Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.     

Influence of cytoskeleton organization on recombinant protein expression by CHO cells

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.     

Mammalian proapoptotic factor ChaC1 and its homologues function as γ‐glutamyl cyclotransferases acting specifically on glutathione

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ‐glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ‐glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E>Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells.     

Overexpression of TLR2 and TLR4 susceptibility to serum deprivation-induced apoptosis in CHO cells

We examined the effect of overexpression of TLR2 and TLR4 on apoptosis. TLR2 and TLR4 transfected CHO cells were subjected to serum deprivation for 0, 24, and 48 h. CHO cells served as control. The survival was 80.4% and 66.8% in CHO cells, 73.8% and 47.6% in TLR2/CHO, and 70.5% and 53.0% in TLR4/CHO, respectively. Flow cytometry examination suggested that apoptotic cells were 7.17% and 32.91% in control CHO cells, 29.0% and 64.6% in TLR2/CHO, and 41.4% and 64.6% in TLR4/CHO, respectively. The levels of FasL and caspase-8 activity in TLR2/CHO and TLR4/CHO cells were significantly higher than that of CHO cells. Transfection of dominant negative FADD into TLR2/CHO and TLR4/CHO cells significantly reduced apoptosis. Our results suggest that overexpression of TLR2 and TLR4 in CHO cells sensitizes the cells to serum deprivation-induced apoptosis and that the mechanisms are involved in the death receptor-mediated signaling pathway.     

Exploring the correlation between deltamethrin stress and Keap1-Nrf2-ARE pathway from Drosophila melanogaster RNASeq data

Deltamethrin (DM) is widely used in a variety of pest control, resulting in serious drug resistance. Keap1-Nrf2-ARE is the antioxidant stress pathway. We identified 268 genes differentially expressed (DEGs) in Drosophila Kc cells treated with DM, including up-regulated 180 genes and down-regulated 88 genes compared with the control group (fold-change≥2, qValue≤0.001) by RNA-seq, they are mainly linked to metabolic process, stimulation response, immune system process. When the cells are treated with DM in the case of overexpression of the Keap1 gene, the cytochrome P450 family genes were significantly down-regulated, and some diseases-related genes and non-coding genes also changed. Our data shown that Keap1-Nrf2-ARE pathway may play an important role in DM stress, which will provide a new direction for studying the mechanism of insect resistance.     

Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation

Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.     

Overexpression of GRP78 enhances survival of CHO cells in response to serum deprivation and oxidative stress

Chinese hamster ovary (CHO) cells are regarded as one of the most commonly used mammalian hosts, which decreases the productivity due to loss in culture viability. Overexpressing antiapoptosis genes in CHO cells was developed as a means of limiting cell death upon exposure to environmental insults. Glucose‐regulated protein 78 (GRP78) is traditionally regarded as a major ER chaperone that participates in protein folding and other cell processes. It is also a potent antiapoptotic protein and plays a critical role in cell survival, proliferation, and metastasis. In this study, the impact of GRP78 on CHO cells in response to environmental insults such as serum deprivation and oxidative stress was investigated. First, it was confirmed that CHO cells were very sensitive to environmental insults. Then, GRP78 overexpressing CHO cell line was established and exposed to serum deprivation and H₂O₂. Results showed that GRP78 engineering increased the viability and decreased the apoptosis of CHO cells. The survival advantage due to GRP78 engineering could be mediated by suppression of caspase‐3 involved in cell death pathways in stressed cells. Besides, GRP78 engineering also enhanced yields of antibody against transferrin receptor in CHO cells. GRP78 should be a potential application in the biopharmaceutical industries.     

Nogo-B promotes angiogenesis and improves cardiac repair after myocardial infarction via activating Notch1 signaling

Nogo-B (Reticulon 4B) is reportedly a regulator of angiogenesis during the development and progression of cancer. However, whether Nogo-B regulates angiogenesis and post-myocardial infarction (MI) cardiac repair remains elusive. In the present study, we aimed to explore the role and underlying mechanisms of Nogo-B in cardiac repair during MI. We observed an increased expression level of Nogo-B in the heart of mouse MI models, as well as in isolated cardiac microvascular endothelial cells (CMECs). Moreover, Nogo-B was significantly upregulated in CMECs exposed to oxygen-glucose deprivation (OGD). Nogo-B overexpression in the endothelium via cardiotropic adeno-associated virus serotype 9 (AAV9) with the mouse endothelial-specific promoter Tie2 improved heart function, reduced scar size, and increased angiogenesis. RNA-seq data indicated that Notch signaling is a deregulated pathway in isolated CMECs along the border zone of the infarct with Nogo-B overexpression. Mechanistically, Nogo-B activated Notch1 signaling and upregulated Hes1 in the MI hearts. Inhibition of Notch signaling using a specific siRNA and γ-secretase inhibitor abolished the promotive effects of Nogo-B overexpression on network formation and migration of isolated cardiac microvascular endothelial cells (CMECs). Furthermore, endothelial Notch1 heterozygous deletion inhibited Nogo-B-induced cardioprotection and angiogenesis in the MI model. Collectively, this study demonstrates that Nogo-B is a positive regulator of angiogenesis by activating the Notch signaling pathway, suggesting that Nogo-B is a novel molecular target for ischemic disease.Subject terms: Heart failure, Ischaemia     

Maternal Nicotine Exposure Leads to Impaired Disulfide Bond Formation and Augmented Endoplasmic Reticulum Stress in the Rat Placenta

Maternal nicotine exposure has been associated with many adverse fetal and placental outcomes. Although underlying mechanisms remain elusive, recent studies have identified that augmented endoplasmic reticulum (ER) stress is linked to placental insufficiency. Moreover, ER function depends on proper disulfide bond formation—a partially oxygen-dependent process mediated by protein disulfide isomerase (PDI) and ER oxidoreductases. Given that nicotine compromised placental development in the rat, and placental insufficiency has been associated with poor disulfide bond formation and ER stress, we hypothesized that maternal nicotine exposure leads to both placental ER stress and impaired disulfide bond formation. To test this hypothesis, female Wistar rats received daily subcutaneous injections of either saline (vehicle) or nicotine bitartrate (1 mg/kg) for 14 days prior to mating and during pregnancy. Placentas were harvested on embryonic day 15 for analysis. Protein and mRNA expression of markers involved in ER stress (e.g., phosphorylated eIF2α, Grp78, Atf4, and CHOP), disulfide bond formation (e.g., PDI, QSOX1, VKORC1), hypoxia (Hif1α), and amino acid deprivation (GCN2) were quantified via Western blot and/or Real-time PCR. Maternal nicotine exposure led to increased expression of Grp78, phosphorylated eIF2α, Atf4, and CHOP (p<0.05) in the rat placenta, demonstrating the presence of augmented ER stress. Decreased expression of PDI and QSOX1 (p<0.05) reveal an impaired disulfide bond formation pathway, which may underlie nicotine-induced ER stress. Finally, elevated expression of Hif1α and GCN2 (p<0.05) indicate hypoxia and amino acid deprivation in nicotine-exposed placentas, respectively, which may also cause impaired disulfide bond formation and augmented ER stress. This study is the first to link maternal nicotine exposure with both placental ER stress and disulfide bond impairment in vivo, providing novel insight into the mechanisms underlying nicotine exposure during pregnancy on placental health.     

Genetic favouring of pheomelanin‐based pigmentation limits physiological benefits of coloniality in lesser kestrels Falco naumanni

Pheomelanin contributes to the pigmentation phenotype of animals by producing orange and light brown colours in the integument. However, pheomelanin synthesis in melanocytes requires consumption of glutathione (GSH), the most important intracellular antioxidant. Therefore, a genetic control favouring the production of large amounts of pheomelanin for pigmentation may lead to physiological costs under environmental conditions that promote oxidative stress. We investigated this possibility in the context of breeding coloniality, a reproductive strategy that may affect oxidative stress. We found in lesser kestrel Falco naumanni nestlings that the GSH:GSSG ratio, which decreases with systemic oxidative stress, increased with the size of the colony where they were reared, but the expression in feather melanocytes of five genes involved in pheomelanin synthesis (Slc7a11, Slc45a2, CTNS, MC1R and AGRP) did not vary with colony size. The antioxidant capacity (TEAC) of lesser kestrel nestlings also increased with colony size, but in a manner that depended on Slc7a11 expression and not on the expression of the other genes. Thus, antioxidant capacity increased with colony size only in nestlings least expressing Slc7a11, a gene with a known role in mediating cysteine (a constituent amino acid of GSH) consumption for pheomelanin production. The main predictor of the intensity of pheomelanin‐based feather colour was Slc45a2 expression followed in importance by Slc7a11 expression, hence suggesting that the genetic regulation of the pigmentation phenotype mediated by Slc7a11 and a lack of epigenetic lability in this gene limits birds from benefiting from the physiological benefits of coloniality.