Optimizing conditions for calcium phosphate mediated transient transfection.

BACKGROUND: Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. RESULTS: In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate-DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells. CONCLUSIONS: Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

KEY POINTS: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+ , ATP, caffeine and luminal Ca2+ . In the presence of Mg2+ , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of ß-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+ -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+ -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca2+ -release in skeletal muscle. ABSTRACT: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca2+ , ATP, adenine, caffeine and to luminal Ca2+ . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+ , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC-A.

TRIM72 modulates caveolar endocytosis in repair of lung cells.

Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.

TRIM72 is required for effective repair of alveolar epithelial cell wounding.

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.

Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischaemia-reperfusion and overventilation-induced injury to the lung when compared with wild-type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases.

PRDM14 promotes active DNA demethylation through the ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells.

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

Facilitation of DNA damage-induced apoptosis by endoplasmic reticulum protein mitsugumin23.

The endoplasmic reticulum (ER) emanates context-dependent signals, thereby mediating cellular response to a variety of stresses. However, the underlying molecular mechanisms have been enigmatic. To better understand the signaling capacity of the ER, we focused on roles played by mitsugumin23 (MG23), a protein residing predominantly in this organelle. Overexpression of MG23 in human embryonic kidney 293T cells specifically enhanced apoptosis triggered by etoposide, a DNA-damaging anti-cancer drug. Conversely, genetic deletion of MG23 reduced susceptibility of thymocytes to DNA damage-induced apoptosis, which was demonstrated by whole-body irradiation experiments. In this setting, induction of the tumor-suppressor gene p53 was attenuated in MG23-knockout thymocytes as compared with their wild-type counterparts, consistent with the elevated radioresistance. It is therefore suggested that MG23 is an essential component of ER-generated lethal signals provoked upon DNA damage, specifying cell fate under pathophysiological conditions.

Essential role of the TRIC-B channel in Ca2+ handling of alveolar epithelial cells and in perinatal lung maturation.

TRIC channels function as monovalent cation-specific channels that mediate counter ion movements coupled with ryanodine receptor-mediated Ca(2+) release from intracellular stores in muscle cells. Mammalian tissues differentially contain two TRIC channel subtypes: TRIC-A is abundantly expressed in excitable cells, whereas TRIC-B is ubiquitously expressed throughout tissues. Here, we report the physiological role of TRIC-B channels in mouse perinatal development. TRIC-B-knockout neonates were cyanotic owing to respiratory failure and died shortly after birth. In the mutant neonates, the deflated lungs exhibited severe histological defects, and alveolar type II epithelial cells displayed ultrastructural abnormalities. The metabolic conversion of glycogen into phospholipids was severely interrupted in the mutant type II cells, and surfactant phospholipids secreted into the alveolar space were insufficient in the mutant neonates. Moreover, the mutant type II cells were compromised for Ca(2+) release mediated by inositol-trisphosphate receptors, despite Ca(2+) overloading in intracellular stores. Our results indicate that TRIC-B channels take an active part in Ca(2+) signalling to establish specialised functions in type II cells and are thus essential for perinatal lung maturation.

Collaborative work on evaluation of ovarian toxicity. 5) Two- or four-week repeated-dose studies and fertility study of busulfan in female rats.

Busulfan, an antineoplastic agent that targets small follicles (primordial and primary follicles), was given orally to female Sprague-Dawley rats (0, 0.1, 0.5, or 1.5 mg/kg/day; n = 10 in each group) for 2 or 4 weeks to assess the optimal administration period for detection of the toxic effects on ovarian morphology. Isolated ovaries were used for histopathological analysis and follicle counts. In addition, a female fertility study was conducted by giving the same dose levels of busulfan from 2 weeks before mating to day 7 of pregnancy to determine the non-observed-adverse-effect-level (NOAEL) for female reproduction. In the 2-week study, all rats treated with busulfan showed normal estrous cyclicity and no toxicological changes in weight or histopathology of the ovaries. In the 4-week study, a decrease in small follicles was found histopathologically in 1 rat, even at 0.5 mg/kg, and in 4 rats at 1.5 mg/kg. Proliferating cell nuclear antigen immunohistochemistry of the follicles confirmed the above decrease in number of small follicles at 1.5 mg/kg. In the female fertility study, increases in dead embryos and post-implantation loss were found in rats at 1.5 mg/kg. Taken together, the NOAELs were 1.5 mg/kg for reproductive performance and 0.5 mg/kg for early embryonic development. In conclusion, the present study indicates that a 4-week administration period and appropriate assessment, including careful histopathological analysis of stage-based follicles are needed to detect small follicle depletion in a general toxicity study used as a first-titer screen.

Augmentation of drug-induced cell death by ER protein BRI3BP.

To determine the contribution of the endoplasmic reticulum (ER) to cell fate decision, we focused on BRI3-binding protein (BRI3BP) residing in this organelle. BRI3BP, when overexpressed, augmented the apoptosis of human embryonic kidney 293T cells challenged with drugs including the anti-cancer agent etoposide. In contrast, the knockdown of BRI3BP reduced the drug-triggered apoptosis. BRI3BP overexpression enhanced both mitochondrial cytochrome c release and caspase-3 activity in etoposide-treated cells. In response to etoposide, the ER reorganized into irregularly shaped lamellae in mock-transfected cells, whereas in BRI3BP-overexpressing cells, such reorganization was not observed. These observations suggest that BRI3BP is involved in the structural dynamics of the ER and affects mitochondrial viability. Taken together, BRI3BP, widely expressed in animal cell types, seems to possess a pro-apoptotic property and can potentiate drug-induced apoptosis.

Co-expression of MG29 and ryanodine receptor leads to apoptotic cell death: effect mediated by intracellular Ca2+ release.

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.