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Neuregulin-1β (NRG-1β) is critical for cardiac development and repair, and recombinant forms are currently being assessed as possible therapeutics for systolic heart failure. We previously demonstrated that recombinant NRG-1β reduces cardiac fibrosis in an animal model of cardiac remodeling and heart failure, suggesting that there may be direct effects on cardiac fibroblasts. Here we show that NRG-1β receptors (ErbB2, ErbB3, and ErbB4) are expressed in normal human cardiac ventricular (NHCV) fibroblast cell lines. Treatment of NHCV fibroblasts with recombinant NRG-1β induced activation of the AKT pathway, which was phosphoinositide 3-kinase (PI3K)-dependent. Moreover, the NRG-1β-induced PI3K/AKT signaling in these cells required phosphorylation of both ErbB2 and ErbB3 receptors at tyrosine (Tyr)1248 and Tyr1289 respectively. RNASeq analysis of NRG-1β-treated cardiac fibroblasts obtained from three different individuals revealed a global gene expression signature consistent with cell growth and survival. We confirmed enhanced cellular proliferation and viability in NHCV fibroblasts in response to NRG-1β, which was abrogated by PI3K, ErbB2, and ErbB3 inhibitors. NRG-1β also induced production and secretion of cytokines (interleukin-1α and interferon-γ) and pro-reparative factors (angiopoietin-2, brain-derived neurotrophic factor, and crypto-1), suggesting a role in cardiac repair through the activation of paracrine signaling.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
Gastrin-releasing peptide (GRP) is a proangiogenic ligand secreted by tumors and acts directly upon binding to GRP receptor in endothelial cells. Angiogenesis plays a critical role in the pathology of various diseases, including cancer, as the formation of new blood vessels potentiates the rate of tumor growth and dissemination. GRP increases the migration of endothelial cells, but much is unknown about its role on endothelial cell proliferation and survival, as well as the signaling pathways involved. In the present study, we showed that GRP increases endothelial cell proliferation and tubule formation. There was a time-dependent increase in the levels of phosphorylated AKT, mammalian target of rapamycin (mTOR), and S6R in human umbilical vein endothelial cells treated with GRP. Interestingly, GRP treatment decreased the expression of proautophagic factors, ATG5, BECN1, and LC3 proteins. GRP also attenuated rapamycin-induced formation of autophagosomes. Moreover, overexpression of ATG5 or BECN1 significantly decreased tubule formation induced by exogenous GRP, whereas siRNA against ATG5 or BECN1 resulted in increased tubule formation with GRP treatment. Our results show that GRP inhibits the process of autophagy in vascular endothelial cells, thereby increasing endothelial cell proliferation and tubule formation. Here, we describe a novel role of GRP in the regulation of autophagy of endothelial cells, thereby providing a potential new therapeutic strategy in targeting angiogenesis during cancer progression.
BACKGROUND - Stromal-epithelial interactions are important in both development and prostate cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. We investigated whether stromal cathepsin D (CathD) overexpression affects prostate tumorigenesis through a paracrine mechanism.
METHODS - Normal prostate fibroblasts (NPF) were retrovirally transduced to overexpress cyclin D1 (CD1) and were designated NPF(CD1) . Cathepsin D expression was knocked down using shRNA in cancer associated fibroblasts (CAF) and NPF(CD1) . We analyzed these stromal cell lines using immunohistochemistry, Western blot, and tissue recombination.
RESULTS - An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue. Overexpression of CD1 in normal prostate fibroblasts (NPF(CD1) ) produced a phenotype similar to, but more moderate than, CAF in a tissue recombination model. Knockdown studies revealed that CathD is required for NPF(CD1) motility and invasive growth in vitro. BPH-1 cell proliferation was found to be induced when cultured with NPF(CD1) conditioned medium, this effect was inhibited when CathD was knocked down in NPF(CD1) cells. Overexpression of CathD in prostate stromal cells induced malignancy in adjacent epithelium, and this transformation was inhibited when stromal CathD expression was knocked down in CAF.
CONCLUSIONS - The study presented here demonstrates increased CathD expression is seen in human CAF. The upregulation of CD1 results in concomitant increases in CathD expression. Elevated CathD expression in the stroma contributes to tumor promotion.
Copyright © 2012 Wiley Periodicals, Inc.
The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1(+) cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31(-) and endothelial CD31(+) cells. The population of Sca-1(+)CD31(+) endothelial cells was significantly reduced, whereas the population of Sca-1(+)CD31(-) stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1(+)CD31(-) stromal cells but not in Sca-1(+)CD31(+) endothelial cells. We found that mouse cardiac Sca-1(+)CD31(-) stromal cells predominantly express mRNA encoding A(2B) adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1(+)CD31(-) stromal cells obtained from wild-type and A(2B) receptor knockout mouse hearts, we demonstrated that A(2B) receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1(+)CD31(-) stromal cells that increase release of IL-6, IL-8, and VEGF in response to A(2B) receptor stimulation. Thus, our study identified A(2B) adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart.
The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase β in airway epithelium (IKTA (IKKβ trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.
AIMS/HYPOTHESIS - We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors.
METHODS - Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations.
RESULTS - Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function.
CONCLUSIONS/INTERPRETATION - This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.
The Neuregulin-1 gene encodes a family of ligands that act through the ErbB family of receptor tyrosine kinases to regulate morphogenesis of many tissues. Work in isolated cardiac cells as well as genetically altered mice demonstrates that neuregulin-1/ErbB signaling is a paracrine signaling system that functions in endocardial-endothelial/cardiomyocyte interactions to regulate tissue organization during development as well as maintain cardiac function throughout life. Treatment of animals with cardiac dysfunction with recombinant neuregulin-1beta improves cardiac function. This has led to ongoing early phase clinical studies examining neuregulin-1beta as a potential novel therapeutic for heart failure. In this review we synthesize the literature behind this rapidly evolving area of translational research. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."
Copyright © 2011 Elsevier Ltd. All rights reserved.
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN-expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN-expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.