Abstract BACKGROUND Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults, characterized by poor patient survival rates. The glycoproteins Integrin αV (IαV), and the Urokinase-type plasminogen activator receptor (uPAR) are key contributors to tumor malignancy in GBM, and although their interaction is well-described, the role of glycans in this process has been scarcely evaluated. Better understanding this interaction could enhance our knowledge of the disease and lead to potential new therapeutics.METHODS We investigated the interaction between IαV and uPAR in human GBM, A172 and LN229, and low-grade glioma, SW1088, cell lines. Expression of these proteins was confirmed via confocal microscopy and co-immunoprecipitation. The role of N-glycosylation was evaluated using the inhibitor Swainsonine (SW) and glycosidase PNGase F. Glycoproteomic analysis by mass spectrometry identified glycosylation sites and differential structures on IαV. The impact of sialic acids and specific glycan structures was assessed using Neuraminidase (NeuA) and lectin binding assays.RESULTS The expression of IαV and uPAR, as well as their interaction, was confirmed in GBM cells but not in low-grade glioma cells, even when uPAR was overexpressed. SW and PNGase treatments markedly reduced IαV/uPAR interaction, highlighting the importance of N-glycosylation. Mass spectrometry analysis showed six glycosylation sites on IαV in GBM cells, with complex and hybrid N-glycans, while only oligomannose N-glycans were detected in low-grade glioma cells. NeuA treatment also reduced IαV/uPAR interaction, underscoring the role of sialic acids. Lectin assays suggested β1–6 branched glycans at specific sites are crucial for this interaction. Inhibition of N-glycosylation and sialic acid removal both decreased AKT phosphorylation, indicating a significant role of these glycans in integrin/uPAR signaling.CONCLUSIONS Our results demonstrate for the first time the interaction between IαV and uPAR in GBM cells, highlighting the critical role of N-glycosylation, particularly β1–6 branched glycans and sialic acids.

Acting as GTPase activating proteins promoting the silencing of activated G-proteins, regulators of G protein signaling (RGSs) are generally considered negative modulators of cell signaling. In the CNS, the expression of RGS4 is altered in diverse pathologies and its upregulation was reported in astrocytes exposed to an inflammatory environment. In a model of cultured cortical astrocytes, we herein investigate the influence of RGS4 on intracellular calcium signaling mediated by type 5 metabotropic glutamate receptor (mGluR5), which is known to support the bidirectional communication between neurons and glial cells. RGS4 activity was manipulated by exposure to the inhibitor CCG 63802 or by infecting the cells with lentiviruses designed to achieve the silencing or overexpression of RGS4. The pharmacological inhibition or silencing of RGS4 resulted in a decrease in the percentage of cells responding to the mGluR5 agonist DHPG and in the proportion of cells showing typical calcium oscillations. Conversely, RGS4-lentivirus infection increased the percentage of cells showing calcium oscillations. While the physiological implication of cytosolic calcium oscillations in astrocytes is still under investigation, the fine-tuning of calcium signaling likely determines the coding of diverse biological events. Indirect signaling modulators such as RGS4 inhibitors, used in combination with receptor ligands, could pave the way for new therapeutic approaches for diverse neurological disorders with improved efficacy and selectivity.

The incidence of gestational diabetes mellitus (GDM) and metabolic disorders during pregnancy are increasing globally. This has resulted in increased use of therapeutic interventions such as metformin to aid in glycemic control during pregnancy. Even though metformin can cross the placental barrier, its impact on offspring brain development remains poorly understood. As metformin promotes AMPK signaling, which plays a key role in axonal growth during development, we hypothesized that it may have an impact on hypothalamic signaling and the formation of neuronal projections in the hypothalamus, the key regulator of energy homeostasis. We further hypothesized that this is dependent on the metabolic and nutritional status of the mother at the time of metformin intervention. Using mouse models of maternal overnutrition, we aimed to assess the effects of metformin exposure on offspring physiology and hypothalamic neuronal circuits during key periods of development.
Female C57BL/6N mice received either a control diet or a high-fat diet (HFD) during pregnancy and lactation periods. A subset of dams was fed a HFD exclusively during the lactation. Anti-diabetic treatments were given during the first postnatal weeks. Body weights of male and female offspring were monitored daily until weaning. Circulating metabolic factors and molecular changes in the hypothalamus were assessed at postnatal day 16 using ELISA and Western Blot, respectively. Hypothalamic innervation was assessed by immunostaining at postnatal days 16 and 21.
We identified alterations in weight gain and circulating hormones in male and female offspring induced by anti-diabetic treatment during the early postnatal period, which were critically dependent on the maternal metabolic state. Furthermore, hypothalamic agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neuronal innervation outcomes in response to anti-diabetic treatment were also modulated by maternal metabolic state. We also identified sex-specific changes in hypothalamic AMPK signaling in response to metformin exposure.
We demonstrate a unique interaction between anti-diabetic treatment and maternal metabolic state, resulting in sex-specific effects on offspring brain development and physiological outcomes. Overall, based on our findings, no positive effect of metformin intervention was observed in the offspring, despite ameliorating effects on maternal metabolic outcomes. In fact, the metabolic state of the mother drives the most dramatic differences in offspring physiology and metformin had no rescuing effect. Our results therefore highlight the need for a deeper understanding of how maternal metabolic state (excessive weight gain versus stable weight during GDM treatment) affects the developing offspring. Further, these results emphasize that the interventions to treat alterations in maternal metabolism during pregnancy need to be reassessed from the perspective of the offspring physiology.
Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.

Hepatocellular carcinoma (HCC) is associated with chronic inflammation caused by different factors; especially, the interaction of inflammatory pathways and bile acids (BAs) can affect hepatocyte proliferation, death, and regeneration, but whether BAs promote HCC progression through inflammatory pathways and the mechanisms is still unclear.
By examining cancer and tumor-adjacent tissue BA levels and genes associated with BA homeostasis in 37 HCC patients, we found that total bile acids (TBAs) were decreased by 36% and varying degrees of changes in factors regulating BA homeostasis (p < 0.05). In addition, we found that BA homeostasis was disturbed in diethylnitrosamine-induced HCC mouse models, and TBA was correlated with inflammasome activation during HCC progression (6-24 W) (p < 0.05). Similarly, the inflammasome and chenodeoxycholic acid (CDCA) content were suppressed in cholestasis model mice (Mrp2-deficient mice) (p < 0.05). In vitro, CDCA significantly promoted the malignant transformation of hepatocytes (p < 0.001), activated the inflammasome by triggering the release of mitochondrial reactive oxygen species and mitochondrial DNA, and ultimately induced pyroptosis. Furthermore, we found that CDCA has a targeted binding effect with HO-1 through molecular docking and Cellular Thermal Shift Assay experiments.
In conclusion, we found that CDCA can trigger the excessive accumulation of mitochondrial reactive oxygen species by targeting HO-1 to promote the activation of the inflammasome and ultimately promote the progression of HCC. Our study provides a novel mechanism by which BAs promote HCC by activating the inflammasome and establishes the important role of BA homeostasis imbalance in the progression of HCC from the aspect of inflammation.
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival and autophagy. Less known is its involvement in the differentiation of cardiomyocytes. As a consequence, mechanisms by which Bcl-2 contributes to cardiac differentiation remain to be elucidated. To address this, we used CRISPR/Cas9 to knockout (KO) BCL2 in human induced pluripotent stem cells (hiPSCs) and investigated the consequence of this KO for differentiation towards cardiomyocytes. Our results indicate that differentiation of hiPSCs to cardiomyocytes was delayed following BCL2 KO. This was not related to the canonical anti-apoptotic function of Bcl-2. This delay led to reduced expression and activity of the cardiomyocyte Ca2+ toolkit. Finally, Bcl-2 KO reduced c-Myc expression and nuclear localization in the early phase of the cardiac differentiation process, which accounts at least in part for the observed delay in the cardiac differentiation. These results suggest that there is a central role for Bcl-2 in cardiomyocyte differentiation and maturation.
© 2023. Published by The Company of Biologists Ltd.