Antagonist Of Signaling Research Articles

Discovery of small molecule antagonists of chemokine receptor CXCR6 that arrest tumor growth in SK-HEP-1 mouse xenografts as a model of hepatocellular carcinoma

The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

Effect of primary cilia expression rate on Wnt/β signaling pathway in tibial growth plate chondrocytes from chronic renal insufficiency young rats

Objective: To observe the effect of primary cilia expression rate on Wnt/β signaling pathway in tibial growth plate chondrocytes from chronic renal insufficiency (CRI) young rats. Methods: Male 2-week-old SD rats were randomly divided into two groups: (1) Sham group: only the left ureter was exposed (n=6); (2) CRI group: the left ureter was ligated (n=6). Rats were sacrificed 2 weeks after the operation and the primary cilia expression rate of growth plate chondrocytes and key protein β-catenin in Wnt/β signaling pathway were observed in histological section of tibia specimen. Chondrocytes isolated from growth plate in two groups were cultured in vitro to P3 generation. The primary cilia expression rate and the level of β-catenin were measured. The primary cilia expression rate was detected by agonists and antagonists Wnt/β signaling pathway in chondrocytes of CRI group. The level of β-catenin was detected by using serum-free culture and chloral hydrate to intervene chondrocytes in CRI group. Results: The primary cilia expression rate of growth plate chondrocytes in histological section of tibia specimen in CRI group was higher than that in Sham group [(17.5±7.7)% vs (8.7±3.6)%, t=3.237, P=0.005], and the level of β-catenin was higher in CRI group (5.1±0.7 vs 1.9±0.8, t=6.731, P<0.001). The primary cilia expression rate of growth plate chondrocytes cultured in vitro in CRI group was higher than that in Sham group [(20.9±8.1)% vs (11.8±4.7)%, t=3.073, P=0.007], and the level of β-catenin was higher in CRI group (0.49±0.12 vs 0.25±0.11, t=3.297, P=0.011). There was no significant change in primary cilia expression rate after intervention by using Wnt/β signaling agonists and antagonists to change the level of β-catenin [agonists group: (21.3±7.6)%, control: (20.6±6.8)%, antagonists group: (22.4±6.2)%, F=0.173, P=0.842]. The level of β-catenin was significantly changed after intervention by using serum-free culture, chloral hydrate to alter the primary cilia expression rate (serum-free culture group: 0.61±0.23, control: 0.39±0.24, chloral hydrate group: 0.15±0.11, F=6.476, P=0.012). There was a positive correlation between the level of β-catenin and primary cilia expression rate. Conclusion: The primary cilia expression rate and the level of Wnt/β signaling pathway were higher in tibial growth plate chondrocytes in CRI rats model, and primary cilia might have positive regulatory effects on the Wnt/β signaling pathway.

HMGB1 and sRAGE: What are Their Links to Acute Myeloid Leukemia?

Introduction: Acute myeloid leukemia (AML) is a disparate hematopoietic malignancy, with many subtypes and variable responsiveness to therapy. Identification of a targetable biomarker can be used to improve survival outcome of this disease. High mobility group box 1 (HMGB1), contributes to the progression of AML by inducing various cytokines production and angiogenic vascular endothelial growth factor (VEGF). Receptor for advanced glycation end products (RAGE) exists as both a membrane and soluble receptor (sRAGE). HMGB1 is a ligand for RAGE, with sRAGE serving as a natural antagonist of RAGE signaling. Accumulating evidence suggests that HMGB1/sRAGE axis is critically involved in many cancers.Objective: The aim of this study was to investigate whether there is a difference in the plasma levels of HMGB1/sRAGE between AML patients with good and poor prognosis and those who go into relapse.Methods: A total of 97 plasma samples from peripheral blood were analysed for the study (47 AML, 50 healthy subjects). HMGB1 and sRAGE levels were measured using ELISA and analysed using GraphPad Prism 5.0.Results: HMGB1 levels were found to be significantly higher in relapsed AML cases as compared to newly diagnosed cases (49.81 ± 25.25 (N=11) vs 9.461 ± 4.770 (N=36), pg/ml, p = 0.0161). HMGB1 was also significantly higher among new AML cases classified as having poor prognosis as compared to good prognosis (26.11 ± 13.31 (N=12) vs 1.135 ± 0.7147 (N=24), pg/ml, p = 0.0114). Although sRAGE levels were also high where HMBG1 levels were high, the differences were not significant.Conclusion: HMGB1 may be used as a prognostic biomarker, as it appears to promote AML progression with higher levels seen in more malignant forms. Unravelling the mechanisms of HMGB1 in the pathogenesis of AML could provide means to regulate its expression and activities and serve as an effective treatment for more aggressive subtypes.International Journal of Human and Health Sciences Supplementary Issue: 2019 Page: 59

Secreted Frizzled-related proteins (sFRPs) in osteo-articular diseases: much more than simple antagonists of Wnt signaling?

Osteo-articular diseases are characterized by a dysregulation of joint and/or bone homeostasis. These include diseases affecting the joints originally, such as osteoarthritis and rheumatoid arthritis, or the bone, such as osteoporosis. Inflammation and the involvement of Wingless-related integration site (Wnt) signaling pathways are key pathophysiological features of these diseases resulting in tissue degradation by matrix-degrading enzymes, namely matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTs), secreted by the joint resident cells and/or by infiltrating immune cells. Activation of Wnt signaling pathways is modulated by different families of proteins, including Dickkopfs and the secreted Frizzled-related proteins (sFRPs). The sFRP family is composed of five secreted glycoproteins in mammals that regulate Wnt signaling in the extracellular compartment. Indeed, sFRPs are able to bind both to the soluble Wnt ligands and to their cell membrane receptors, the Frizzled proteins. Their expression profile is altered in osteo-articular diseases, suggesting that they could account for the abnormal activation of Wnt pathways. In the present article, we review how sFRPs are more than simple antagonists of the Wnt signaling pathways and discuss their pathophysiological relevance in the context of osteo-articular diseases. We detail their Wnt-dependent and their Wnt-independent roles, with a particular emphasis on their ability to modulate the inflammatory response and extracellular matrix (ECM) remodeling. We also discuss their potential therapeutic use with a focus on bone remodeling, osteo-articular cancers, and tissue engineering.

LncRNA SPRY4‑IT1 promotes progression of osteosarcoma by regulating ZEB1 and ZEB2 expression through sponging of miR‑101 activity.

Long non-coding (lnc)RNA sprouty receptor tyrosine kinase signalling antagonist 4-intronic transcript 1 (SPRY4-IT1) has been demonstrated to serve a critical role in the tumorigenesis of osteosarcoma (OS); however, the specific underlying mechanism remains unclear. The aim of the present study was to examine the interactions between SPRY4-IT1 and its downstream effectors, to determine if any of the interactions contributed to SPRY4-IT1-mediated proliferation, migration and invasion in cancer cells. A signalling cascade which involved SPRY4-IT1, miR-101 and zinc finger E-box-binding homeoboxes (ZEBs) was examined in the present study. Intracellular SPRY4-IT1 and miR-101 expression levels were altered through transfection to assess their effect on proliferation, cell cycle progression, survival, migration and invasion. A dual-luciferase assay was utilized to determine the association between SPRY4-IT1/miR-101 and ZEBs/miR-101 and nude mouse xenograft experiments were performed to determine the effect of SPRY4-IT1 in vivo. The results indicated that the SPRY4-IT1 levels were negatively associated with miR-101 expression levels in OS cells, an association which was not observed in the normal osteoblast cells. SPRY4-IT1 knockdown or miR-101 overexpression reduced proliferation, cell cycle progression, survival, migration and invasion of MG-63 and U2OS cells. SPRY4-IT1 knockdown was accompanied by increased expression of miR-101 and E-cadherin levels, as well as decreased expression levels of ZEB1/2 and other epithelial-mesenchymal transition-associated proteins. Simultaneous knockdown of SPRY4-IT1 and inhibition of miR-101 partially reversed the anti-tumour effects of SPRY4-IT1 inhibition in vitro. Consistent with these findings, short hairpin RNA targeting SPRY4-IT1 also hindered xenograft tumour growth and altered the levels of miR-101, ZEB1/2 and E-cadherin in vivo. Dual-luciferase reporter assays demonstrated that SPRY4-IT1 may have regulated the expression of ZEB1 and ZEB2 by sponging miR-101. In conclusion, SPRY4-IT1 inhibition increased miR-101 levels, resulting in downregulation of ZEB1/2 expression and thus exerting anti-tumour effects in OS.

A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling

Estrogen exerts extensive and diverse effects throughout the body of women. In addition to the classical nuclear estrogen receptors (ERα and ERβ), the G protein-coupled estrogen receptor GPER is an important mediator of estrogen action. Existing ER-targeted therapeutic agents act as GPER agonists. Here, we report the identification of a small molecule, named AB-1, with the previously unidentified activity of high selectivity for binding classical ERs over GPER. AB-1 also possesses a unique functional activity profile as an agonist of transcriptional activity but an antagonist of rapid signaling through ERα. Our results define a class of small molecules that discriminate between the classical ERs and GPER, as well as between modes of signaling within the classical ERs. Such an activity profile, if developed into an ER antagonist, could represent an opportunity for the development of first-in-class nuclear hormone receptor-targeted therapeutics for breast cancer exhibiting reduced acquired and de novo resistance.

Trpc1 as the Missing Link Between the Bmp and Ca2+ Signalling Pathways During Neural Specification in Amphibians

In amphibians, the inhibition of bone morphogenetic protein (BMP) in the dorsal ectoderm has been proposed to be responsible for the first step of neural specification, called neural induction. We previously demonstrated that in Xenopus laevis embryos, the BMP signalling antagonist, noggin, triggers an influx of Ca2+ through voltage-dependent L-type Ca2+ channels (LTCCs), mainly via CaV1.2, and we showed that this influx constitutes a necessary and sufficient signal for triggering the expression of neural genes. However, the mechanism linking the inhibition of BMP signalling with the activation of LTCCs remained unknown. Here, we demonstrate that the transient receptor potential canonical subfamily member 1, (Trpc1), is an intermediate between BMP receptor type II (BMPRII) and the CaV1.2 channel. We show that noggin induces a physical interaction between BMPRII and Trpc1 channels. This interaction leads to the activation of Trpc1 channels and to an influx of cations, which depolarizes the plasma membrane up to a threshold sufficient to activate Cav1.2. Together, our results demonstrate for the first time that during neural induction, Ca2+ entry through the CaV1.2 channel results from the noggin-induced interaction between Trpc1 and BMPRII.

Dissimilar interaction between dopaminergic and cholinergic systems in the initiation of emission of 50-kHz and 22-kHz vocalizations

Rats emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) to signal their emotional state to other conspecifics. The 22-kHz USVs signal a negative emotional state while 50-kHz USVs reflect a positive affective state. The initiation of 22-kHz USVs is dependent on the activity of cholinergic neurons within the laterodorsal tegmental nucleus that release acetylcholine along the medial cholinoceptive vocalization strip. Emission of 50-kHz USVs is dependent upon the activation of dopaminergic neurons located within the ventral tegmental area that release dopamine into the medial shell of the nucleus accumbens. There have been reports that showed an antagonistic interaction between acetylcholine and dopamine during the expression of emotional states, and dopamine agonists decreased carbachol-induced emission of 22-kHz USVs. The current study tests the hypothesis that initial antagonism of dopamine receptors by systemic haloperidol or intraacumbens raclopride should increase the subsequent emission of 22 kHz USVs induced by carbachol from the lateral septum. Our findings showed that antagonism of dopaminergic signaling either via systemic haloperidol or via intracerebral raclopride did not alter the number of emitted 22-kHz USVs. Thus, inhibition of the mesolimbic dopamine system did not increase the magnitude of a negative emotional state. It was found, however, that prolonged emission of 22-kHz USVs initiated by carbachol caused a delayed rebound emission (R) of 50-kHz USVs appearing after 300 s of emission of 22-kHz USVs, i.e., when the response was subsiding. The R-50-kHz USVs were predominantly frequency modulated (FM) USVs and their number was directly proportional to the number of recorded 22-kHz USVs. The emission of R-50-kHz USVs was reversed by systemic pretreatment with haloperidol or intraacumbens injection of raclopride. It is argued that the R-50-kHz USVs represent a rebound emotional state that is opposite in valence and arousal induced by carbachol. Importantly, prolonged emission of amphetamine-induced 50 kHz USVs failed to show any vocalization rebound effect.

Dysregulation in IL-6 receptors is associated with upregulated IL-17A related signaling in CD4+ T cells of children with autism

Autism spectrum disorder (ASD) is a heterogeneous syndrome characterized by dysregulations in speech and social interactions as well as repetitive and stereotypical behavioral patterns in which immune system plays a significant role. IL-6, an essential cytokine for polarization of Th0 cells into Th17 cells has been demonstrated to be crucial in the etiology of ASD in past studies both in humans and mice. Th17 cells are also believed to be central players in the pathogenesis of ASD through release of IL-17A. However, there is still insufficient data regarding identification of Th17 cells with respect to IL-6 signaling in ASD subjects. Therefore, this study explored IL-6 receptors (IL-6R/sIL-6R) and Th17 (p-STAT3/IL-17A/IL-23R) related markers comprehensively in the blood of typically-developing control (TDC, n = 35) and ASD children (n = 45). Our data show that there is enhanced sIL-6R levels in plasma and CD4+ T cells of ASD subjects as compared to TDC group. Increased sIL-6R signaling is associated with upregulated Th17 development in ASD subjects. Further, severe ASD subjects have higher inflammation in terms of IL-6/IL-17A related signaling as compared to moderate ASD patients. Furthermore, treatment of CD4 + T cells in vitro with IL-6 leads to much greater upregulation of p-STAT3, and IL-17A in ASD subjects than similarly treated CD4+ T cells in TDC group. Antagonism of IL-6 signaling by SC144 in vitro led to blockade of IL-6 mediated effects on CD4+ T cells. These data display unequivocally that IL-6 signaling components are dysregulated which play a crucial in enhancement of Th17 development in ASD subjects.

Epigenetic changes in fibroblasts drive cancer metabolism and differentiation.

Genomic changes that drive cancer initiation and progression contribute to the co-evolution of the adjacent stroma. The nature of the stromal reprogramming involves differential DNA methylation patterns and levels that change in response to the tumor and systemic therapeutic intervention. Epigenetic reprogramming in carcinoma-associated fibroblasts are robust biomarkers for cancer progression and have a transcriptional impact that support cancer epithelial progression in a paracrine manner. For prostate cancer, promoter hypermethylation and silencing of the RasGAP, RASAL3 that resulted in the activation of Ras signaling in carcinoma-associated fibroblasts. Stromal Ras activity initiated a process of macropinocytosis that provided prostate cancer epithelia with abundant glutamine for metabolic conversion to fuel its proliferation and a signal to transdifferentiate into a neuroendocrine phenotype. This epigenetic oncogenic metabolic/signaling axis seemed to be further potentiated by androgen receptor signaling antagonists and contributed to therapeutic resistance. Intervention of stromal signaling may complement conventional therapies targeting the cancer cell.

TLR9 acts as a sensor for tumor-released DNA to modulate anti-tumor immunity after chemotherapy

The tumor microenvironment exists in a state of dynamic equilibrium, in which a balance of agonist and antagonist signals govern the anti-tumor immune responses. Previous studies have shown that chemotherapy could shift this balance in favor of agonistic signals for the anti-tumor immune responses mounted by CD8+ cytotoxic T lymphocytes (CTL), providing sufficiently high antigen density within the tumor. We undertook the current study to characterize the anti-tumor immune response following chemotherapy and its underlying mechanisms. We show that this ‘adjuvant effect’ of chemotherapy is, at least partially, mediated by the release of tumor DNA and acts through the Toll-like receptor 9 (TLR9) pathway. We found that tumor-released DNA causes accumulation, antigen uptake, and maturation of dendritic cells (DCs) in the tumor in a TLR9-dependent manner. These DCs subsequently migrate into the draining lymph nodes and prime tumor-specific CTLs. Our study provides novel insights to the molecular and cellular mechanisms by which chemotherapy converts the tumor microenvironment into a site permissive for the activation of a potent tumor-specific adaptive immune response.

P5991Cellular mechanisms of aortic valve calcification in primary human valvular interstitial cell culture

Abstract Background Aortic stenosis (AS) is the most common acquired valvular heart disease. Calcification of the aortic valve (AV) cusps is the main pathogenetic mechanism of AS formation, however triggering and progression mechanisms of it are not fully understood. Aortic valve interstitial cells (AVIC) are one of the main cell populations responsible for the AV structure and homeostasis. The aim of the study was to characterize the ability of AVIC to osteogenic differentiation using the model of the primary culture of AVIC of patients with aortic stenosis and assess the expression of genes involved in osteogenesis (RUNX2, BMPG2, SPRY1, SOX9, CTNNB1, POSTN, OPG and OPN). Materials and methods The study was carried out on primary human AVIC culture obtained by enzymatic dissociation of experimental valve specimens from patients with AS (n=15) and the control specimens from the orthotopic heart transplantation recipients (n=10). To assess the osteogenic potential of AVIC, the routine stem cell osteodifferentiation protocol based on the culture medium with addition of inductors (10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 50 μg/ml ascorbic acid) was used. Calcium deposits were demonstrated by Alizarin Red staining. Analysis of the expression of osteogenic differentiation genes, such as RUNX2 (runtrelated transcription factor 2), BMP2 (bone morphogenetic protein 2), SPRY1 (Sprouty RTK signaling antagonist 1), SOX-9 (SRY-box9), CTNNB1 (β-catenin1), POSTN (periostin), OPG (osteoprotegerin), and OPN (osteopontin), was performed by real-time PCR. Results The inductors of osteogenic differentiation provoked greater mineralization of AVIC cultures derived from the patients with AS than that observed in control group (p=0.0003). The expression of RUNX2 and SPRY1 in non-differentiated cells was reduced compared with control cells in patients with bicuspid AV (p=0.02). After 21 days of the osteogenic induction, the expression of RUNX2 and SPRY1 increased in all three groups. At the same time, the expression of SPRY1 was lower in the group with bicuspid AV compared with tricuspid AV (p=0.007). The expression level of BMP2 did not differ between groups in unstimulated AVIC, however, it increased after osteogenic differentiation in the group of patients with tricuspid AV (p=0.017). OPN expression was higher in cells from tricuspid AV, while OPG expression was reduced in patients with both bicuspid and tricuspid AV (p&lt;0.01), No differences were found between the groups) for the remaining genes (POSTIN, CTNNB1, SOX9) before and after stimulation with an osteogenic medium. Conclusions The osteogenic potential of AVIC is increased in patients with aortic stenosis. The gene expression profile of osteogenic differentiation differs in patients with a bicuspid and tricuspid aortic valve. Damage of protective mechanisms may be a potential mechanism for accelerated valve calcification mostly in patients with tricuspid aortic valve. Acknowledgement/Funding Russian Foundation for Basic Research, project 18-015-00016

LncRNA UCA1 Predicts a Poor Prognosis and Regulates Cell Proliferation and Migration by Repressing p21 and SPRY1 Expression in GC

Dysregulated expression of long non-coding RNAs (lncRNAs) has been reported in many types of cancers, indicating that it has important regulatory roles in human cancer biology. Recently, lncRNA urothelial cancer-associated 1 (UCA1) was shown to be dysregulated in many cancer types, but the detailed mechanisms remain largely unknown. In our study, we found that upregulated UCA1 is associated with poor prognosis in gastric cancer patients. Further experiments revealed that UCA1 knockdown significantly repressed the proliferation and migration both in vitro and in vivo. Moreover, RNA sequencing (RNA-seq) analysis revealed that UCA1 knockdown preferentially affected genes that are linked to cell proliferation, cell cycle, and cell migration. Mechanistically, UCA1 promotes cell proliferation progression through repressing p21 and Sprouty RTK signaling antagonist 1 (SPRY1) expression by binding to EZH2. We found that UCA1 could mediate the trimethylation of H3K27 in promoters of p21 and SPRY1. To our knowledge, this is the first report showing the global gene profile of downstream targets of UCA1 in the progression of gastric cancer. Collectively, our data reveal the important roles of UCA1 in gastric cancer (GC) oncogenesis.

Conceptualization of a Parasympathetic Endocrine System.

We here propose a parasympathetic endocrine system (PES) comprised of circulating peptides released from secretory cells in the gut, significantly modulated by vagal projections from the dorsal motor nucleus of the vagus (DMV). While most of these gut peptides mediate well-described satiety and digestive effects that increase parasympathetic control of digestion (Lee et al., 1994; Gutzwiller et al., 1999; Klok et al., 2007), they also have actions that are far-reaching and increase parasympathetic signaling broadly throughout the body. The actions beyond satiety that peptides like somatostatin, cholecystokinin, glucagon-like peptide 1, and vasoactive intestinal peptide have been well-examined, but not in a systematic way. Consideration has been given to the idea that these and other gut-derived peptides are part of an endocrine system has been partially considered (Rehfeld, 2012; Drucker, 2016), but that it is coordinated through parasympathetic control and may act to increase the actions of parasympathetic projections has not been formalized before. Here only gut-derived hormones are included although there are potentially other parasympathetically mediated factors released from other sites like lung and liver (Drucker, 2016). The case for the existence of the PES with the DMV as its integrative controller will be made through examination of an anatomical substrate and evidence of physiological control mechanisms as well as direct examples of PES antagonism of sympathetic signaling in mammals, including humans. The implications for this conceptual understanding of a PES reframe diseases like metabolic syndrome and may help underscore the role of the autonomic nervous system in the associated symptoms.

Effects of Alcohol and Estrogen Receptor Blockade Using ICI 182,780 on Bone in Ovariectomized Rats.

Estrogen signaling is essential for the sexual dimorphism of the skeleton, is required for normal bone remodeling balance in adults, and may influence the skeletal response to alcohol. High levels of alcohol consumption lower bone mass in ovary-intact but not ovariectomized (ovx) rats. However, the extremely rapid rate of bone loss immediately following ovx may obscure the effects of alcohol. We therefore determined (i) whether heavy alcohol consumption (35% caloric intake) influences bone in sexually mature ovx rats with established cancellous osteopenia and (ii) whether ICI 182,780 (ICI), a potent estrogen receptor signaling antagonist, alters the skeletal response to alcohol. Three weeks following ovx, rats were randomized into 5 groups, (i) baseline, (ii) control+vehicle, (iii) control+ICI, (iv) ethanol (EtOH)+vehicle, or (v) EtOH+ICI, and treated accordingly for 4weeks. Dual-energy X-ray absorptiometry, microcomputed tomography, blood measurements of markers of bone turnover, and gene expression in femur and uterus were used to evaluate response to alcohol and ICI. Rats consuming alcohol had lower bone mass and increased fat mass. Bone microarchitecture of the tibia and gene expression in femur were altered; specifically, there was reduced accrual of cortical bone, net loss of cancellous bone, and differential expression of 19/84 genes related to bone turnover. Furthermore, osteocalcin, a marker of bone turnover, was lower in alcohol-fed rats. ICI had no effect on weight gain, body composition, or cortical bone. ICI reduced cancellous bone loss and serum CTX-1, a biochemical marker of bone resorption; alcohol antagonized the latter 2 responses. Neither alcohol nor ICI affected uterine weight or gene expression. Alcohol exaggerated bone loss in ovx rats in the presence or absence of estrogen receptor blockade with ICI. The negligible effect of alcohol on uterus and limited effects of ICI on bone in alcohol-fed ovx rats suggest that estrogen receptor signaling plays a limited role in the action of alcohol on bone in a rat model for chronic alcohol abuse.

Highly expressed BMP9/GDF2 in postnatal mouse liver and lungs may account for its pleiotropic effects on stem cell differentiation, angiogenesis, tumor growth and metabolism

Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFβ/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood.

Interleukin-36 cytokines alter the intestinal microbiome and can protect against obesity and metabolic dysfunction

Members of the interleukin-1 (IL-1) family are important mediators of obesity and metabolic disease and have been described to often play opposing roles. Here we report that the interleukin-36 (IL-36) subfamily can play a protective role against the development of disease. Elevated IL-36 cytokine expression is found in the serum of obese patients and negatively correlates with blood glucose levels among those presenting with type 2 diabetes. Mice lacking IL-36Ra, an IL-36 family signalling antagonist, develop less diet-induced weight gain, hyperglycemia and insulin resistance. These protective effects correlate with increased abundance of the metabolically protective bacteria Akkermansia muciniphila in the intestinal microbiome. IL-36 cytokines promote its outgrowth as well as increased colonic mucus secretion. These findings identify a protective role for IL-36 cytokines in obesity and metabolic disease, adding to the current understanding of the role the broader IL-1 family plays in regulating disease pathogenesis.

Soluble fms-Like Tyrosine Kinase 1 Localization in Renal Biopsies of CKD

IntroductionSoluble fms-like tyrosine kinase 1 (sFLT1) is a splice variant of the vascular endothelial growth factor (VEGF) receptor lacking the transmembrane and cytoplasmic domains and acts as a powerful antagonist of VEGF signaling. Plasma sFLT1 levels are higher in patients with chronic kidney disease (CKD) and correlate with renal dysfunction. The source of plasma sFLT1 in CKD is unclear.MethodsFifty-two renal biopsies were studied for sFLT1 expression using immunohistochemistry and evaluated on a 0–4 grading scale of positive cells within inflammatory infiltrates. These included drug-induced interstitial nephritis (6); allografts (12), with polyomavirus nephritis (3); diabetes mellitus (10); lupus glomerulonephritis (6); pauci-immune vasculitis (7); IgA nephropathy (6); and miscellaneous CKD (5).ResultsForty-seven biopsies had inflammatory infiltrates of which 37 had sFLT1-positive cells: of these biopsies, 3 were grade 4, i.e., had cells that constituted more than 50% of the inflammatory infiltrate, 9 were grade 3 (25%–50%), 5 were grade 2 (10%–25%), 3 were grade 1 (10%), and 17 were grade 0.5 (<10%). There was a robust correlation (r2 = 0.89) between degree of inflammation and sFLT1-positive cells. CD68/sFLT1 co-immunostaining studies indicated that sFLT1-positive cells were histiocytes. The surrounding capillary network was reduced.ConclusionsFLT1-positive histiocytes are generally part of the inflammatory infiltrates noted in CKD and are particularly abundant in forms of interstitial nephritis. Their presence promotes an anti-angiogenic state locally in the tubulointerstitium that could inhibit capillary repair, contribute to peritubular capillary loss, and enhance fibrosis in CKD.

Secreted frizzled-related protein 4 and its implication in obesity and type-2 diabetes.

Secreted frizzled-related protein 4 (SFRP4) is a member of secreted protein family with sequence similarity to frizzled receptors of wingless-related integration site (Wnt) signaling pathways. These proteins control diverse functions from embryonic development to adults in many organisms including humans. Initially, SFRPs were recognized as antagonists of Wnt signaling and supposed to interact with Wnts. Further research demonstrated their interactions to frizzled receptors and a functional diversity was related to these proteins, Wnt signaling potentiation in addition to modulation. SFRP4 is the largest member of SFRP family and is implicated in many diseases including obesity, type 2 diabetes (T2D), and cancer. SFRP4 acts as a biomarker for T2D and was expressed several years before clinical diagnosis of disease. This review mainly focusses on the role of SFRP4 in obesity and how it can lead to β-cell failure and ultimately to T2D. The role of SFRP4 in adipose tissues causing increased production of adipokines lead to the oxidative stress in pancreas that particularly have low amount of antioxidant enzymes in pancreatic β-cells leading to failure in exocytosis of insulin containing granules causing T2D. Obesity-induced inflammation is a principal factor in pathogenesis of insulin resistance as well as metabolic syndrome. Pro-inflammatory cytokines have potential to cause insulin resistance in skeletal muscles, adipose tissue, and liver via inhibition of insulin signal transduction. Secretion of SFRP4 is mediated by interleukin 1-β (IL1-β). This review highlights the molecular mechanisms by which SFRP4 leads to T2D. Understanding of molecular mechanism and targeting SFRP4 could help to eradicate or reduce chances of developing T2D.

Abstract 1201: IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer

Abstract IL-4 has a number of well-established functions in immune cells including proliferation, and survival of lymphocytes, polarization of macrophages to the pro-tumor M2 tumor associated macrophage phenotype. Another role of IL-4 signaling is induction of metabolic changes in immune cells including increased glycolysis in B-lymphocytes, and increased expression of arginase 1 with a switch to fatty acid oxidation in macrophages. However, the effects of IL-4 signaling in tumor cells that express IL-4 receptor is not well understood. Previous work from our lab has shown that loss of IL-4 receptor (IL-4R) significantly abrogates metastasis in experimental metastasis models in mice. Thus, we wondered whether changes in metabolism downstream of IL-4 receptor activation may contribute to metastatic phenotypes. Since altered metabolic pathways have been shown to regulate the epigenetic state of cancer cells, we are pursuing histone modifications as a mechanism for the connection between altered metabolism and increased metastatic capability in IL-4R expressing breast cancer cells. MDA-MB-231 and BT549 triple negative breast cancer cells were treated with IL-4 for 2,4,6, and 8 days. Samples of media were assayed for glutamine and glucose consumption, as well as lactate and glutamate secretion. IL-4 decreased consumption of glucose and glutamine, yet concomitantly increased the release of lactate and glutamate. These data suggest that IL-4 alters the utilization of glucose and glutamine in cancer cells. To study potential links to epigenetic regulation cells were treated for 18, or 48 hours with IL-4 before biochemical fractionation was performed in order to obtain nuclear and cytosolic lysates. The nuclear fractions were assessed for histone acetyltransferase (HAT) activity using commercially available assays, and showed that IL-4 increased the activity of histone acetyltransferases. We also performed pull down of acetylated lysine residue from the nuclear fractions of IL-4 treated cells. The cells were then assessed for abundance of acetylated histone 3. In addition, we used western blotting to measure the abundance of specific histone modifications such as histone 3 lysine 9 acetylation and histone 3 lysine 27 acetylation. Overall, histone acetyltransferase activity and levels of histone acetylation were increased by IL-4. Additionally, we found that IL4 increases the acetylation of histone 3 lysine 9. In order to determine the effects of IL-4 signaling on metabolism in vivo we have initiated PET imaging studies in humanized mice that underwent tail vein injections of BT549 cell lines. These mice were treated with MDNA413, an antagonist of IL-4 signaling, and are being assessed for uptake of glucose and glutamine. Together these suggest that IL-4 alters the metabolism and epigenetic state of cancer cells and may be a viable target for therapy in metastatic breast cancer. Citation Format: Demond Williams, Adam Miranda, Chilesi Uririri, Barbara Fingleton. IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1201.