefly luciferase gene under the control of the promoter for the IFN-induced gene Mx1. After 148 passages, the luciferase expression was still highly induced by polyinosinic:polycytidylic acid (poly I:C) in RTG-P1 cells. Different IFN inducers (dsRNA, viral hemorrhagic septicaemia virus or conditioned medium containing rainbow trout antiviral activity) were able to stimulate the IFN-reporter system in RTG-P1, showing that this cell line can be used to study the activation of the IFN pathway in various contexts. Pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor, significantly blocked poly I:C induced luciferase accumulation in RTG-P1 at intermediate doses (1-10 microM), suggesting that Mx1 induction through the IFN signalling pathway is NF-kappaB-dependent in fish. This inhibition was not observed for doses of 50 microM or higher. The RTG-P1 reporter system constitutes an interesting tool to study the induction and regulation of IFN signalling in teleost fish.     

Validation of an Mx/CAT reporter gene assay for the quantification of bovine type-I interferon

We describe here a specific and sensitive assay for biologically active bovine type-I interferon (IFN) in an Mx/CAT reporter gene assay. The assay is based on Madin-Darby Bovine Kidney cells transfected with a plasmid, containing a human MxA promoter driving a chloramphenicol acetyltransferase (CAT) cDNA. CAT expression was quantified in a commercially available enzyme linked immunosorbant assay. The response to recombinant bovine INF-alpha(1) was dose dependent between 0.25 and 125.0 iu/ml and was shown to be specific for type-I IFN as no significant effect was seen with a number of other cytokines, including IFN-gamma. This Mx/CAT reporter assay also has advantages in terms of simplicity and reliability over conventional cytopathic effect reduction assays used to quantify the IFN activity in bovine samples. The Mx/CAT reporter assay was used successfully to measure trophoblast derived type-1 IFN activity (IFN-tau) in uterine flushings collected from pregnant cows. IFN-tau is the pregnancy recognition signal produced in ruminants by pre-implantation embryos and was shown to increase markedly between the 12th (0.7+/-0.14 iu/ml) and 18th (44085.0+/-14414.2 iu/ml) day of pregnancy. In contrast, IFN-tau activity remained basal (0.5-0.7 iu/ml) in inseminated non-pregnant animals. Duplicate samples analysed using a cytopathic effect reduction assay correlated well (P<0.001; r(2)=0.945) with IFN levels obtained using the Mx/CAT reporter assay, confirming the reporter assay as a reliable substitute for the standard anti-viral IFN assay.     

Intracellular staining of Mx proteins in cells from peripheral blood, bone marrow and skin.

AIM/BACKGROUND: The Mx proteins are known to be specifically and dose dependently induced in mononuclear cells (MNC) by type I interferons (IFN). The aim of this study was to establish a staining method for the human intracellular Mx proteins, MxA and MxB, in leucocytes and bone marrow and skin cells. METHODS: Several monoclonal antibodies directed against the MxA and MxB proteins were generated. These antibodies were used to stain Mx proteins in both frozen and paraffin wax sections using the standard alkaline phosphatase anti-alkaline phosphatase (APAAP) method. RESULTS: Granulocytes, monocytes and lymphocytes extracted from freshly collected blood from 21 healthy subjects did not stain. After incubating MNC from these subjects with IFN alpha 2b for 48 hours, Mx proteins were detected in monocytes and lymphocytes. Within two days of starting treatment with subcutaneous IFN alpha 2b, granulocytes, monocytes and lymphocytes of 16 patients with cancer stained strongly for Mx proteins. The intensity of staining was correlated with the Mx content of whole blood measured using a specific ELISA. Prior to IFN treatment, cells from bone marrow and skin tissue specimens were negative for Mx proteins with the exception of endothelial cells. During treatment with IFN alpha 2b, nearly all cells from bone marrow and skin stained intensely. CONCLUSIONS: These new monoclonal antibodies facilitate the detection of Mx positive cells in peripheral blood and in frozen or paraffin wax specimens. The advantage of this staining method is that individual cells which have responded to viruses or biologically active IFN alpha, beta or omega can be identified.     

Mx genes show weaker primary response to virus than other interferon-regulated genes.

Some interferon (IFN)-regulated genes are induced as a primary response to virus as well as secondarily through virus-induced IFN. Here we investigated whether this dual control mechanism would also regulate the activity of the human and mouse Mx genes that encode proteins with intrinsic antiviral potentials. To distinguish between a primary response to virus and a secondary response to virus-induced IFN, we studied virus-induced Mx gene expression in cell lines that lack a functional IFN system and in cells with blocked protein synthesis. In contrast to the two IFN-regulated human genes ISG56 and ISG15, the human MxA gene showed almost no primary response to Newcastle disease virus (NDV) or influenza virus. Similarly, direct activation of the mouse Mx1 gene by NDV or influenza virus was not significant in mouse embryo cells or explanted peritoneal macrophages. A moderate primary Mx1 response to NDV was observed in the permanent cell line L1210. Lack of a strong IFN-independent Mx response to virus indicates that this mode of gene regulation does not play a significant role in Mx-mediated resistance to viral disease.     

干扰素刺激反应元件Ⅰ/Ⅱ在维甲酸诱导基因G表达调控中的作用

目的 深入研究维甲酸诱导基因G(retinoic acid-induced gene G,RIG-G)启动子上所含的干扰素刺激反应元件(interferon-stimulated response elements,ISRE)对RIG-G基因表达的调控作用.方法 根据RIG-G基因启动子所包含的ISRE序列,利用定点突变技术分别构建野生型和位点突变型的报告基因质粒,然后采用报告基因转染实验检测RIG-G基因启动子中ISRE序列的功能活性.结果 研究发现单独突变RIG-G基因启动子上的ISRE Ⅱ元件不影响报告基因的表达,而单独突变ISRE Ⅰ则会对报告基因的表达产生明显的抑制作用;同时突变ISRE Ⅰ和ISRE Ⅱ元件则会使报告基因完全失去对转录因子的反应性.结论 RIG-G基因启动子所包含的ISRE Ⅰ和ISRE Ⅱ元件是诱导该基因表达的转录因子复合物的作用位点,是该基因表达的分子基础,且ISRE Ⅰ元件的作用要优先于ISRE Ⅱ.     

Characterisation of gamma-interferon responsive promoters in fish

Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.