scholarly journals Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways

2021 ◽  
Vol 118 (11) ◽  
pp. e2009647118
Author(s):  
Judith Prado ◽  
Remco H. S. Westerink ◽  
Jelena Popov-Celeketic ◽  
Cristine Steen-Louws ◽  
Aridaman Pandit ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Signaling Pathways ◽  
Sensory Neurons ◽  
Inflammatory Pain ◽  
Biological Effects ◽  
Cytokine Receptor ◽  
Cytokine Receptors ◽  
Signal Pathways ◽  
Regulatory Cytokine

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4–10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4–10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4–10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4–10 FP. In vitro, IL4–10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4–10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4–10 FP. Thus, IL4–10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

scholarly journals Positive Crosstalk Between Hedgehog and NF-κB Pathways Is Dependent on KRAS Mutation in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuqiong Wang ◽  
Dan Wang ◽  
Yanmiao Dai ◽  
Xiangyu Kong ◽  
Xian Zhu ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Signaling Pathways ◽  
Kras Mutation ◽  
Biological Effects ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Hh Signaling

It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.

scholarly journals Sialic Acid-Engineered IL4–10 Fusion Protein is Bioactive and Rapidly Cleared from the Circulation

2019 ◽  
Vol 37 (2) ◽  
Author(s):  
Cristine Steen-Louws ◽  
Peter Boross ◽  
Judith Prado ◽  
Jan Meeldijk ◽  
Jurgen B. Langenhorst ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Inflammatory Pain ◽  
Intrathecal Injection ◽  
Systemic Exposure ◽  
Pharmacokinetic Parameters ◽  
Systemic Clearance ◽  
Mice Model ◽  
Whole Blood Assay

Abstract Purpose Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4–10 fusion protein (IL4–10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. Methods CHO cell lines producing low (IL4–10 FP lowSA) and high sialylated (IL4–10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4–10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. Results IL4–10 FP lowSA and IL4–10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4–10 FP lowSA, whereas the calculated half-life of both IL4–10 FP lowSA and IL4–10 FP highSA was 20.7 min. Finally, both IL4–10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. Conclusions Differential sialylation of IL4–10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4–10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.

scholarly journals In Vitro Evidence That Cytokine Receptor Signals Are Required for Differentiation of Double Positive Thymocytes into Functionally Mature CD8+ T Cells

2003 ◽  
Vol 197 (4) ◽  
pp. 475-487 ◽  
Author(s):  
Qing Yu ◽  
Batu Erman ◽  
Avinash Bhandoola ◽  
Susan O. Sharrow ◽  
Alfred Singer
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Glucose Transporter ◽  
Experimental System ◽  
Cytokine Receptor ◽  
Cytokine Receptors ◽  
Double Positive ◽  
Thymocyte Differentiation ◽  
Tcr Signals

CD4+8+ double positive (DP) thymocytes differentiate into CD4+ and CD8+ mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4−8+ thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8+ thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8+ T cells.

Differential Gene Signature and Signaling Pathways in Childhood Burkitt (BL) vs Diffuse Large B-Cell Lymphoma (DLBCL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4142-4142
Author(s):  
Nancy S. Day ◽  
Janet Ayello ◽  
Ian Waxman ◽  
Evan Shereck ◽  
Catherine McGuinn ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Cytokine Receptor ◽  
Receptor Interaction ◽  
Signal Pathways ◽  
Type I

Abstract The prognosis and treatment of both major forms of advanced childhood B-NHL (BL and DLBCL) is similar with short and intensive multi-agent chemotherapy (Cairo/Patte et al., Blood, 2007 and Patte/Cairo et al., Blood, 2007). Despite both BL and DLBCL being germinal center derived, our recent cytogenetic results of BL vs DLBCL in the FAB LMB 96 study have demonstrated significant differences in secondary chromosomal aberrations in BL vs DLBCL and a differential prognosis based on secondary cytogenetic findings (Poirel/Cairo/Patte, Blood, 2003a). Thus, we sought to identify genes that could uniquely differentiate childhood BL vs DLBCL and discover potential genetic mechanisms of differential molecular pathogenesis and to determine the signal pathways that contribute to the genetic disparity between these two histological types of childhood B-NHL. Nine BL (7 patient samples and 2 cell lines, Raji and Ramos) and 3 DLBCL (1 patient sample and 2 cell lines, Pfeiffer and DB) were compared. Total RNA was isolated, reverse transcribed to cDNA biotinylated cRNA and hybridized to Affymetrix U133A_2 as we have previously described (Jiang/Cairo et al., Journal of Immunology, 2004). Data were analyzed using Agilent GeneSpring 7.3. Signal intensities were compared using one way ANOVA and Welch Test for statistical analysis. Two-fold changes between BL and DLBCL were considered as significant (p<0.05). KEGG Pathways were evaluated for the genes identified. There were 120 genes over-expressed and 217 genes under-expressed in BL vs DLBCL. BL expressed significantly higher level of Ki-67 (a measure of lymphoma-cell proliferation) than DLBCL (2.68F). BL also expressed higher level of the pro-apoptotic gene, p53 compared to DLBCL (1.46F). Over-expressed genes in BL vs DLBCL included TNFSF10 (11.87F), RHOQ (3.16F), PIP5K1B (5.22F) among many others. The genes significantly under-expressed in BL vs DLBCL included PIGL (0.45F), Inositol (myo)-1 (or 4)-monophosphatase 1 (IMPA1; 0.28F), cAMP-dependent regulatory type I, alpha protein kinase (PRKAR1A; 0.37F) among many others. TNFSF10 induces apoptosis in transformed and tumor cells and is known to participate in pathways including cytokine-cytokine receptor interaction and induction of apoptosis through DR3 and DR4/5 death receptors. PIP5K1B is involved in the Rho signaling pathway and PIGL catalyzes the second step of glycosylphosphatidylinositol (GPI) biosynthesis. Since activation of IL3R-mediated cAMP-dependent protein kinase leads to increased cell survival, we searched gene expression profiles in BL vs DLBCL that were involved in IL signaling pathways. The genes that were identified to be over-expressed in BL vs DLBCL included IL2RG (2.24F), IL8RB, IL18 receptor accessory protein (IL18RAP), IL18, IL18R1, and IL1R2 (natural log values of 11.11, 22.95, 2.16, 1.73 and 11.84, respectively in BL vs non-detectable values in DLBCL). Taken together, since IL1, IL2, IL8, and IL18 all belong to IL1 super family, these results suggest significant involvement of TNF (TRAIL) and IL1 super family via cytokine-cytokine receptor interaction and activation of the Rho signaling pathway in Burkitt vs DLBCL lymphomagenesis.

scholarly journals Effects of Axotomy on Cultured Sensory Neurons of Aplysia: Long-Term Injury-Induced Changes in Excitability and Morphology Are Mediated by Different Signaling Pathways

2008 ◽  
Vol 100 (6) ◽  
pp. 3209-3224 ◽  
Author(s):  
Supinder S. Bedi ◽  
Diancai Cai ◽  
David L. Glanzman
Keyword(s):  
Signaling Pathways ◽  
Sensory Neurons ◽  
Specific Inhibitor ◽  
Distinct Pattern ◽  
Extracellular Signal ◽  
Vitro Model ◽  
Induced Changes ◽  
And Control

To facilitate an understanding of injury-induced changes within the nervous system, we used a single-cell, in vitro model of axonal injury. Sensory neurons were individually dissociated from the CNS of Aplysia and placed into cell culture. The major neurite of some neurons was then transected (axotomized neurons). Axotomy in hemolymph-containing culture medium produced long-term hyperexcitability (LTH-E) and enhanced neuritic sprouting (long-term hypermorphogenesis [LTH-M]). Axotomy in the absence of hemolymph induced LTH-E, but not LTH-M. Hemolymph-derived growth factors may activate tyrosine receptor kinase (Trk) receptors in sensory neurons. To examine this possibility, we treated uninjured (control) and axotomized sensory neurons with K252a, an inhibitor of Trk receptor activity. K252a depressed the excitability of both axotomized and control neurons. K252a also produced a distinct pattern of arborizing outgrowth of neurites in both axotomized and control neurons. Protein kinase C (PKC) is an intracellular signal downstream of Trk; accordingly, we tested the effects of bisindolylmaleimide I (Bis-I), a specific inhibitor of PKC, on the axotomy-induced cellular changes. Bis-I blocked LTH-E, but did not disrupt LTH-M. Finally, because Trk activates the extracellular signal regulated kinase pathway in Aplysia sensory neurons, we examined whether this pathway mediates the injury-induced changes. Sensory neurons were axotomized in the presence of U0126, an inhibitor of mitogen-activated/extracellular receptor-regulated kinase. U0126 blocked the LTH-M due to axotomy, but did not impair LTH-E. Therefore distinct cellular signaling pathways mediate the induction of LTH-E and LTH-M in the sensory neurons.

scholarly journals Conserved Expression of Nav1.7 and Nav1.8 Contribute to the Spontaneous and Thermally Evoked Excitability in IL-6 and NGF-Sensitized Adult Dorsal Root Ganglion Neurons In Vitro

2020 ◽  
Vol 7 (2) ◽  
pp. 44
Author(s):  
Rahul R. Atmaramani ◽  
Bryan J. Black ◽  
June Bryan de la Peña ◽  
Zachary T. Campbell ◽  
Joseph J. Pancrazio
Keyword(s):  
Sodium Channels ◽  
Sensory Neurons ◽  
Inflammatory Pain ◽  
Dorsal Root Ganglion Neurons ◽  
Electrode Arrays ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Ganglion Neurons

Sensory neurons respond to noxious stimuli by relaying information from the periphery to the central nervous system via action potentials driven by voltage-gated sodium channels, specifically Nav1.7 and Nav1.8. These channels play a key role in the manifestation of inflammatory pain. The ability to screen compounds that modulate voltage-gated sodium channels using cell-based assays assumes that key channels present in vivo is maintained in vitro. Prior electrophysiological work in vitro utilized acutely dissociated tissues, however, maintaining this preparation for long periods is difficult. A potential alternative involves multi-electrode arrays which permit long-term measurements of neural spike activity and are well suited for assessing persistent sensitization consistent with chronic pain. Here, we demonstrate that the addition of two inflammatory mediators associated with chronic inflammatory pain, nerve growth factor (NGF) and interleukin-6 (IL-6), to adult DRG neurons increases their firing rates on multi-electrode arrays in vitro. Nav1.7 and Nav1.8 proteins are readily detected in cultured neurons and contribute to evoked activity. The blockade of both Nav1.7 and Nav1.8, has a profound impact on thermally evoked firing after treatment with IL-6 and NGF. This work underscores the utility of multi-electrode arrays for pharmacological studies of sensory neurons and may facilitate the discovery and mechanistic analyses of anti-nociceptive compounds.

Kaempferol inhibits pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways

2020 ◽  
Author(s):  
Xu Chen ◽  
Yaqin Chen ◽  
Zhongqiong Yin ◽  
Rui Wang ◽  
Huaiyue Hu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Virus Replication ◽  
Pseudorabies Virus ◽  
Target Genes ◽  
Control Measure ◽  
Control Measures ◽  
Economic Losses ◽  
Signal Pathways ◽  
Dependent Manner

Abstract Pseudorabies virus (PRV), belonging to the family Herpesviridae, is a pathogen of Aujeszky’s disease leading great economic losses to pig industry. Re-outburst of pseudorabies implies that new control measures are urgent needed. The present study provides a candidate drug for PRV infection that kaempferol possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro. Kaempferol at a concentration of 52.40 μM could decrease PRV-induced cell death by 90%. Kaempferol with a IC50 of 25.57μM is more effective than acyclovir (Positive control) with a IC 50 of 54.97 μM. Mode of action study indicated that kaempferol inhibited viral penetration and replication stages and virus load was decreased by 4-fold and 30-fold, respectively. Addition of kaempferol within 16 hours post infection (hpi) could significantly inhibit virus replication, and the DNA copies were decreased by almost 15-fold when kaempferol was added at 2 hpi. Kaempferol could regulate NF-κB and MAPKs signal pathways involved in PRV infection and change the levels of the target genes of MAPKs (ATF-2 and c-Jun) and NF-κB (IL-1α, IL-1β and IL-2) signaling pathways. All the results indicated that kaempferol has the ability to be an alternative control measure for PRV infection.¶ These authors contribute equally to this work and should be considered as the first author.

Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A

2004 ◽  
Vol 84 (1) ◽  
pp. 137-167 ◽  
Author(s):  
KJETIL TASKÉN ◽  
EINAR MARTIN AANDAHL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Biological Effects ◽  
Organ Specificity ◽  
Complex Signal ◽  
Signal Pathways ◽  
Pka Pathway ◽  
Kinase A

Taskén, Kjetil, and Einar Martin Aandahl. Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A. Physiol Rev 84: 137–167, 2004; 10.1152/physrev.00021.2003.—More than 20% of the human genome encodes proteins involved in transmembrane and intracellular signaling pathways. The cAMP-protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells and is involved in regulation of cellular functions in almost all tissues in mammals. Various extracellular signals converge on this signal pathway through ligand binding to G protein-coupled receptors, and the cAMP-PKA pathway is therefore tightly regulated at several levels to maintain specificity in the multitude of signal inputs. Ligand-induced changes in cAMP concentration vary in duration, amplitude, and extension into the cell, and cAMP microdomains are shaped by adenylyl cyclases that form cAMP as well as phosphodiesterases that degrade cAMP. Different PKA isozymes with distinct biochemical properties and cell-specific expression contribute to cell and organ specificity. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein signaling complexes that serve as crossroads for different paths of cell signaling. Targeting of PKA and integration of a wide repertoire of proteins involved in signal transduction into complex signal networks further increase the specificity required for the precise regulation of numerous cellular and physiological processes.

scholarly journals Signaling via Tumor Necrosis Factor Receptor 1 but Not Toll-Like Receptor 2 Contributes Significantly to Hydrosalpinx Development following Chlamydia muridarum Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1833-1839 ◽  
Author(s):  
Xiaohua Dong ◽  
Yuanjun Liu ◽  
Xiaotong Chang ◽  
Lei Lei ◽  
Guangming Zhong
Keyword(s):  
Pattern Recognition ◽  
Signaling Pathways ◽  
Chlamydial Infection ◽  
Pattern Recognition Receptor ◽  
Cytokine Receptor ◽  
Interleukin 17 ◽  
Similar Time ◽  
Deficient Mice ◽  
Recognition Receptor

ABSTRACTChlamydial infection in the lower genital tract can lead to hydrosalpinx, which is accompanied by activation of both pattern recognition receptor TLR2- and inflammatory cytokine receptor TNFR1-mediated signaling pathways. In the current study, we compared the relative contributions of these two receptors to chlamydial induction of hydrosalpinx in mice. We found that mice with or without deficiencies in TLR2 or TNFR1 displayed similar time courses of live organism shedding from vaginal swabs, suggesting that these receptor-mediated signaling pathways are not required for controlling chlamydial lower genital infection. However, mice deficient in TNFR1 but not TLR2 developed significantly reduced hydrosalpinx. The decreased pathogenicity correlated with a significant reduction in interleukin-17 byin vitro-restimulated splenocytes of TNFR1-deficient mice. Although TLR2-deficient mice developed hydrosalpinx as severe as that of wild-type mice, peritoneal macrophages from mice deficient in TLR2 but not TNFR1 produced significantly reduced cytokines upon chlamydial stimulation, suggesting that reduced macrophage responses to chlamydial infection do not always lead to a reduction in hydrosalpinx. Thus, we have demonstrated that the signaling pathways triggered by the cytokine receptor TNFR1 play a more significant role in chlamydial induction of hydrosalpinx than those mediated by the pattern recognition receptor TLR2, which has laid a foundation for further revealing the chlamydial pathogenic mechanisms.

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