Membrane Translocation Research Articles

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCβ-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

A20 promotes colorectal cancer immune evasion by upregulating STC1 expression to block “eat-me” signal

Immune checkpoint inhibitors (ICIs) have induced durable clinical responses in a subset of patients with colorectal cancer (CRC). However, the dis-satisfactory response rate and the lack of appropriate biomarkers for selecting suitable patients to be treated with ICIs pose a major challenge to current immunotherapies. Inflammation-related molecule A20 is closely related to cancer immune response, but the effect of A20 on “eat-me” signal and immunotherapy efficacy remains elusive. We found that A20 downregulation prominently improved the antitumor immune response and the efficacy of PD-1 inhibitor in CRC in vitro and in vivo. Higher A20 expression was associated with less infiltration of immune cells including CD3 (+), CD8 (+) T cells and macrophages in CRC tissues and also poorer prognosis. Gain- and loss-A20 functional studies proved that A20 could decrease the “eat-me” signal calreticulin (CRT) protein on cell membrane translocation via upregulating stanniocalcin 1 (STC1), binding to CRT and detaining in mitochondria. Mechanistically, A20 inhibited GSK3β phosphorylating STC1 at Thr86 to slow down the degradation of STC1 protein. Our findings reveal a new crosstalk between inflammatory molecule A20 and “eat-me” signal in CRC, which may represent a novel predictive biomarker for selecting CRC patients most likely to benefit from ICI therapy.

Cryo-EM structures of metazoan AAA protein Bcs1L captured in ATP hydrolysis suggested a concerted membrane translocation mechanism of folded substrate

Cryo-EM structures of metazoan AAA protein Bcs1L captured in ATP hydrolysis suggested a concerted membrane translocation mechanism of folded substrate

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia

BackgroundThe urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. MethodsThe effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model.Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. ResultsWe initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1β, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. ConclusionCrebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

TRIM21-Promoted FSP1 Plasma Membrane Translocation Confers Ferroptosis Resistance in Human Cancers.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive accumulation of lipid peroxides, has become a promising strategy in cancer treatment. Cancer cells exploit antioxidant proteins, including Ferroptosis Suppressor Protein 1 (FSP1), to prevent ferroptosis. In this study, it is found that the E3 ubiquitin ligase TRIM21 bound to FSP1 and mediated its ubiquitination on K322 and K366 residues via K63 linkage, which is essential for its membrane translocation and ferroptosis suppression ability. It is further verified the protective role of the TRIM21-FSP1 axis in RSL3-induced ferroptosis in cancer cells and a subcutaneous tumor model. Moreover, TRIM21 is highly expressed in multiple gastrointestinal (GI) tumors, and its expression is further stimulated upon ferroptosis induction in cancer cells and the KPC mouse model. In summary, This study identifies TRIM21 as a negative regulator of ferroptosis through K63 ubiquitination of FSP1, which can serve as a therapeutic target to enhance the chemosensitivity of tumors based on ferroptosis induction.

Nuclear Transport of the Molecular Drug via Nanocarrier-Based Nonendocytic Cellular Uptake.

Although subcellular targeting can enhance the therapeutic performance of most drugs, such targeting requires appropriate carrier-based delivery that can bypass endosomal/lysosomal trafficking. Recent works show that nanocarriers can be designed for direct cell membrane translocation and nonendocytic uptake, bypassing the usual endocytosis processes. Here we show that this approach can be adapted for the rapid cell nucleus delivery of molecular drugs. In particular, a guanidinium-terminated nanocarrier is used to create a weak interaction-based carrier-drug nanoassembly for direct membrane translocation into the cytosol. The rapid and extensive entry of a drug-loaded nanocarrier into the cell without any vesicular coating and affinity of the drug to the nucleus allows their nucleus labeling. Compared to endocytotic uptake that requires more than hours for cell uptake followed by predominant lysosomal entrapment, this nonendocytic uptake labels the nucleus within a few minutes without any lysosomal trafficking. This approach may be utilized for nanocarrier-based subcellular targeting of drugs for more effective therapy.

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

In Search of a Molecular View of Peptide-Lipid Interactions in Membranes.

Lipid bilayer membranes are often represented as a continuous nonpolar slab with a certain thickness bounded by two more polar interfaces. Phenomena such as peptide binding to the membrane surface, folding, insertion, translocation, and diffusion are typically interpreted on the basis of this view. In this Perspective, I argue that this membrane representation as a hydrophobic continuum solvent is not adequate to understand peptide-lipid interactions. Lipids are not small compared to membrane-active peptides: their sizes are similar. Therefore, peptide diffusion needs to be understood in terms of free volume, not classical continuum mechanics; peptide solubility or partitioning in membranes cannot be interpreted in terms of hydrophobic mismatch between membrane thickness and peptide length; peptide folding and translocation, often involving cationic peptides, can only be understood if realizing that lipids adapt to the presence of peptides and the membrane may undergo considerable lipid redistribution in the process. In all of those instances, the detailed molecular interactions between the peptide residues and the lipid components are essential to understand the mechanisms involved.

New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energise their transport into cells and catalyse their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Impaired Insulin Signaling Mediated by the Small GTPase Rac1 in Skeletal Muscle of the Leptin-Deficient Obese Mouse.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

EDA ligand triggers plasma membrane trafficking of its receptor EDAR via PKA activation and SNAP23-containing complexes

BackgroundEctodysplasin-A (EDA), a skin-specific TNF ligand, interacts with its membrane receptor EDAR to trigger EDA signaling in skin appendage formation. Gene mutations in EDA signaling cause Anhidrotic/Hypohidrotic Ectodermal Dysplasia (A/HED), which affects the formation of skin appendages including hair, teeth, and several exocrine glands.ResultsWe report that EDA triggers the translocation of its receptor EDAR from a cytosolic compartment into the plasma membrane. We use protein affinity purification to show that upon EDA stimulation EDAR associates with SNAP23-STX6-VAMP1/2/3 vesicle trafficking complexes. We find that EDA-dependent PKA activation is critical for the association. Notably, either of two HED-linked EDAR mutations, T346M and R420W, prevents EDA-induced EDAR translocation; and both EDA-induced PKA activation and SNAP23 are required for Meibomian gland (MG) growth in a skin appendage model.ConclusionsOverall, in a novel regulatory mechanism, EDA increases plasma membrane translocation of its own receptor EDAR, augmenting EDA-EDAR signaling in skin appendage formation. Our findings also provide PKA and SNAP23 as potential targets for the intervention of HED.

In situ structures of secretins from bacterial type II secretion system reveal their membrane interactions and translocation process

<i>In situ</i> structures of secretins from bacterial type II secretion system reveal their membrane interactions and translocation process

Cancer Drug Delivery Systems Using Bacterial Toxin Translocation Mechanisms.

Recent advances in targeted cancer therapy hold great promise for both research and clinical applications and push the boundaries in finding new treatments for various currently incurable cancers. However, these therapies require specific cell-targeting mechanisms for the efficient delivery of drug cargo across the cell membrane to reach intracellular targets and avoid diffusion to unwanted tissues. Traditional drug delivery systems suffer from a limited ability to travel across the barriers posed by cell membranes and, therefore, there is a need for high doses, which are associated with adverse reactions and safety concerns. Bacterial toxins have evolved naturally to specifically target cell subtypes via their receptor binding module, penetrating the cell membrane efficiently through the membrane translocation process and then successfully delivering the toxic cargo into the host cytosol. They have, thus, been harnessed for the delivery of various drugs. In this review, we focus on bacterial toxin translocation mechanisms and recent progress in the targeted delivery systems of cancer therapy drugs that have been inspired by the receptor binding and membrane translocation processes of the anthrax toxin protective antigen, diphtheria toxin, and Pseudomonas exotoxin A. We also discuss the challenges and limitations of these studies that should be addressed before bacterial toxin-based drug delivery systems can become a viable new generation of drug delivery approaches in clinical translation.

The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro, p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis.

Domperidone Inhibits Clostridium botulinum C2 Toxin and Bordetella pertussis Toxin.

Bordetella pertussis toxin (PT) and Clostridium botulinum C2 toxin are ADP-ribosylating toxins causing severe diseases in humans and animals. They share a common translocation mechanism requiring the cellular chaperones Hsp90 and Hsp70, cyclophilins, and FK506-binding proteins to transport the toxins' enzyme subunits into the cytosol. Inhibitors of chaperone activities have been shown to reduce the amount of transported enzyme subunits into the cytosol of cells, thus protecting cells from intoxication by these toxins. Recently, domperidone, an approved dopamine receptor antagonist drug, was found to inhibit Hsp70 activity. Since Hsp70 is required for cellular toxin uptake, we hypothesized that domperidone also protects cells from intoxication with PT and C2. The inhibition of intoxication by domperidone was demonstrated by analyzing the ADP-ribosylation status of the toxins' specific substrates. Domperidone had no inhibitory effect on the receptor-binding or enzyme activity of the toxins, but it inhibited the pH-driven membrane translocation of the enzyme subunit of the C2 toxin and reduced the amount of PTS1 in cells. Taken together, our results indicate that domperidone is a potent inhibitor of PT and C2 toxins in cells and therefore might have therapeutic potential by repurposing domperidone to treat diseases caused by bacterial toxins that require Hsp70 for their cellular uptake.

1631-P: scEMC10 Suppresses Glucose Uptake in Skeletal Muscle via the Inhibition of AMPK Signaling

Skeletal muscle is one of the most important organs involved in the glucose metabolism, and is mainly responsible for postprandial glucose uptake. We have previously shown that overexpression of the secreted isoform of endoplasmic reticulum protein complex subunit 10 (scEMC10) impairs, while Emc10 knockout (KO) or antibody neutralization of circulating scEMC10 improves glucose tolerance in mice. In this study, we presented evidence the regulation of glucose metabolism is achieved through the impacts of scEMC10 on skeletal muscle. PET-CT analyses showed Emc10 KO increased, whereas overexpression of scEMC10 decreased glucose uptake of skeletal muscle in mice, which can be accounted for by the corresponding changes in GLUT4 expression in their skeletal muscle tissues. Recombinant scEMC10 protein reduced both expression and membrane translocation of GLUT4, thus inhibiting glucose uptake in L6-GLUT4myc skeletal muscle cells. Mechanistically, scEMC10 decreased phosphorylations of both AMPK and its downstream target TBC1D1 in both skeletal muscle cells and tissues. Intraperitoneal administration of a scEMC10 neutralizing antibody increased both expression and membrane translocation of GLUT4 accompanied by increased uptake of glucose in gastrocnemius in mice. These data support scEMC10 as an inhibitor of glucose uptake in skeletal muscle and suggest the inhibition of scEMC10 will be a promising way to lower blood glucose in diabetes. Disclosure S.Jin: None. Y.Wang: None. K.Chen: None. J.Dai: None. L.Chen: None. S.Liu: None. X.Wang: None. Funding National Natural Science Foundation of China (81873645); Science and Technology Commission of Shanghai Municipality (22140902700)

Apolipoprotein E ε4 triggers neurotoxicity via cholesterol accumulation, acetylcholine dyshomeostasis, and PKCε mislocalization in cholinergic neuronal cells

The Apolipoprotein E (ApoE) has been known to regulate cholesterol and β-amyloid (Aβ) production, redistribution, and elimination, in the central nervous system (CNS). The ApoE ε4 polymorphic variant leads to impaired brain cholesterol homeostasis and amyloidogenic pathway, thus representing the major risk factor for Alzheimer's Disease (AD). Currently, less is known about the molecular mechanisms connecting ApoE ε4-related cholesterol metabolism and cholinergic system degeneration, one of the main AD pathological features. Herein, in vitro cholinergic neuron models were developed in order to study ApoE neuronal expression and investigate the possible interplay between cholesterol metabolism and cholinergic pathway impairment prompted by ε4 isoform. Particularly, alterations specifically occurring in ApoE ε4-carrying neurons (i.e. increased intracellular ApoE, amyloid precursor protein (APP) and Aβ levels, elevated apoptosis, and reduced cell survival) were recapitulated. ApoE ε4 expression was found to increase intracellular cholesterol accumulation, by regulating the related gene expression, while reducing cholesterol precursor acetyl-CoA, which in turn fuels the acetylcholine (ACh) synthesis route. In parallel, although the ACh intracellular signalling was activated, as demonstrated by the boosted extracellular ACh as well as increased IP3 and Ca2+, the PKCε activation via membrane translocation was surprisingly suppressed, probably explained by the cholesterol overload in ApoE ε4 neuron-like cells. Consequently, the PKC-dependent anti-apoptotic and neuroprotective roles results impaired, reliably adding to other causes of cell death prompted by ApoE ε4. Overall, the obtained data open the way to further critical considerations of ApoE ε4-dependent cholesterol metabolism dysregulation in the alteration of cholinergic pathway, neurotoxicity, and neuronal death.

Deoxyelephantopin—a novel PPARγ agonist regresses pressure overload-induced cardiac fibrosis via IL-6/STAT-3 pathway in crosstalk with PKCδ

Pathological cardiac hypertrophy is associated with ventricular fibrosis leading to heart failure. The use of thiazolidinediones as Peroxisome Proliferator-Activated Receptor-gamma (PPARγ)-modulating anti-hypertrophic therapeutics has been restricted due to major side-effects. The present study aims to evaluate the anti-fibrotic potential of a novel PPARγ agonist, deoxyelephantopin (DEP) in cardiac hypertrophy. AngiotensinII treatment in vitro and renal artery ligation in vivo were performed to mimic pressure overload-induced cardiac hypertrophy. Myocardial fibrosis was evaluated by Masson's trichrome staining and hydroxyproline assay. Our results showed that DEP treatment significantly improves the echocardiographic parameters by ameliorating ventricular fibrosis without any bystander damage to other major organs. Following molecular docking, all-atomistic molecular dynamics simulation, reverse transcription-polymerase chain reaction and immunoblot analyses, we established DEP as a PPARγ agonist stably interacting with the ligand-binding domain of PPARγ. DEP specifically downregulated the Signal Transducer and Activator of Transcription (STAT)-3-mediated collagen gene expression in a PPARγ-dependent manner, as confirmed by PPARγ silencing and site-directed mutagenesis of DEP-interacting PPARγ residues. Although DEP impaired STAT-3 activation, it did not have any effect on the upstream Interleukin (IL)-6 level implying possible crosstalk of the IL-6/STAT-3 axis with other signaling mediators. Mechanistically, DEP increased the binding of PPARγ with Protein Kinase C-delta (PKCδ) which impeded the membrane translocation and activation of PKCδ, downregulating STAT-3 phosphorylation and resultant fibrosis. This study, therefore, for the first time demonstrates DEP as a novel cardioprotective PPARγ agonist. The therapeutic potential of DEP as an anti-fibrotic remedy can be exploited against hypertrophic heart failure in the future.

Syntaxin4, P-cadherin, and CCAAT enhancer binding protein β as signaling elements in the novel differentiation pathway for cultured embryonic stem cells

Pluripotent stem cells possess the potential to differentiate into all three germ layers. However, upon removal of the stemness factors, pluripotent stem cells, such as embryonic stem cells (ESCs), exhibit EMT-like cell behavior and lose stemness signatures. This process involves the membrane translocation of the t-SNARE protein syntaxin4 (Stx4) and the expression of the intercellular adhesion molecule P-cadherin. The forced expression of either of these elements induces the emergence of such phenotypes even in the presence of stemness factors. Interestingly, extracellular Stx4, but not P-cadherin, appears to induce a significant upregulation of the gastrulation-related gene brachyury, along with a slight upregulation of the smooth muscle cell-related gene ACTA2 in ESCs. Furthermore, our findings reveal that extracellular Stx4 plays a role in preventing the elimination of CCAAT enhancer binding protein β (C/EBPβ). Notably, the forced overexpression of C/EBPβ led to the downregulation of brachyury and a significant upregulation of ACTA2 in ESCs. These observations suggest that extracellular Stx4 contributes to early mesoderm induction while simultaneously activating an element that alters the differentiation state. The fact that a single differentiation cue can elicit multiple differentiation responses may reflect the challenges associated with achieving sensitive and directed differentiation in cultured stem cells.