Phospholipid Scramblase 4 (PLSCR4) Regulates Adipocyte Differentiation via PIP3-Mediated AKT Activation.

Phospholipid scramblase 4 (PLSCR4) is a member of a conserved enzyme family with high relevance for the remodeling of phospholipid distribution in the plasma membrane and the regulation of cellular signaling. While PLSCR1 and -3 are involved in the regulation of adipose-tissue expansion, the role of PLSCR4 is so far unknown. PLSCR4 is significantly downregulated in an adipose-progenitor-cell model of deficiency for phosphatase and tensin homolog (PTEN). PTEN acts as a tumor suppressor and antagonist of the growth and survival signaling phosphoinositide 3-kinase (PI3K)/AKT cascade by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP3). Patients with PTEN germline deletion frequently develop lipomas. The underlying mechanism for this aberrant adipose-tissue growth is incompletely understood. PLSCR4 is most highly expressed in human adipose tissue, compared with other phospholipid scramblases, suggesting a specific role of PLSCR4 in adipose-tissue biology. In cell and mouse models of lipid accumulation, we found PLSCR4 to be downregulated. We observed increased adipogenesis in PLSCR4-knockdown adipose progenitor cells, while PLSCR4 overexpression attenuated lipid accumulation. PLSCR4 knockdown was associated with increased PIP3 levels and the activation of AKT. Our results indicated that PLSCR4 is a regulator of PI3K/AKT signaling and adipogenesis and may play a role in PTEN-associated adipose-tissue overgrowth and lipoma formation.

Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84.

BACKGROUND: Medium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria. They activate G protein-coupled receptors, including GPR84 and HCA3, which regulate metabolism and immune functions. Although both receptors are coupled to Gi proteins, share at least one agonist and show overlapping tissue expression, GPR84 exerts pro-inflammatory effects whereas HCA3 is involved in anti-inflammatory responses. Here, we analyzed signaling kinetics of both HCA3 and GPR84, to unravel signal transduction components that may explain their physiological differences. METHODS: To study the signaling kinetics and components involved in signal transduction of both receptors we applied the label-free dynamic mass redistribution technology in combination with classical cAMP, ERK signaling and ß-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models. RESULTS: We present strong evidence for a natural biased signaling of structurally highly similar agonists at HCA3 and GPR84. We show that HCA3 signaling and trafficking depends on dynamin-2 function. Activation of HCA3 by 3-hydroxyoctanoic acid but not 3-hydroxydecanoic acid leads to ß-arrestin-2 recruitment, which is relevant for cell-cell adhesion. GPR84 stimulation with 3-hydroxydecanoic acid causes a sustained ERK activation but activation of GPR84 is not followed by ß-arrestin-2 recruitment. CONCLUSIONS: In summary, our results highlight that biased agonism is a physiological property of HCA3 and GPR84 with relevance for innate immune functions potentially to differentiate between endogenous, non-pathogenic compounds and compounds originating from e.g. pathogenic bacteria. Video Abstract.

Nanoparticle-complexed antimiRs for inhibiting tumor growth and metastasis in prostate carcinoma and melanoma.

BACKGROUND: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI's ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models. RESULTS: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well. CONCLUSIONS: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological activity and cellular uptake kinetics.

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation.

Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium.

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins--including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein--was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl-interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.

Fine mapping of the 1p36 deletion syndrome identifies mutation of PRDM16 as a cause of cardiomyopathy.

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM.

Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression.

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

Phospholipase C epsilon (PLCε) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca(2+) concentration [Ca(2+) ]i . Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca(2+) influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCß and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes.

Role of PTEN in modulation of ADP-dependent signaling pathways in vascular endothelial cells.

ADP plays critical signaling roles in the vascular endothelium. ADP receptors are targeted by several cardiovascular drugs, yet the intracellular pathways modulated by ADP are incompletely understood. These studies have identified important roles for the phosphatase PTEN in ADP-dependent modulation of the endothelial isoform of nitric oxide synthase (eNOS) as well as of lipid and protein kinase pathways in endothelial cells. We find that ADP-promoted eNOS activation as well as phosphorylation of p38 MAPK are enhanced by siRNA-mediated PTEN knockdown. However, the increase in ADP-dependent eNOS activation promoted by PTEN knockdown is abrogated by siRNA-mediated knockdown of p38 MAPK. These findings indicate that PTEN tonically suppresses both p38 phosphorylation as well as ADP-stimulated eNOS activity. A key enzymatic activity of PTEN is its role as a lipid phosphatase, catalyzing the dephosphorylation of phosphoinositol-3,4,5-trisphosphate (PIP3) to phosphoinositol-4,5-bisphosphate (PIP2). We performed biochemical analyses of cellular phospholipids in endothelial cells to show that siRNA-mediated PTEN knockdown leads to a marked increase in PIP3. Because these complex lipids activate the small GTPase Rac1, we explored the role of PTEN in ADP-modulated Rac1 activation. We used a FRET biosensor for Rac1 to show that ADP-dependent Rac1 activation is blocked by siRNA-mediated PTEN knockdown. We then exploited a FRET biosensor for PIP3 to show that the striking ADP-dependent increase in intracellular PIP3 is entirely blocked by PTEN knockdown. These studies identify a key role for PTEN in the modulation of lipid mediators involved in ADP receptor-regulated endothelial signaling pathways involving eNOS activation in vascular endothelial cells.

15-Epi-lipoxin A4 inhibits human neutrophil superoxide anion generation by regulating polyisoprenyl diphosphate phosphatase 1.

Regulation of leukocyte activation is critical to limit unintended tissue injury during acute inflammation. On neutrophil activation, polyisoprenyl diphosphate phosphatase 1 (PDP1) rapidly converts presqualene diphosphate to presqualene monophosphate to facilitate cell activation. Lipoxins are potent anti-inflammatory mediators for neutrophils, yet their counterregulatory signaling mechanisms remain to be determined. 15-Epi-lipoxin A4 (15-epi-LXA4) blocked agonist-initiated association of the nicotinamide adenine dinucleotide phosphate oxidase components p47(PHOX) and p22(PHOX) in human neutrophils. 15-Epi-LXA4 (0.1-100 nM) inhibited neutrophil superoxide anion (O2(-)) generation in a concentration- and ALX/FPR2 receptor-dependent manner that was disrupted by PDP1-specific antibodies. In differentiated HL60 cells, a myeloid cell line, agonist-initiated O2(-) generation was inhibited by PDP1 siRNA. Recombinant human PDP1 was directly phosphorylated in vitro by select protein kinase C (PKC) isoforms, including PKCßII. When neutrophils were exposed to formyl-methionyl-leucyl-phenylalanine (fMLP), PKCßII was rapidly phosphorylated and physically associated with PDP1. Agonist-initiated conversion of neutrophil presqualene diphosphate to presqualene monophosphate was blocked by PKCßII inhibition. Neutrophil exposure to 15-epi-LXA4 attenuated fMLP triggered PKCßII phosphorylation and its interactions with PDP1. Together, these findings indicate that PDP1 serves an integral signaling role in neutrophil proinflammatory responses and as a target for counter-regulatory mediators.

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu(2)(FL2E)] to study adult cardiac myocytes from wild-type, eNOS(null), and nNOS(null) mice, we discovered that physiological concentrations of H(2)O(2) activate eNOS but not nNOS. H(2)O(2)-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H(2)O(2) biosensor HyPer2 show marked H(2)O(2) synthesis when stimulated by angiotensin II, but not following ß-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H(2)O(2), whereas ß-adrenergic contractile responses occur independently of H(2)O(2) signaling. These studies establish differential roles for H(2)O(2) in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.

GFRAL Is Widely Distributed in the Brain and Peripheral Tissues of Mice.

In 2017, four independent publications described the glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as receptor for the growth differentiation factor 15 (GDF15, also MIC-1, NAG-1) with an expression exclusively in the mice brainstem area postrema (AP) and nucleus tractus solitarii (NTS) where it mediates effects of GDF15 on reduction of food intake and body weight. GDF15 is a cell stress cytokine with a widespread expression and pleiotropic effects, which both seem to be in contrast to the reported highly specialized localization of its receptor. This discrepancy prompts us to re-evaluate the expression pattern of GFRAL in the brain and peripheral tissues of mice. In this detailed immunohistochemical study, we provide evidence for a more widespread distribution of this receptor. Apart from the AP/NTS region, GFRAL-immunoreactivity was found in the prefrontal cortex, hippocampus, nucleus arcuatus and peripheral tissues including liver, small intestine, fat, kidney and muscle tissues. This widespread receptor expression, not taken into consideration so far, may explain the multiple effects of GDF-15 that are not yet assigned to GFRAL. Furthermore, our results could be relevant for the development of novel pharmacological therapies for physical and mental disorders related to body image and food intake, such as eating disorders, cachexia and obesity.

Angiotensin-II and MARCKS: a hydrogen peroxide- and RAC1-dependent signaling pathway in vascular endothelium.

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.

The MARCKS protein plays a critical role in phosphatidylinositol 4,5-bisphosphate metabolism and directed cell movement in vascular endothelial cells.

The MARCKS protein (myristoylated alanine-rich C kinase substrate) is an actin- and calmodulin-binding protein that is expressed in many mammalian tissues. The role of MARCKS in endothelial signaling responses is incompletely understood. We found that siRNA-mediated knockdown of MARCKS in cultured endothelial cells abrogated directed cell movement in a wound healing assay. We used biochemical and cell imaging approaches to explore the role of MARCKS in endothelial signal transduction pathways activated by insulin. Insulin treatment of vascular endothelial cells promoted the dose- and time-dependent phosphorylation of MARCKS. Cell imaging and hydrodynamic approaches revealed that MARCKS is targeted to plasmalemmal caveolae and undergoes subcellular translocation in response to insulin. Insulin treatment promoted an increase in levels of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) in plasmalemmal caveolae. The insulin-stimulated increase in caveolar PIP(2) was blocked by siRNA-mediated knockdown of MARCKS, as determined using both biochemical assays and imaging studies using FRET-based PIP(2) biosensors. The critical role of PIP(2) in MARCKS responses was explored by examining the PIP(2)- and actin-binding proteins Arp2/3 and N-WASP. Insulin promoted the rapid and robust phosphorylation of both N-WASP and Arp2/3, but these phosphorylation responses were markedly attenuated by siRNA-mediated MARCKS knockdown. Moreover, MARCKS knockdown effectively abrogated N-WASP activation in response to insulin, as determined using a FRET-based N-WASP activity biosensor. Taken together, these studies show that MARCKS plays a key role in insulin-dependent endothelial signaling to PIP(2) and is a critical determinant of actin assembly and directed cell movement in the vascular endothelium.

Therapeutic miR-506-3p Replacement in Pancreatic Carcinoma Leads to Multiple Effects including Autophagy, Apoptosis, Senescence, and Mitochondrial Alterations In Vitro and In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality. Considering its very poor prognosis, novel treatment options are urgently needed. MicroRNAs (miRNAs) are involved in the regulation of various physiological and pathological processes. In tumors, aberrant downregulation of given miRNAs may result in pathological overexpression of oncogenes, rendering miRNA replacement as a promising therapeutic strategy. In different tumor entities, miRNA-506-3p (miR506-3p) has been ambivalently described as tumor suppressing or oncogenic. In PDAC, miR-506 is mainly considered as a tumor-suppressing miRNA. In this study, we extensively analyze the cellular and molecular effects of miRNA-506-3p replacement in different PDAC cell lines. Beyond profound antiproliferation and induction of cell death and autophagy, we describe new cellular miR506-3p effects, i.e., induction of senescence and reactive oxygen species (ROS), as well as alterations in mitochondrial potential and structure, and identify multiple underlying molecular effects. In a preclinical therapy study, PDAC xenograft-bearing mice were treated with nanoparticle-formulated miRNA-506 mimics. Profound tumor inhibition upon systemic miRNA-506 administration was associated with multiple cellular and molecular effects. This demonstrates miRNA replacement as a potential therapeutic option for PDAC patients. Due to its broad mechanisms of action on multiple relevant target genes, miR506-3p is identified as a particularly powerful tumor-inhibitory miRNA.

Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration.

The aim of this study was the evaluation of cross-linked gelatin microparticles (cGM) as substrates for osteogenic cell culture to assemble 3D microtissues and their use as delivery system for siRNA to cells in these assemblies. In a 2D transwell cultivation system, we found that cGM are capable to accumulate calcium ions from the surrounding medium. Such a separation of cGM and SaOS-2 â€‹cells consequently led to a suppressed matrix mineral formation in the SaOS-2 culture on the well bottom of the transwell system. Thus, we decided to use cGM as component in 3D microtissues and get a close contact between calcium ion accumulating microparticles and cells to improve matrix mineralization. Gelatin microparticles were cross-linked with a N,N-diethylethylenediamine-derivatized (DEED) maleic anhydride (MA) containing oligo (pentaerythritol diacrylate monostearate-co-N-isopropylacrylamide-co-MA) (oPNMA) and aggregated with SaOS-2 or human mesenchymal stem cells (hMSC) to microtissue spheroids. We systematically varied the content of cGM in microtissues and observed cell differentiation and tissue formation. Microtissues were characterized by gene expression, ALP activity and matrix mineralization. Mineralization was detectable in microtissues with SaOS-2 â€‹cells after 7 days and with hMSC after 24-28 days in osteogenic culture. When we transfected hMSC via cGM loaded with Lipofectamine complexed chordin siRNA, we found increased ALP activity and accelerated mineral formation in microtissues in presence of BMP-2. As a model for positive paracrine effects that indicate promising in vivo effects of these microtissues, we incubated pre-differentiated microtissues with freshly seeded hMSC monolayers and found improved mineral formation all over the well in the co-culture model. These findings may support the concept of microtissues from hMSC and siRNA-loaded cGM for bone regeneration.

The N Terminus of Adhesion G Protein-Coupled Receptor GPR126/ADGRG6 as Allosteric Force Integrator.

The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study, we provide this information by combining classic second-messenger detection with single-cell atomic force microscopy. We established a monoclonal antibody targeting the N terminus to stimulate GPR126 and compared it to the activation through its known ECM ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be regarded as an allosteric module that can fine-tune receptor activation in a context-specific manner.

Extracellular Inflammasome Particles Are Released After Marathon Running and Induce Proinflammatory Effects in Endothelial Cells.

Objectives: The intracellular NLRP3 inflammasome is an important regulator of sterile inflammation. Recent data suggest that inflammasome particles can be released into circulation. The effects of exercise on circulating extracellular apoptosis-associated speck-like protein (ASC) particles and their effects on endothelial cells are not known. Methods: We established a flow cytometric method to quantitate extracellular ASC specks in human serum. ASC specks were quantitated in 52 marathon runners 24-72 h before, immediately after, and again 24-58 h after the run. For mechanistic characterization, NLRP3 inflammasome particles were isolated from a stable mutant NLRP3 (p.D303N)-YFP HEK cell line and used to treat primary human coronary artery endothelial cells. Results: Athletes showed a significant increase in serum concentration of circulating ASC specks immediately after the marathon (+52% compared with the baseline, p < 0.05) and a decrease during the follow-up after 24-58 h (12% reduction compared with immediately after the run, p < 0.01). Confocal microscopy revealed that human endothelial cells can internalize extracellular NLRP3 inflammasome particles. After internalization, endothelial cells showed an inflammatory response with a higher expression of the cell adhesion molecule ICAM1 (6.9-fold, p < 0.05) and increased adhesion of monocytes (1.5-fold, p < 0.05). Conclusion: These findings identify extracellular inflammasome particles as novel systemic mediators of cell-cell communication that are transiently increased after acute extensive exercise with a high mechanical muscular load.

A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the Caco-2 intestinal barrier model.

Bovine milk-derived extracellular vesicles (EVs) hold promises as oral drug delivery systems. Since EV bioavailability studies are difficult to compare, key factors regarding EV uptake and intestinal permeability remain little understood. This work aims to critically study uptake and transport properties of milk-derived EVs across the intestinal barrier in vitro by standardization approaches. Therefore, uptake properties were directly compared to liposomes in intestinal Caco-2 cells. Reliable staining results were obtained by the choice of three distinct EV labeling sites, while non-specific dye transfer and excess dye removal were carefully controlled. A novel fluorescence correction factor was implemented to account for different labeling efficiencies. Both EV and liposome uptake occurred mainly energy dependent with the neonatal Fc receptor (FcRn) providing an exclusive active pathway for EVs. Confocal microscopy revealed higher internalization of EVs whereas liposomes rather remained attached to the cell surface. Internalization could be improved when changing the liposomal formulation to resemble the EV lipid composition. In a Caco-2/HT29-MTX co-culture liposomes and EVs showed partial mucus penetration. For transport studies across Caco-2 monolayers we further established a standardized protocol considering the distinct requirements for EVs. Especially insert pore sizes were systematically compared with 3 µm inserts found obligatory. Obtained apparent permeability coefficients (Papp) reflecting the transport rate will allow for better comparison of future bioavailability testing.