Project description:The ligand for the c-Kit receptor, KitL, exists as a membrane-associated (mKitL) and a soluble form (sKitL). KitL functions outside c-Kit activation have not been identified. We show that co-culture of c-Kit– and mKitL–expressing NIH3T3 cells results in signaling through mKitL: c-Kit–bound mKitL recruits calcium-modulating cyclophilin ligand (CAML) to selectively activate Akt, leading to CREB phosphorylation, mTOR pathway activation, and increased cell proliferation. Activation of mKitL in thymic vascular endothelial cells (VECs) induces mKitL- and Akt-dependent proliferation, and genetic ablation of mKitL in thymic VECs blocks their c-Kit responsiveness and proliferation during neonatal thymic expansion. Therefore, mKitL–c-Kit form a bi-directional signaling complex that acts in the developing thymus to coordinate thymic VEC and early thymic progenitor (ETP) expansion by simultaneously promoting ETP survival and VEC proliferation. This mechanism may be relevant to both normal tissues and malignant tumors that depend on KitL–c-Kit signaling for their proliferation.
Project description:Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2-4 years. Injury to and/or dysfunction of alveolar epithelium are strongly implicated in IPF disease initiation, but what factors determine why fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that ZEB1-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments TGF-β-induced profibrogenic responses in underlying lung fibroblasts by paracrine signalling. Here we investigated bi-directional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA sequencing (RNA-seq) of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced EMT identified many differentially expressed genes including those involved in cell migration and extracellular matrix (ECM) regulation. We confirmed that paracrine signalling between AS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a ZEB1-tissue plasminogen activator (tPA) axis. In a reciprocal fashion, paracrine signalling from TGF-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially via the secreted protein, SPARC. Together these data identify that aberrant bi-directional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining pro-fibrotic signals.
Project description:Emerging studies reveal that neuropeptides play critical role in regulating anti-helminth immune responses, hinting at the potential of intrinsic enteric neurons (iENs) in orchestrating intestinal immunity. However, whether and how the iENs get activated during infection, and whether they engage in a bi-directional communication with the immune cells, remain poorly defined. Here we show that iENs became activated in response to helminth infection. Single-nucleus RNA sequencing of the iENs revealed significant alterations in gene expression in IL-13R+ intrinsic primary afferent neurons (IPANs), including the upregulation of the neuropeptide -CGRP. Using genetic mouse models, we demonstrated that both group 2 innate lymphoid cells (ILC2s) and neuronal IL-13R signaling are indispensable for optimal iEN activation, which subsequently inhibit ILC2 responses and anti-helminth immunity. Together, these results reveal a previously unrecognized bi-directional neuro-immune crosstalk in the intestine.
Project description:Circadian rhythms are daily physiological and behavioral changes governed by an internal molecular clock, and dysfunctions in circadian rhythms have long been associated with various neurodegenerative diseases. Abnormal sleep-wake cycle often precedes the onset of cognitive and motor symptoms in patients, while the pathological changes may further exacerbate the disturbance in circadian cycle. It is unclear whether dysregulated circadian rhythm is a consequence of, or a contributing factor for, neurodegeneration. In addition, the evidence directly connecting the neurodegeneration-associated proteins to core circadian clock gene expression remains sparse. Here we show that FUS, a RNA-binding protein implicated in the pathogenesis of ALS and frontotemporal dementia, exhibits a bi-directional regulation with circadian rhythm. Our meta-analysis of RNAseq datasets and subsequent biochemical analysis revealed FUS as a gene regulated by circadian oscillation. Furthermore, NR1D1 binds the FUS promoter and regulates the amplitude of FUS oscillation. Meanwhile, FUS is recruited by transcriptional co-repressor PSF, and is found in the same complex as Bmal-Clock to repress Per2 expression. More strikingly, in cells and brain tissues from homozygous knock-in rats, the pathogenic R521C mutant FUS significantly alters the oscillation patterns of core circadian genes even at young age. Therefore, our results have revealed a novel bi-directional mechanism whereby dysregulated circadian clock and FUS expression may exacerbate neurodegeneration via mutual influence.
Project description:Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signalling pathway and molecular changes after inhibition. Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes representing 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signalling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.
Project description:Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signalling pathway and molecular changes after inhibition. Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes representing 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signalling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect. The present study aimed at identifying the transcriptional and translational responses of neoplastic canine mast cells to the tyrosine kinase inhibitor masitinib. To this end, C2 cells, a cell line with a tandem duplication in the juxtamembrane unit and thus constitutively activated KIT, were treated with masitinib and changes in the global mRNA and protein expression levels were characterized.