Probes for INS

Neuronal circuits involving the central amygdala (CeA) are gaining prominence as important centers for regulation of metabolic functions. As a part of the subcortical food motivation circuitry, CeA is associated with food motivation and hunger. We have previously shown that interleukin-6 (IL-6) can act as a downstream mediator of the metabolic effects of glucagon-like peptide-1 receptor (GLP-1R) stimulation in the brain, but the sites of these effects are largely unknown. We here used the newly generated and validated RedIL6 reporter mouse strain to investigate the presence of IL-6 in the CeA, as well as possible interactions between IL-6 and GLP-1 in this nucleus. IL-6 was present in the CeA, mostly in cells in the medial and lateral parts of this structure, and a majority of IL-6-containing cells also co-expressed GLP-1R. Triple staining showed GLP-1 containing fibers co-staining with synaptophysin close to or overlapping with IL-6 containing cells. GLP-1R stimulation enhanced IL-6 mRNA levels. IL-6 receptor-alpha was found to a large part in neuronal CeA cells. Using electrophysiology, we determined that cells with neuronal properties in the CeA could be rapidly stimulated by IL-6 administration in vitro. Moreover, microinjections of IL-6 into the CeA could slightly reduce food intake in vivo in overnight fasted rats. In conclusion, IL-6 containing cells in the CeA express GLP-1R, are close to GLP-1-containing synapses, and get increased IL-6 mRNA in response to GLP-1R agonist treatment. IL-6, in turn, exerts biological effects in the CeA, possibly via IL-6 receptor-alpha present in this nucleus.

Abstract

Inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), are elevated in ovarian cancer. Differences in cytokine expression by histologic subytpe or ovarian cancer risk factors can provide useful insight into ovarian cancer risk and etiology. We used ribonucleic acid (RNA) in-situ hybridization to assess TNF-α and IL-6 expression on tissue microarray slides from 78 epithelial ovarian carcinomas (51 serous, 12 endometrioid, 7 clear cell, 2 mucinous, 6 other) from a population-based case control study. Cytokine expression was scored semi-quantitatively and odds ratios (OR) and 95% confidence intervals (CI) were calculated using polytomous logistic regression. TNF-α was expressed in 46% of the tumors while sparse IL-6 expression was seen only 18% of the tumors. For both markers, expression was most common in high grade serous carcinomas followed by endometrioid carcinomas. Parity was associated with a reduced risk of TNF-α positive (OR = 0.3, 95% CI: 0.1-0.7 for 3 or more children versus none) but not TNF-α negative tumors (p-heterogeneity = 0.02). In contrast, current smoking was associated with a nearly three fold increase in risk of TNF-α negative (OR = 2.8, 95% CI: 1.2, 6.6) but not TNF-α positive tumors (p-heterogeneity = 0.06). Our data suggests that TNF-α expression in ovarian carcinoma varies by histologic subtype and provides some support for the role of inflammation in ovarian carcinogenesis. The novel associations detected in our study need to be validated in a larger cohort of patients in future studies.

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.

Abstract

BACKGROUND & AIMS:

The present study aimed to elucidate the clinicopathological significance of IL-6 and IL-33 expression in intrahepatic cholangiocarcinomas (iCCAs) and perihilar cholangiocarcinomas (pCCAs).

METHODS:

IL-6 and IL-33 mRNA expression was examined in iCCAs (n=55) and pCCAs (n=32) using quantitative real-time PCR and a highly sensitive in situ hybridization protocol (RNAscope™ ), and expression values were correlated with clinicopathological features. According to a recently proposed classification scheme, iCCAs were separated into small- (n=33) and large-duct types (n=22).

RESULTS:

IL-6 and IL-33 expression levels were higher in large-duct iCCAs and pCCAs than in small-duct iCCAs, with a positive correlation between the values of these cytokines. In double in situ hybridization/immunostaining, IL-6 mRNA was expressed in actin-positive (myo)fibroblasts, while IL-33 was mainly produced by CD31-positive endothelial cells. Based on the average expression value as a cut-off point, cases were classified as IL-6high and IL-6low or IL-33high and IL-33low . In the combined cohort of large-duct iCCAs and pCCAs, IL-6high and IL-6low cholangiocarcinomas shared many features, while IL-33high cases had less aggressive characteristics than IL-33low cases as evidenced by lower tumour marker concentrations, smaller tumour sizes, less common vascular invasion, lower pT stages, and higher lymphocyte-to-monocyte ratios in blood. KRAS mutations were slightly less common in IL-33high cases than in IL-33low cancers (9% vs 29%; p=0.061). The strong expression of IL-33 in tissue appeared to be an independent favourable prognostic factor.

CONCLUSIONS:

IL-33high cholangiocarcinomas may represent a unique, less aggressive carcinogenetic process of the large bile ducts.

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC withoutJAK2amplification. Detection ofJAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates ofJAK2amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available,JAK2amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines withJAK2copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore,JAK2amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.