The inotropic agent digitoxin strengthens desmosomal adhesion in cardiac myocytes in an ERK1/2-dependent manner.

Desmosomal proteins are components of the intercalated disc and mediate cardiac myocyte adhesion. Enhancement of cardiac myocyte cohesion, referred to as "positive adhesiotropy", was demonstrated to be a function of sympathetic signaling and to be relevant for a sufficient inotropic response. We used the inotropic agent digitoxin to investigate the link between inotropy and adhesiotropy. In contrast to wild-type hearts, digitoxin failed to enhance pulse pressure in perfused mice hearts lacking the desmosomal protein plakoglobin which was paralleled with abrogation of plaque thickening indicating that positive inotropic response requires intact desmosomal adhesion. Atomic force microscopy revealed that digitoxin increased the binding force of the adhesion molecule desmoglein-2 at cell-cell contact areas. This was paralleled by enhanced cardiac myocyte cohesion in both HL-1 cardiac myocytes and murine cardiac slices as determined by dissociation assays as well as by accumulation of desmosomal proteins at cell-cell contact areas. However, total protein levels or cytoskeletal anchorage were not affected. siRNA-mediated depletion of desmosomal proteins abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2. In line with this, inhibition of ERK1/2 signaling abrogated the effects of digitoxin on cell-cell adhesion and desmosomal reorganization. These results show that the positive inotropic agent digitoxin enhances cardiac myocyte cohesion with reorganization of desmosomal proteins in an ERK1/2-dependent manner. Desmosomal adhesion seems to be important for a sufficient positive inotropic response of digitoxin treatment, which can be of medical relevance for the treatment of heart failure.

Phosphorylation of PFKL regulates metabolic reprogramming in macrophages following pattern recognition receptor activation.

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.

Oxysterole-binding protein targeted by SARS-CoV-2 viral proteins regulates coronavirus replication.

Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.

Adrenergic Signaling-Induced Ultrastructural Strengthening of Intercalated Discs via Plakoglobin Is Crucial for Positive Adhesiotropy in Murine Cardiomyocytes.

Intercalated discs (ICDs), which connect adjacent cardiomyocytes, are composed of desmosomes, adherens junctions (AJs) and gap junctions (GJs). Previous data demonstrated that adrenergic signaling enhances cardiac myocyte cohesion, referred to as positive adhesiotropy, via PKA-mediated phosphorylation of plakoglobin (PG). However, it was unclear whether positive adhesiotropy caused ultrastructural modifications of ICDs. Therefore, we further investigated the role of PG in adrenergic signaling-mediated ultrastructural changes in the ICD of cardiomyocytes. Quantitative transmission electron microscopy (TEM) analysis of ICD demonstrated that cAMP elevation caused significant elongation of area composita and thickening of the ICD plaque, paralleled by enhanced cardiomyocyte cohesion, in WT but not PG-deficient cardiomyocytes. STED microscopy analysis supported that cAMP elevation ex vivo enhanced overlap of desmoglein-2 (Dsg2) and N-cadherin (N-cad) staining in ICDs of WT but not PG-deficient cardiomyocytes. For dynamic analyses, we utilized HL-1 cardiomyocytes, in which cAMP elevation induced translocation of Dsg2 and PG but not of N-cad to cell junctions. Nevertheless, depletion of N-cad but not of Dsg2 resulted in a decrease in basal cell cohesion whereas positive adhesiotropy was abrogated in monolayers depleted for either Dsg2 or N-cad. In the WT mice, ultrastrutural changes observed after cAMP elevation were paralleled by phosphorylation of PG at serine 665. Our data demonstrate that in murine hearts adrenergic signaling enhanced N-cad and Dsg2 in the ICD paralleled by ultrastrutural strengthening of ICDs and that effects induced by positive adhesiotropy were strictly dependent on Pg.

Novel mouse mutants with primary cellular immunodeficiencies generated by genome-wide mutagenesis.

BACKGROUND: Primary cellular immunodeficiencies are a group of genetic disorders in which 1 or more components of the cellular immune system are lacking or dysfunctional. OBJECTIVE: We sought to identify novel mouse mutants that display primary cellular immunodeficiencies. METHODS: Genome-wide N-ethyl-N-nitrosourea mutagenesis was performed in mice, followed by a phenotype screen of immunologic blood parameters. RESULTS: We identified novel mouse mutants with isolated B-cell deficiency, combined block in early B- and T-cell development, combined T-cell and natural killer cell reduction, and 3 different forms of T-cell deficiencies. One of the mutants, designated DeltaT3, displayed a combined phenotype of increased IgE, absence of peripheral T cells, and block in late thymocyte differentiation. In addition, DeltaT3 mice were unable to mount specific humoral immune responses. Chromosomal mapping and sequencing of candidate genes revealed a novel point mutation in the kinase domain of the T-cell receptor zeta chain-associated protein kinase (Zap70). In contrast to Zap70-deficient mice, DeltaT3 mutants displayed normal Zap70 mRNA and residual Zap70 protein levels. Complementation studies with Zap70-deficient mice confirmed that the point mutation found in Zap70 was causative for the DeltaT3 phenotype, including increased IgE plasma levels, a phenotype that has not been associated with altered Zap70 function in the past. CONCLUSION: Random genome-wide mutagenesis combined with a phenotype screen can be used to generate novel mouse mutants with primary cellular immunodeficiencies.

Specific transgene expression in mouse pancreatic beta-cells under the control of the porcine insulin promoter.

The availability of regulatory sequences directing tissue-specific expression of transgenes in genetically modified mice and large animals is a prerequisite for the development of adequate models for human diseases. The rat insulin 2 gene (Ins2) promoter, widely used to achieve transgene expression in pancreatic beta-cells of mice, also directs expression to extrapancreatic tissues and performs poorly in isolated pancreatic islets of human, mouse, and pig. To evaluate whether the full 5' untranslated region (UTR) of the porcine insulin gene (INS) confers robust and specific expression in beta-cells we generated an expression cassette containing 1500bp of the porcine INS 5' UTR and the 3' UTR of the bovine growth hormone gene (GH). The cassette was designed to allow easy exchange of the sequences to be expressed and easy removal of the vector backbone from the expression cassette. To evaluate the properties of the cassette, we initially inserted a cDNA encoding human betacellulin, a growth factor known to affect structural and functional parameters of beta-cells. After confirming the functionality and specificity of the construct in vitro, transgenic mouse lines were generated by pronuclear DNA microinjection. Using RT-PCR, immunohistochemistry and immunofluorescence, we show that transgenic mice expressed human betacellulin exclusively in beta-cells. Confirming the proposed insulinotropic effect of betacellulin, transgenic mice showed improved glucose tolerance. We conclude that the newly designed expression cassette containing 1500bp of the porcine insulin promoter 5' UTR confers robust and specific transgene expression to beta-cells in vitro and in transgenic mice.

Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos.

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.