The SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFκB intracrine senescent noncanonical pathway.

Senescent cells produce a Senescence-Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin-6 (IL-6), has the potential to amplify cellular senescence in the SASP-producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL-6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL-6 sustains intracrine senescence interacting with the intracellular IL-6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS-STING, and NFκB activation. This pathway triggered by intracellular IL-6 significantly contributes to cell-autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL-6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re-expression of IL-6 restores senescence control of tumor growth. The intracrine senescent IL-6 pathway is further evidenced in three human cellular models of therapy-induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL-6 to sustain cell-autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence-based therapies.

Evidence for reduced plasmodesmata callose accumulation in Nicotiana benthamiana leaves with increased symplastic cell-to-cell communication caused by RNA processing defects of chloroplasts.

RNA processing defects in chloroplasts were previously associated with increased plasmodesmata (PD) permeability. However, the underlying mechanisms for such association are still unknown. To provide insight into this, we silenced the expression of chloroplast-located INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) RNA helicase in Nicotiana benthamiana leaves and determined an increase in PD permeability which is caused by a reduction of PD callose deposition. Moreover, the silencing of two other nuclear genes encoding chloroplastic enzymes involved in RNA processing, RH3, and CLPR2, also increased PD permeability accompanied by reduced callose accumulation at PD. In addition, we quantified the plastidic hydrogen peroxide levels using the chloroplast-targeted fluorescent sensor, HyPer, in ISE2, RH3, and CLPR2 silenced N. benthamiana leaves. The levels of chloroplastic hydrogen peroxide were not correlated with the increased cell-to-cell movement of the marker protein GFP2X. We, therefore, propose that defects in chloroplast RNA metabolism mediate PD gating by suppressing PD callose deposition, and hydrogen peroxide levels in the organelles are not directly linked to this process.

Interplay between potato virus X and RNA granules in Nicotiana benthamiana.

Cytoplasmic RNA granules consist of microscopic agglomerates of mRNAs and proteins and occur when the translation is reversibly and temporally halted (stress granules, SGs) or mRNAs are targeted for decapping (processing bodies, PBs). The induction of RNA granules formation by virus infection is a common feature of mammalian cells. However, plant-virus systems still remain poorly characterized. In this work, the SG marker AtUBP1b was expressed in Nicotiana benthamiana plants to decipher how the virus infection of plant cells affects SG dynamics. We found that the hypoxia-induced SG assembly was substantially inhibited in Potato virus X (PVX)-infected cells. Furthermore, we determined that the expression of PVX movement protein TGBp1 by itself, mimics the inhibitory effect of PVX on SG formation under hypoxia. Importantly, overexpression of AtUBP1b showed inhibition of the PVX spreading, whereas the overexpression of the dominant negative AtUBP1brrm enhanced PVX spreding, indicating that AtUBP1b negatively affects PVX infection. Notably, PVX infection did not inhibit the formation of processing bodies (PBs), indicating PVX has distinct effects depending on the type of RNA granule. Our results suggest that SG inhibition could be part of the virus strategy to infect the plant.

Identification of MRP4/ABCC4 as a Target for Reducing the Proliferation of Pancreatic Ductal Adenocarcinoma Cells by Modulating the cAMP Efflux.

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P < 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P < 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENT: ABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.

Low-molecular-weight heparin inhibition in classical complement activation pathway during pregnancy.

INTRODUCTION: Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications. MATERIALS AND METHODS: The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH(50) assay). RESULTS: The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03+/-1.20 microg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 microg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG. CONCLUSIONS: We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.