Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche

Role of the microbiome in the normal and aberrant glycemic response

Multiple studies in the recent years suggest that the microbiome is critically important for normal host functions, while impaired host microbiome interactions contribute to the pathogenesis of numerous common disorders. Of these, much attention is recently given to the involvement of the microbiome in the pathogenesis of impaired glucose tolerance, type II diabetes mellitus (T2DM), and other metabolic disorders comprising the 'metabolic syndrome', including obesity, non-alcoholic fatty liver disease and their complications. In addition, alterations in the microbiome have been linked to the pathogenesis of type 1 diabetes mellitus (T1DM), an autoimmune disorder affecting the glycemic response, of distinct pathogenesis than T2DM. In this chapter we will discuss the roles of the microbiome in regulating the normal and impaired glycemic response in both mice and humans, and outline examples of underlying mechanisms by which the microbiome is contributing to diabetes mellitus. We will further discuss means by which the microbiome can be manipulated to develop future therapeutic interventions for hyperglycemia and its adverse effects

Non-alcoholic fatty liver and the gut microbiota

Background: Non-alcoholic fatty liver (NAFLD) is a common, multi-factorial, and poorly understood liver disease whose incidence is globally rising. NAFLD is generally asymptomatic and associated with other manifestations of the metabolic syndrome. Yet, up to 25% of NAFLD patients develop a progressive inflammatory liver disease termed non-alcoholic steatohepatitis (NASH) that may progress towards cirrhosis, hepatocellular carcinoma, and the need for liver transplantation. In recent years, several lines of evidence suggest that the gut microbiome represents a significant environmental factor contributing to NAFLD development and its progression into NASH. Suggested microbiome-associated mechanisms contributing to NAFLD and NASH include dysbiosis-induced deregulation of the gut endothelial barrier function, which facilitates systemic bacterial translocation, and intestinal and hepatic inflammation. Furthermore, increased microbiome-modulated metabolites such as lipopolysaccharides, short chain fatty acids (SCFAs), bile acids, and ethanol, may affect liver pathology through multiple direct and indirect mechanisms. Scope of review: Herein, we discuss the associations, mechanisms, and clinical implications of the microbiome's contribution to NAFLD and NASH. Understanding these contributions to the development of fatty liver pathogenesis and its clinical course may serve as a basis for development of therapeutic microbiome-targeting approaches for treatment and prevention of NAFLD and NASH. Major conclusions: Intestinal host–microbiome interactions play diverse roles in the pathogenesis and progression of NAFLD and NASH. Elucidation of the mechanisms driving these microbial effects on the pathogenesis of NAFLD and NASH may enable to identify new diagnostic and therapeutic targets of these common metabolic liver diseases. This article is part of a special issue on microbiota

Inflammation and adipose tissue macrophages in lipodystrophic mice

Lipodystrophy and obesity are opposites in terms of a deficiency versus excess of adipose tissue mass, yet these conditions are accompanied by similar metabolic consequences, including insulin resistance, dyslipidemia, hepatic steatosis, and increased risk for diabetes and atherosclerosis. Hepatic and myocellular steatosis likely contribute to metabolic dysregulation in both states. Inflammation and macrophage infiltration into adipose tissue also appear to participate in the pathogenesis of obesity-induced insulin resistance, but their contributions to lipodystrophy-induced insulin resistance have not been evaluated. We used aP2-nSREBP-1c transgenic (Tg) mice, an established model of lipodystrophy, to ask this question. Circulating cytokine elevations suggested systemic inflammation but even more dramatic was the number of infiltrating macrophages in all white and brown adipose tissue depots of the Tg mice; in contrast, there was no evidence of inflammatory infiltrates or responses in any other tissue including liver. Despite there being overt evidence of adipose tissue inflammation, antiinflammatory strategies including salicylate treatment and genetic suppression of myeloid NF-kappaB signaling that correct insulin resistance in obesity were ineffective in the lipodystrophic mice. We further showed that adipose tissue macrophages (ATMs) in lipodystrophy and obesity are very different in terms of activation state, gene expression patterns, and response to lipopolysaccharide. Although ATMs are even more abundant in lipodystrophy than in obesity, they have distinct phenotypes and likely roles in tissue remodeling, but do not appear to be involved in the pathogenesis of insulin resistance

Interleukin-1β regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity

Circulating Blood Monocyte Subclasses and Lipid-Laden Adipose Tissue Macrophages in Human Obesity

<div><p>Background</p><p>Visceral adipose tissue foam cells are increased in human obesity, and were implicated in adipose dysfunction and increased cardio-metabolic risk. In the circulation, non-classical monocytes (NCM) are elevated in obesity and associate with atherosclerosis and type 2 diabetes. We hypothesized that circulating NCM correlate and/or are functionally linked to visceral adipose tissue foam cells in obesity, potentially providing an approach to estimate visceral adipose tissue status in the non-surgical obese patient.</p><p>Methods</p><p>We preformed <i>ex-vivo</i> functional studies utilizing sorted monocyte subclasses from healthy donors. Moreover, we assessed circulating blood monocyte subclasses and visceral fat adipose tissue macrophage (ATM) lipid content by flow-cytometry in paired blood and omental-fat samples collected from patients (n = 65) undergoing elective abdominal surgery.</p><p>Results</p><p><i>Ex-vivo</i>, NCM and NCM-derived macrophages exhibited lower lipid accumulation capacity compared to classical or intermediate monocytes/-derived macrophages. Moreover, of the three subclasses, NCM exhibited the lowest migration towards adipose tissue conditioned-media. In a cohort of n = 65, increased %NCM associated with higher BMI (r = 0.250,p<0.05) and ATM lipid content (r = 0.303,p<0.05). Among patients with BMI≥25Kg/m², linear regression models adjusted for age, sex or BMI revealed that NCM independently associate with ATM lipid content, particularly in men.</p><p>Conclusions</p><p>Collectively, although circulating blood NCM are unlikely direct functional precursor cells for adipose tissue foam cells, their increased percentage in the circulation may clinically reflect higher lipid content in visceral ATMs.</p></div

Monocyte subclasses association with clinical and adipose tissue parameters.

<p><b>A)</b> Spearman Correlation analysis between clinical parameters and omental adipose tissue parameter and % circulating monocyte subclass (CM-classical monocytes; IM-intermediate monocytes; NCM-non-classical monocytes). <b>B)</b> Distribution of the patients' cohort to %NCM-low and–high according to the median value (13.2%). Omental ATM lipid content was compared between %NCM-low and %NCM-high <b>C)</b> in the whole cohort (n = 32 vs. n = 33, respectively), or separately <b>D)</b> in men (left graph, n = 13 vs. n = 11, respectively) and in women (right graph, n = 19 vs. n = 22, respectively). Adipose tissue chemokine expression was compared between %NCM-low and %NCM-high <b>E)</b> in the whole cohort (n = 11 vs. n = 15, respectively), or separately <b>F)</b> in men (left graph, n = 4 vs. n = 6, respectively) and in women (right graph, n = 7 vs. n = 9, respectively). Statistical comparison was obtained by the Mann-Whitney U test. *p< 0.05, **p<0.01.</p

Non-classical and intermediate monocytes migrate less towards conditioned medium of omental adipose tissue.

<p><b>A)</b> Following isolation of PBMCs from peripheral blood of healthy male donors, monocytes were enriched by CD14+ magnetic separation and stained for CD14 and CD16. 1–1.5*10⁶ cells were placed in the upper part of a migration chamber, placed on top of RPMI media containing 10% FBS or 10% human omental adipose tissue conditioned media (hOAT-CM). After 2.5 hours in 37°C, cells in the lower chamber were collected, counted, and <b>B)</b> analyzed by FACS, and the percentage of migrating cells of each subclass from the original fraction cultured was calculated. Summary of the results obtained from 6 independent donors are shown in <b>C</b>. Statistical comparison was obtained by the Wilcoxon matched pairs non-parametric test, *p<0.05.</p