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The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca 2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca 2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca 2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca 2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP 3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca 2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca 2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system., (© 2019 Wiley Periodicals, Inc.)

Leptin is produced in the adipocytes and plays a pivotal role in regulation of energy balance by controlling appetite and metabolism. Leptin receptors are widely distributed in the brain, especially in the hypothalamus, hippocampus, and neocortex. The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. However, it is still an open issue whether and how leptin modulates IC neural activities. Our paired whole-cell patch-clamp recordings using IC slice preparations demonstrated that unitary inhibitory postsynaptic currents (uIPSCs) but not uEPSCs were potentiated by leptin in the connections between pyramidal (PNs) and fast-spiking neurons (FSNs). The leptin-induced increase in uIPSC amplitude was accompanied by a decrease in paired-pulse ratio. Under application of inhibitors of JAK2-PI3K but not MAPK pathway, leptin did not change uIPSC amplitude. Variance-mean analysis revealed that leptin increased the release probability but not the quantal size and the number of release site. These electrophysiological findings suggest that the leptin-induced uIPSC increase is mediated by activation of JAK2-PI3K pathway in presynaptic FSNs. An in vivo optical imaging revealed that leptin application decreased excitatory propagation in IC induced by electrical stimulation of IC. These leptin-induced effects were not observed under the low energy states: low glucose concentration (2.5 mM) in vitro and one-day-fasting condition in vivo. However, leptin enhanced uIPSCs under application of low glucose with an AMPK inhibitor. These results suggest that leptin suppresses IC excitation by facilitating GABA release in FSN→PN connections, which may not occur under a hunger state., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Skin atrophy is a major adverse effect of topical glucocorticoids. We recently reported that REDD1 (regulated in development and DNA damage 1) and FKBP51 (FK506 binding protein 5), negative regulators of mTOR/Akt signaling, are induced by glucocorticoids in mouse and human skin and are central drivers of steroid skin atrophy. Thus, we hypothesized that REDD1/FKBP51 inhibitors could protect skin against catabolic effects of glucocorticoids., Methods: Using drug repurposing approach, we screened LINCS library (http://lincsproject.org/LINCS/) to identify repressors of REDD1/FKBP51 expression. Candidate compounds were tested for their ability to inhibit glucocorticoid-induced REDD1/FKBP51 expression in human primary/immortalized keratinocytes and in mouse skin. Reporter gene expression, microarray, and chromatin immunoprecipitation were employed to evaluate effect of these inhibitors on the glucocorticoid receptor (GR) signaling., Findings: Bioinformatics analysis unexpectedly identified phosphoinositide-3-kinase (PI3K)/mTOR/Akt inhibitors as a pharmacological class of REDD1/FKBP51 repressors. Selected PI3K/mTOR/Akt inhibitors-Wortmannin (WM), LY294002, AZD8055, and two others indeed blocked REDD1/FKBP51expression in human keratinocytes. PI3K/mTOR/Akt inhibitors also modified global effect of glucocorticoids on trascriptome, shifting it towards therapeutically important transrepression; negatively impacted GR phosphorylation; nuclear translocation; and GR loading on REDD1/FKBP51 gene promoters. Further, topical application of LY294002 together with glucocorticoid fluocinolone acetonide (FA) protected mice against FA-induced proliferative block and skin atrophy but did not alter the anti-inflammatory activity of FA in ear edema test., Interpretation: Our results built a strong foundation for development of safer GR-targeted therapies for inflammatory skin diseases using combination of glucocorticoids with PI3K/mTOR/Akt inhibitors. FUND: Work is supported by NIH grants R01GM112945, R01AI125366, and HESI-THRIVE foundation., (Copyright © 2019. Published by Elsevier B.V.)

Cisplatin is regularly used in the treatment of lung cancer. However, its efficacy is limited because of drug resistance. In this study, we found that Akt expression and activity was increased in lung cancer cells with acquired cisplatin resistance (A549/DDP cells and H460/DDP cells) when compared to their parental cells. Inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt kinase activity by its natural inhibitor, Wortmannin, could sensitize DDP-resistant cells to DDP and reverse DDP resistance. Combination treatment of Wortmannin with cisplatin is capable of increasing the mortality rate of both A549/DDP cells and H460/DDP cells. The present study also demonstrated that hyperactivation of PI3K/Aktpathway is closely associated with cisplatin resistance by regulating the Bax-mitochondria-mediated apoptosis pathway in human lung cancer. Inhibition of PI3K/Aktactivity in A549/DDP cells and H460/DDP cells could reverse cisplatin resistance by enhancing the effect of cisplatin on Bax oligomerization and release of Cytochrome C, allowing activation of the caspase-mediated apoptosis pathway. In conclusion, cisplatin resistance of lung cancer can be reversed via the inhibition of the PI3K/Akt signaling pathway. Therefore, both PI3K and Akt may be potential targets for overcoming cisplatin resistance in lung cancer.