International journal of surgical pathology
Uehara, T;Sato, K;Iwaya, M;Asaka, S;Nakajima, T;Nagaya, T;Kitazawa, M;Ota, H;
PMID: 37306249 | DOI: 10.1177/10668969231177705
Background. Tumor budding is a poor prognostic factor in colorectal adenocarcinoma, but the underlying mechanism remains unclear. Interleukin-6 (IL6) is one of the main cytokines produced by cancer-associated fibroblasts. IL6 is linked with cancer progression and poor prognosis by activating cancer cells and modifying the cancer microenvironment. However, little is known about the expression of IL6 in tumor budding and its association with tumor budding in colorectal adenocarcinoma. Methods. The clinicopathological and prognostic significance of IL6 in tumor budding was examined using a tissue microarray consisting of 36 patient samples of tumor budding in colorectal adenocarcinoma. IL6 mRNA was detected by RNAscope. Patients were stratified into negative and positive IL6 expression groups. Results. IL6 expression was overwhelmingly observed in cancer stroma but was negligible in cancer cells. Tumor budding grade was higher in the IL6-positive group in cancer stroma than in the IL6-negative group (P = .0161), while the IL6-positive group significantly exhibited the epithelial-mesenchymal transition phenotype compared with the IL6-negative group in cancer stroma (P = .0301). There was no significant difference in overall survival between colorectal adenocarcinoma patients in the IL6-positive and -negative groups in cancer stroma. Conclusion. Tumor budding may be affected by IL6 expression, and IL6 expression in cancer stroma at tumor budding may be an important prognostic marker.
Journal of molecular endocrinology, 50(3), 325–336.
Boess F, Bertinetti-Lapatki C, Zoffmann S, George C, Pfister T, Roth A, Lee SM, Thasler WE, Singer T, Suter L (2013).
PMID: 23463748 | DOI: 10.1530/JME-12-0186.
Glucagon-like peptide 1 (GLP1) analogs have been associated with an increased incidence of thyroid C-cell hyperplasia and tumors in rodents. This effect may be due to a GLP1 receptor (GLP1R)-dependent mechanism. As the expression of GLP1R is much lower in primates than in rodents, the described C-cell proliferative lesions may not be relevant to man. Here, we aimed to establish primary thyroid cell cultures of rat and human to evaluate the expression and function of GLP1R in C-cells. In our experiments, GLP1R expression was observed in primary rat C-cells (in situ hybridization) but was not detected in primary human C-cells (mRNA and protein levels). The functional response of the cultures to the stimulation with GLP1R agonists is an indirect measure of the presence of functional receptor. Liraglutide and taspoglutide elicited a modest increase in calcitonin release and in calcitonin expression in rat primary thyroid cultures. Contrarily, no functional response to GLP1R agonists was observed in human thyroid cultures, despite the presence of few calcitonin-positive C-cells. Thus, the lack of functional response of the human cultures adds to the weight of evidence indicating that healthy human C-cells have very low levels or completely lack GLP1R. In summary, our results support the hypothesis that the GLP1R agonist-induced C-cell responses in rodents may not be relevant to primates. In addition, the established cell culture method represents a useful tool to study the physiological and/or pathological roles of GLP1 and GLP1R agonists on normal, non-transformed primary C-cells from rats and man.
bioRxiv : the preprint server for biology
Sun, Q;van de Lisdonk, D;Ferrer, M;Gegenhuber, B;Wu, M;Tollkuhn, J;Janowitz, T;Li, B;
PMID: 36711916 | DOI: 10.1101/2023.01.12.523716
Interleukin-6 (IL-6) has been long considered a key player in cancer-associated cachexia 1-15 . It is believed that sustained elevation of IL-6 production during cancer progression causes brain dysfunctions, which ultimately result in cachexia 16-20 . However, how peripheral IL-6 influences the brain remains poorly understood. Here we show that neurons in the area postrema (AP), a circumventricular structure in the hindbrain, mediate the function of IL-6 in cancer-associated cachexia in mice. We found that circulating IL-6 can rapidly enter the AP and activate AP neurons. Peripheral tumor, known to increase circulating IL-6 1-5,15,18,21-23 , leads to elevated IL-6 and neuronal hyperactivity in the AP, and causes potentiated excitatory synaptic transmission onto AP neurons. Remarkably, neutralization of IL-6 in the brain of tumor-bearing mice with an IL-6 antibody prevents cachexia, reduces the hyperactivity in an AP network, and markedly prolongs lifespan. Furthermore, suppression of Il6ra , the gene encoding IL-6 receptor, specifically in AP neurons with CRISPR/dCas9 interference achieves similar effects. Silencing of Gfral-expressing AP neurons also ameliorates the cancer-associated cachectic phenotypes and AP network hyperactivity. Our study identifies a central mechanism underlying the function of peripheral IL-6, which may serve as a target for treating cancer-associated cachexia.
Zhang, L;Koller, J;Gopalasingam, G;Qi, Y;Herzog, H;
PMID: 35691527 | DOI: 10.1016/j.molmet.2022.101525
Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons.We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons.Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control.NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.
Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells
Grunddal, KV;Tonack, S;Egerod, KL;Thompson, JJ;Petersen, N;Engelstoft, MS;Vagne, C;Keime, C;Gradwohl, G;Offermanns, S;Schwartz, TW;
PMID: 33831593 | DOI: 10.1016/j.molmet.2021.101231
GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.
Ronn J, Jensen EP, Wewer Albrechtsen NJ, Holst JJ, Sorensen CM.
PMID: 29233907 | DOI: 10.14814/phy2.13503
Glucagon-like peptide-1 (GLP-1) is an incretin hormone increasing postprandial insulin release. GLP-1 also induces diuresis and natriuresis in humans and rodents. The GLP-1 receptor is extensively expressed in the renal vascular tree in normotensive rats where acute GLP-1 treatment leads to increased mean arterial pressure (MAP) and increased renal blood flow (RBF). In hypertensive animal models, GLP-1 has been reported both to increase and decrease MAP. The aim of this study was to examine expression of renal GLP-1 receptors in spontaneously hypertensive rats (SHR) and to assess the effect of acute intrarenal infusion of GLP-1. We hypothesized that GLP-1 would increase diuresis and natriuresis and reduce MAP in SHR. Immunohistochemical staining and in situ hybridization for the GLP-1 receptor were used to localize GLP-1 receptors in the kidney. Sevoflurane-anesthetized normotensive Sprague-Dawley rats and SHR received a 20 min intrarenal infusion of GLP-1 and changes in MAP, RBF, heart rate, dieresis, and natriuresis were measured. The vasodilatory effect of GLP-1 was assessed in isolated interlobar arteries from normo- and hypertensive rats. We found no expression of GLP-1 receptors in the kidney from SHR. However, acute intrarenal infusion of GLP-1 increased MAP, RBF, dieresis, and natriuresis without affecting heart rate in both rat strains. These results suggest that the acute renal effects of GLP-1 in SHR are caused either by extrarenal GLP-1 receptors activating other mechanisms (e.g., insulin) to induce the renal changes observed or possibly by an alternative renal GLP-1 receptor.
Egerod KL, Petersen N ,Timshel PN, Rekling JC, Wang Y, Liu Q, Schwartz TW, Gautron L.
PMID: - | DOI: 10.1016/j.molmet.2018.03.016
Abstract
Objectives
G protein-coupled receptors (GPCRs) act as transmembrane molecular sensors of neurotransmitters, hormones, nutrients, and metabolites. Because unmyelinated vagalafferents richly innervate the gastrointestinal mucosa, gut-derived molecules may directly modulate the activity of vagal afferents through GPCRs. However, the types of GPCRs expressed in vagal afferents are largely unknown. Here, we determined the expression profile of all GPCRs expressed in vagal afferents of the mouse, with a special emphasis on those innervating the gastrointestinal tract.
Methods
Using a combination of high-throughput quantitative PCR, RNA sequencing, and in situhybridization, we systematically quantified GPCRs expressed in vagal unmyelinated Nav1.8-expressing afferents.
Results
GPCRs for gut hormones that were the most enriched in Nav1.8-expressing vagal unmyelinated afferents included NTSR1, NPY2R, CCK1R, and to a lesser extent, GLP1R, but not GHSR and GIPR. Interestingly, both GLP1R and NPY2R were coexpressed with CCK1R. In contrast, NTSR1 was coexpressed with GPR65, a marker preferentially enriched in intestinal mucosal afferents. Only few microbiome-derived metabolite sensors such as GPR35 and, to a lesser extent, GPR119 and CaSR were identified in the Nav1.8-expressing vagal afferents. GPCRs involved in lipid sensing and inflammation (e.g. CB1R, CYSLTR2, PTGER4), and neurotransmitters signaling (CHRM4, DRD2, CRHR2) were also highly enriched in Nav1.8-expressing neurons. Finally, we identified 21 orphan GPCRs with unknown functions in vagal afferents.
Conclusion
Overall, this study provides a comprehensive description of GPCR-dependent sensing mechanisms in vagal afferents, including novel coexpression patterns, and conceivably coaction of key receptors for gut-derived molecules involved in gut-brain communication.
The Journal of clinical investigation
Alter, C;Henseler, AS;Owenier, C;Hesse, J;Ding, Z;Lautwein, T;Bahr, J;Hayat, S;Kramann, R;Kostenis, E;Scheller, J;Schrader, J;
PMID: 36943408 | DOI: 10.1172/JCI163799
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 and how its production is regulated is largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. IL-6, measured in various cell types in post MI hearts by qPCR, RNAscope and at protein level, was preferentially formed by fibroblasts (CFs). scRNAseq in infarcted mouse and human hearts confirmed this finding. Adenosine stimulated fibroblast IL-6 formation via A2bR in a Gq-dependent manner. CFs highly expressed Adora2b, rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans Adora2B was also mainly expressed by fibroblasts (scRNAseq). Global IL-6 formation was assessed in isolated hearts in mice lacking CD73 on T-cells (CD4CD73-/-) a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4CD73-/- mice, suggesting adenosine-mediated modulation. Together this demonstrates that post-MI IL-6 is mainly derived from activated CFs and is controlled by T-cell derived adenosine. Purinergic metabolic cooperation between CFs and T-cells is a novel mechanism with therapeutic potential which modulates IL6 formation by the heart.
The induction of preterm labor in rhesus macaques is determined by the strength of immune response to intrauterine infection
Cappelletti, M;Presicce, P;Feiyang, M;Senthamaraikannan, P;Miller, LA;Pellegrini, M;Sim, MS;Jobe, AH;Divanovic, S;Way, SS;Chougnet, CA;Kallapur, SG;
PMID: 34495952 | DOI: 10.1371/journal.pbio.3001385
Intrauterine infection/inflammation (IUI) is a major contributor to preterm labor (PTL). However, IUI does not invariably cause PTL. We hypothesized that quantitative and qualitative differences in immune response exist in subjects with or without PTL. To define the triggers for PTL, we developed rhesus macaque models of IUI driven by lipopolysaccharide (LPS) or live Escherichia coli. PTL did not occur in LPS challenged rhesus macaques, while E. coli-infected animals frequently delivered preterm. Although LPS and live E. coli both caused immune cell infiltration, E. coli-infected animals showed higher levels of inflammatory mediators, particularly interleukin 6 (IL-6) and prostaglandins, in the chorioamnion-decidua and amniotic fluid (AF). Neutrophil infiltration in the chorio-decidua was a common feature to both LPS and E. coli. However, neutrophilic infiltration and IL6 and PTGS2 expression in the amnion was specifically induced by live E. coli. RNA sequencing (RNA-seq) analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon (IFN) response, chemotaxis, sumoylation, and iron homeostasis were up-regulated in the E. coli group compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.
Castration-induced down-regulation of SPARC in stromal cells drives neuroendocrine differentiation of prostate cancer
Enriquez, C;Cancila, V;Ferri, R;Sulsenti, R;Fischetti, I;Milani, M;Ostano, P;Gregnanin, I;Mello-Grand, M;Berrino, E;Bregni, M;Renne, G;Tripodo, C;Colombo, MP;Jachetti, E;
PMID: 34185677 | DOI: 10.1158/0008-5472.CAN-21-0163
Fatal neuroendocrine differentiation (NED) of castration-resistant prostate cancer is a recurrent mechanism of resistance to androgen deprivation therapies (ADT) and anti-androgen receptor pathway inhibitors (ARPI) in patients. The design of effective therapies for neuroendocrine prostate cancer (NEPC) is complicated by limited knowledge of the molecular mechanisms governing NED. The paucity of acquired genomic alterations and the deregulation of epigenetic and transcription factors suggest a potential contribution from the microenvironment. In this context, whether ADT/ARPI induces stromal cells to release NED-promoting molecules and the underlying molecular networks are unestablished. Here, we utilized transgenic and transplantable mouse models and co-culture experiments to unveil a novel tumor-stroma crosstalk that is able to induce NED under the pressure of androgen deprivation. Castration induced upregulation of GRP78 in tumor cells, which triggers miR-29b-mediated downregulation of the matricellular protein SPARC in the nearby stroma. SPARC downregulation enabled stromal cells to release IL-6, a known inducer of NED. A drug that targets GRP78 blocked NED in castrated mice. A public, human NEPC gene expression dataset showed that Hspa5 (encoding for GRP78) positively correlates with hallmarks of NED. Finally, prostate cancer specimens from patients developing local NED after ADT showed GRP78 upregulation in tumor cells and SPARC downregulation in the stroma. These results point to GRP78 as a potential therapeutic target and to SPARC downregulation in stromal cells as a potential early biomarker of tumors undergoing NED.
Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland
Proceedings of the National Academy of Sciences of the United States of America
Vennekens, A;Laporte, E;Hermans, F;Cox, B;Modave, E;Janiszewski, A;Nys, C;Kobayashi, H;Malengier-Devlies, B;Chappell, J;Matthys, P;Garcia, MI;Pasque, V;Lambrechts, D;Vankelecom, H;
PMID: 34161279 | DOI: 10.1073/pnas.2100052118
Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.
López-Ferreras L, Eerola K, Mishra D, Shevchouk OT, Richard JE, Nilsson FH, Hayes MR, Skibicka KP.
PMID: - | DOI: 10.1016/j.molmet.2018.11.005
Objective
The supramammillary nucleus (SuM) is nestled between the lateral hypothalamus (LH) and the ventral tegmental area (VTA). This neuroanatomical position is consistent with a potential role of this nucleus to regulate ingestive and motivated behavior. Here neuroanatomical, molecular, and behavior approaches are utilized to determine whether SuM contributes to ingestive and food-motivated behavior control.
Methods
Through the application of anterograde and retrograde neural tract tracing with novel designer viral vectors, the current findings show that SuM neurons densely innervate the LH in a sex dimorphic fashion. Glucagon-like peptide-1 (GLP-1) is a clinically targeted neuro-intestinal hormone with a well-established role in regulating energy balance and reward behaviors. Here we determine that GLP-1 receptors (GLP-1R) are expressed throughout the SuM of both sexes, and also directly on SuM LH-projecting neurons and investigate the role of SuM GLP-1R in the regulation of ingestive and motivated behavior in male and female rats.
Results
SuM microinjections of the GLP-1 analogue, exendin-4, reduced ad libitum intake of chow, fat, or sugar solution in both male and female rats, while food-motivated behaviors, measured using the sucrose motivated operant conditioning test, was only reduced in male rats. These data contrasted with the results obtained from a neighboring structure well known for its role in motivation and reward, the VTA, where females displayed a more potent response to GLP-1R activation by exendin-4. In order to determine the physiological role of SuM GLP-1R signaling regulation of energy balance, we utilized an adeno-associated viral vector to site-specifically deliver shRNA for the GLP-1R to the SuM. Surprisingly, and in contrast to previous results for the two SuM neighboring sites, LH and VTA, SuM GLP-1R knockdown increased food seeking and adiposity in obese male rats without altering food intake, body weight or food motivation in lean or obese, female or male rats.
Conclusion
Taken together, these results indicate that SuM potently contributes to ingestive and motivated behavior control; an effect contingent on sex, diet/homeostatic energy balance state and behavior of interest. These data also extend the map of brain sites directly responsive to GLP-1 agonists, and highlight key differences in the role that GLP-1R play in interconnected and neighboring nuclei.
