Molecular cloning and expression analysis of IFN-β promoter stimulator 1 in Tibetan pigs

IFN-β promoter stimulator 1 (IPS-1) is an important adaptor protein linking RIG-I/MDA5 to the downstream signaling molecules and plays the pivotal role in type I interferons induction. In this study, we cloned and characterized Tibetan porcine IPS-1, investigated the tissue distribution, compared different messenger RNA expression for IPS-1 between Tibetan and Crossbred (Duroc × Yorkshire × Landrace) pigs (DLY). The Tibetan porcine IPS-1 gene was first cloned from spleen. The entire open reading frame (ORF) of the IPS-1 is 1,575 bp and encodes for 524 amino acid residues, has 1 putative transmembrane domains, with a higher degree of sequence similarity with common pig (99.37%) and cattle (81.23%) than with human (70.20%) or mouse (63.44%). Real-time quantitative PCR analysis indicated that Tibetan porcine IPS-1 mRNA was most abundant in the liver and kidney. The expression of IPS-1 of Tibetan pigs in most tissues was higher than DLY pigs.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.     

Cellular responses to TGFβ and TGFβ receptor expression in human colonic epithelial cells require CaSR expression and function

CaSR and TGFβ are robust promoters of differentiation in the colonic epithelium. Loss of cellular responses to TGFβ or loss of CaSR expression is tightly linked to malignant progression. Human colonic epithelial CBS cells, originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer a unique opportunity in determining the functional linkage (if any) between CaSR and TGFβ. Knocking down CaSR expression abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca²⁺ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca²⁺ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR; there was no up-regulation of TGFβ receptor. It is concluded that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function.     

Expression profiling in APP23 mouse brain: inhibition of Aβ amyloidosis and inflammation in response to LXR agonist treatment

Background

Recent studies demonstrate that in addition to its modulatory effect on APP processing, in vivo application of Liver X Receptor agonist T0901317 (T0) to APP transgenic and non-transgenic mice decreases the level of Aβ_42. Moreover, in young Tg2576 mice T0 completely reversed contextual memory deficits. Compared to other tissues, the regulatory functions of LXRs in brain remain largely unexplored and our knowledge so far is limited to the cholesterol transporters and apoE. In this study we applied T0 to APP23 mice for various times and examined gene and protein expression. We also performed a series of experiments with primary brain cells derived from wild type and LXR knockout mice subjected to various LXR agonist treatments and inflammatory stimuli.

Results

We demonstrate an upregulation of genes related to lipid metabolism/transport, metabolism of xenobiotics and detoxification. Downregulated genes are involved in immune response and inflammation, cell death and apoptosis. Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Aβ. In primary LXR^wt but not in LXRα^-/-β^-/- microglia and astrocytes LXR agonists suppressed the inflammatory response induced by LPS or fibrillar Aβ.

Conclusion

The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation. An increased understanding of the LXR controlled regulation of Aβ aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.     

Protein kinase Cι expression and oncogenic signaling mechanisms in cancer

Accumulating evidence demonstrates that PKCι is an oncogene and prognostic marker that is frequently targeted for genetic alteration in many major forms of human cancer. Functional data demonstrate that PKCι is required for the transformed phenotype of lung, pancreatic, ovarian, prostate, colon, and brain cancer cells. Future studies will be required to determine whether PKCι is also an oncogene in the many other cancer types that also overexpress PKCι. Studies of PKCι using genetically defined models of tumorigenesis have revealed a critical role for PKCι in multiple stages of tumorigenesis, including tumor initiation, progression, and metastasis. Recent studies in a genetic model of lung adenocarcinoma suggest a role for PKCι in transformation of lung cancer stem cells. These studies have important implications for the therapeutic use of aurothiomalate (ATM), a highly selective PKCι signaling inhibitor currently undergoing clinical evaluation. Significant progress has been made in determining the molecular mechanisms by which PKCι drives the transformed phenotype, particularly the central role played by the oncogenic PKCι-Par6 complex in transformed growth and invasion, and of several PKCι-dependent survival pathways in chemo-resistance. Future studies will be required to determine the composition and dynamics of the PKCι-Par6 complex, and the mechanisms by which oncogenic signaling through this complex is regulated. Likewise, a better understanding of the critical downstream effectors of PKCι in various human tumor types holds promise for identifying novel prognostic and surrogate markers of oncogenic PKCι activity that may be clinically useful in ongoing clinical trials of ATM.     

The Chlamydia muridarum-induced IFN-β response is TLR3-dependent in murine oviduct epithelial cells

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-β synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-β production. To investigate the role of TLR3 in the Chlamydia-induced IFN-β response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-β secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-β synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-β synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-β synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Dietary fiber enhances TGF-β signaling and growth inhibition in the gut

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.     

TGF-β polymorphism and its expression correlated with CXCR4 expression in human breast cancer

The role of chemokines and the growth factors has been extensively analyzed both in cancer risk and tumor progression. The transforming growth factor beta (TGF-β) and chemokine (C-X-C motif) receptor 4 (CXCR4) genes are implicated in several diseases, including breast cancer. Genomic DNA was obtained from 21 samples of peripheral blood or from normal tissue, previously fixed in formalin and embedded in paraffin for TGF-β T869C polymorphism analyses. Total cellular RNA was extracted from the same 21 patients, but from fresh tissue (tumor and adjacent healthy from the same breast) for expression analysis by Real Time PCR. No significant differences were observed in genotype distribution according to clinicopathological characteristics. Transforming growth factor beta (TGF-β) mRNA expression was assessed according to T869C polymorphism and CC patients presented a higher TGF-β expression but not significant when compared to other genotypes (p?=?0.064). A positive correlation was observed in relative mRNA expressions of CXCR4 and TGF-β (p?=?0.020). It is known that overexpression of TGF-β by both tumor and stromal tissue can facilitate the development of metastases, mainly by TGF-β stimulated angiogenesis and increased tumor cell motility. Our findings suggested a role of these genes as progression markers for breast carcinoma.     

Cloning and expression of human IFN-γ in <Emphasis Type="Italic">Leishmania tarentolae</Emphasis>

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-γ), an antiviral pro-inflammatory cytokine, has broadened interest in optimizing methods for its production. We herein describe a unicellular eukaryotic system, Leishmania tarentolae, a Trypanosomatidae protozoan parasite of gecko Tarentola annularis, which has recently been introduced as a candidate for heterologous gene expression. In this study, the hIFN-γ cDNA was amplified from phyto-hemagglutinin-stimulated peripheral blood mononuclear cells of a healthy blood donor using RT–PCR. In order to express, the rhIFN-γ protein, the resulting cDNA was cloned in two expression cassettes (each containing one copy of hIFN-γ cDNA) and integrated into the small subunit of ribosomal RNA gene of L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Western blotting of rhIFN-γ and ELISA confirmed the expression and production of 9.5 mg of rhIFN-γ protein/l respectively.     

IFN-λ1 in CHO cells: its expression and biological activity

Background

Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.

Results

A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 10⁶ IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.

Conclusion

The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

     

Musashi expression in ��-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic β-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates β-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of β-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and β-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and β-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory β-cell proliferation and the loss of β-cell gene expression seen in specific phases of the progression to type 2 diabetes.     

MyD88 and STING signaling pathways are required for IRF3-mediated IFN-β induction in response to Brucella abortus infection

Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis.