Dubois C, Rocks N, Blacher S, Primac I, Gallez A, García-Caballero M, Gérard C, Brouchet L, Noel A, Lenfant F, Cataldo D, Péqueux C.
PMID: 30444717 | DOI: 10.1530/ERC-18-0328
Estrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of estrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and to identify patients who could potentially benefit from anti-estrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grew faster in female mice than in males. Moreover, estradiol (E2) promoted tumour development in female mice and increased lymph/angiogenesis and levels of VEGFA and bFGF in lung tumours of females through an estrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERbeta antagonist was inefficient, ERalpha antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 to 55 years old revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERalpha-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of estrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.
Pascalin eAim�, Saravanan S.Karuppagounder, Apeksha Rao, Yingxin Chen, Robert E.Burke, Rajiv R.Ratan, Lloyd A.Greene
PMID: 31911115 | DOI: 10.1016/j.nbd.2019.104725
Identifying disease-causing pathways and drugs that target them in Parkinson's disease (PD) has remained challenging. We uncovered a PD-relevant pathway in which the stress-regulated heterodimeric transcription complex CHOP/ATF4 induces the neuron prodeath protein Trib3 that in turn depletes the neuronal survival protein Parkin. Here we sought to determine whether the drug adaptaquin, which inhibits ATF4-dependent transcription, could suppress Trib3 induction and neuronal death in cellular and animal models of PD. Neuronal PC12 cells and ventral midbrain dopaminergic neurons were assessed in vitro for survival, transcription factor levels and Trib3 or Parkin expression after exposure to 6-hydroxydopamine or 1-methyl-4-phenylpyridinium with or without adaptaquin co-treatment. 6-hydroxydopamine injection into the medial forebrain bundle was used to examine the effects of systemic adaptaquin on signaling, substantia nigra dopaminergic neuron survival and striatal projections as well as motor behavior. In both culture and animal models, adaptaquin suppressed elevation of ATF4 and/or CHOP and induction of Trib3 in response to 1-methyl-4-phenylpyridinium and/or 6-hydroxydopamine. In culture, adaptaquin preserved Parkin levels, provided neuroprotection and preserved morphology. In the mouse model, adaptaquin treatment enhanced survival of dopaminergic neurons and substantially protected their striatal projections. It also significantly enhanced retention of nigrostriatal function. These findings define a novel pharmacological approach involving the drug adaptaquin, a selective modulator of hypoxic adaptation, for suppressing Parkin loss and neurodegeneration in toxin models of PD. As adaptaquin possesses an oxyquinoline backbone with known safety in humans, these findings provide a firm rationale for advancing it towards clinical evaluation in PD
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
Glover, EJ;Margaret Starr, E;Gascon, A;Clayton-Stiglbauer, K;Amegashie, CL;Selchick, AH;Vaughan, DT;Wayman, WN;Woodward, JJ;Judson Chandler, L;
PMID: 37221326 | DOI: 10.1038/s41386-023-01612-5
The rostromedial tegmental nucleus (RMTg) encodes negative reward prediction error (RPE) and plays an important role in guiding behavioral responding to aversive stimuli. Previous research has focused on regulation of RMTg activity by the lateral habenula despite studies revealing RMTg afferents from other regions including the frontal cortex. The current study provides a detailed anatomical and functional analysis of cortical input to the RMTg of male rats. Retrograde tracing uncovered dense cortical input to the RMTg spanning the medial prefrontal cortex, the orbitofrontal cortex and anterior insular cortex. Afferents were most dense in the dorsomedial subregion of the PFC (dmPFC), an area that is also implicated in both RPE signaling and aversive responding. RMTg-projecting dmPFC neurons originate in layer V, are glutamatergic, and collateralize to select brain regions. In-situ mRNA hybridization revealed that neurons in this circuit are predominantly D1 receptor-expressing with a high degree of D2 receptor colocalization. Consistent with cFos induction in this neural circuit during exposure to foot shock and shock-predictive cues, optogenetic stimulation of dmPFC terminals in the RMTg drove avoidance. Lastly, acute slice electrophysiology and morphological studies revealed that exposure to repeated foot shock resulted in significant physiological and structural changes consistent with a loss of top-down modulation of RMTg-mediated signaling. Altogether, these data reveal the presence of a prominent cortico-subcortical projection involved in adaptive behavioral responding to aversive stimuli such as foot shock and provide a foundation for future work aimed at exploring alterations in circuit function in diseases characterized by deficits in cognitive control over reward and aversion.
Journal of gastroenterology
Sui, Y;Hoshi, N;Ohgaki, R;Kong, L;Yoshida, R;Okamoto, N;Kinoshita, M;Miyazaki, H;Ku, Y;Tokunaga, E;Ito, Y;Watanabe, D;Ooi, M;Shinohara, M;Sasaki, K;Zen, Y;Kotani, T;Matozaki, T;Tian, Z;Kanai, Y;Kodama, Y;
PMID: 36739585 | DOI: 10.1007/s00535-023-01960-5
Amino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood.To investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed.This study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/β-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production.LAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.
Brain : a journal of neurology
Ray, PR;Shiers, S;Caruso, JP;Tavares-Ferreira, D;Sankaranarayanan, I;Uhelski, ML;Li, Y;North, RY;Tatsui, C;Dussor, G;Burton, MD;Dougherty, PM;Price, TJ;
PMID: 35867896 | DOI: 10.1093/brain/awac266
Neuropathic pain is a leading cause of high impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the dorsal root ganglia is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human dorsal root ganglia from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain associated dorsal root ganglia. We sequenced 70 human dorsal root ganglia, and among these 50 met inclusion criteria for sufficient neuronal mRNA signal for downstream analysis. Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14, and OSM in male and including CCL1, CCL21, PENK and TLR3 in female dorsal root ganglia associated with neuropathic pain. Co-expression modules revealed enrichment in members of JUN-FOS signalling in males, and centromere protein coding genes in females. Neuro-immune signalling pathways revealed distinct cytokine signalling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization. Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.
Cellular and molecular life sciences : CMLS
Lebas, H;Guérit, S;Picot, A;Boulay, AC;Fournier, A;Vivien, D;Cohen Salmon, M;Docagne, F;Bardou, I;
PMID: 35633384 | DOI: 10.1007/s00018-022-04340-z
In multiple sclerosis (MS), disturbance of the plasminogen activation system (PAS) and blood brain barrier (BBB) disruption are physiopathological processes that might lead to an abnormal fibrin(ogen) extravasation into the parenchyma. Fibrin(ogen) deposits, usually degraded by the PAS, promote an autoimmune response and subsequent demyelination. However, the PAS disruption is not well understood and not fully characterized in this disorder.Here, we characterized the expression of PAS actors during different stages of two mouse models of MS (experimental autoimmune encephalomyelitis-EAE), in the central nervous system (CNS) by quantitative RT-PCR, immunohistofluorescence and fluorescent in situ hybridization (FISH). Thanks to constitutive PAI-1 knockout mice (PAI-1 KO) and an immunotherapy using a blocking PAI-1 antibody, we evaluated the role of PAI-1 in EAE models and its impact on physiopathological processes such as fibrin(ogen) deposits, lymphocyte infiltration and demyelination.We report a striking overexpression of PAI-1 in reactive astrocytes during symptomatic phases, in two EAE mouse models of MS. This increase is concomitant with lymphocyte infiltration and fibrin(ogen) deposits in CNS parenchyma. By genetic invalidation of PAI-1 in mice and immunotherapy using a blocking PAI-1 antibody, we demonstrate that abolition of PAI-1 reduces the severity of EAE and occurrence of relapses in two EAE models. These benefits are correlated with a decrease in fibrin(ogen) deposits, infiltration of T4 lymphocytes, reactive astrogliosis, demyelination and axonal damage.These results demonstrate that a deleterious overexpression of PAI-1 by reactive astrocytes leads to intra-parenchymal dysfibrinolysis in MS models and anti-PAI-1 strategies could be a new therapeutic perspective for MS.
Cellular and molecular life sciences : CMLS
Gillot, L;Lebeau, A;Baudin, L;Pottier, C;Louis, T;Durré, T;Longuespée, R;Mazzucchelli, G;Nizet, C;Blacher, S;Kridelka, F;Noël, A;
PMID: 35567669 | DOI: 10.1007/s00018-022-04262-w
Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.
Manshouri, T;Veletic, I;Li, P;Yin, CC;Post, SM;Verstovsek, S;Estrov, Z;
PMID: 35595725 | DOI: 10.1038/s41419-022-04932-4
Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1+ fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.
Science translational medicine
Mederacke, I;Filliol, A;Affo, S;Nair, A;Hernandez, C;Sun, Q;Hamberger, F;Brundu, F;Chen, Y;Ravichandra, A;Huebener, P;Anke, H;Shi, H;Martínez García de la Torre, RA;Smith, JR;Henderson, NC;Vondran, FWR;Rothlin, CV;Baehre, H;Tabas, I;Sancho-Bru, P;Schwabe, RF;
PMID: 35385339 | DOI: 10.1126/scitranslmed.abe5795
Fibrosis contributes to ~45% of deaths in western countries. In chronic liver disease, fibrosis is a major factor determining outcomes, but efficient antifibrotic therapies are lacking. Although platelet-derived growth factor and transforming growth factor-β constitute key fibrogenic mediators, they do not account for the well-established link between cell death and fibrosis in the liver. Here, we hypothesized that damage-associated molecular patterns (DAMPs) may link epithelial cell death to fibrogenesis in the injured liver. DAMP receptor screening identified purinergic receptor P2Y14 among several candidates as highly enriched in hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Conversely, P2Y14 ligands uridine 5'-diphosphate (UDP)-glucose and UDP-galactose were enriched in hepatocytes and were released upon different modes of cell death. Accordingly, ligand-receptor interaction analysis that combined proteomic and single-cell RNA sequencing data revealed P2Y14 ligands and P2Y14 receptor as a link between dying cells and HSCs, respectively. Treatment with P2Y14 ligands or coculture with dying hepatocytes promoted HSC activation in a P2Y14-dependent manner. P2Y14 ligands activated extracellular signal-regulated kinase (ERK) and Yes-associated protein (YAP) signaling in HSCs, resulting in ERK-dependent HSC activation. Global and HSC-selective P2Y14 deficiency attenuated liver fibrosis in multiple mouse models of liver injury. Functional expression of P2Y14 was confirmed in healthy and diseased human liver and human HSCs. In conclusion, P2Y14 ligands and their receptor constitute a profibrogenic DAMP pathway that directly links cell death to fibrogenesis.
Takemura, H;Kushimoto, K;Horii, Y;Fujita, D;Matsuda, M;Sawa, T;Amaya, F;
PMID: 34687776 | DOI: 10.1016/j.brainresbull.2021.10.011
Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.
Morphological and neurochemical characterization of glycinergic neurons in laminae I-IV of the mouse spinal dorsal horn
The Journal of comparative neurology
Miranda, CO;Hegedüs, K;Wildner, H;Zeilhofer, HU;Antal, M;
PMID: 34382691 | DOI: 10.1002/cne.25232
A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.
Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease
Pflugers Archiv : European journal of physiology
Neder, TH;Schrankl, J;Fuchs, MAA;Broeker, KAE;Wagner, C;
PMID: 34355294 | DOI: 10.1007/s00424-021-02604-4
Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.
