Targeting Cell Signaling Research Articles

Engineering the RNA-Nanobio Interface.

RNA nanotechnology is attracting a great deal of attention recently. As the multiple roles that RNA plays in molecular biology and physiological regulation become clearer, there are many opportunities for engineering RNA-Nanoparticle Complexes (RNA-NPCs). The high “engineerability” of RNA-NPCs comes from the ability to modify the RNA and NP chemistry. For example, the NP can be derived from materials with anticancer activity and the RNA delivered by it, designed to target cell signaling pathways that contribute to the molecular basis of these diseases. Despite this rapid advancement and the availability of new quantification and characterization techniques, a key challenge is to develop a better understanding of the RNA-nanobio interface; that is, the interactions of RNA with NP (RNA-nanobio interface) and how that impacts the structure, function, delivery, and activity of the RNA. Here, we attempt to summarize the state-of-the-art in this new and exciting field, and to lay out potential directions for bioengineering research on RNA-NPCs.

Chemokines in tumor proximal fluids.

Chemokines are chemotactic cytokines produced by leukocytes and other types of cells including tumor cells. Their action is determined by the expression of cognate receptors and subsequent signaling in target cells, followed by the modulation of cytoskeletal proteins and the induction of other responses. In tumors, chemokines produced by neoplastic/stroma cells control the leukocyte infiltrate influencing tumor growth and progression. Tumor cells also express functional chemokine receptors responding to chemokine signals, promoting cell survival, proliferation and metastasis formation. Chemokines may be detected in serum of cancer patients, but due to the paracrine nature of these molecules, more significant concentrations are found in the tumor adjacent, non-vascular fluids, collectively called tumor proximal fluids. This review summarizes the expression of CC and CXC chemokines in these fluids, namely in interstitial fluid, pleural, ascitic, and cyst fluids, but also in urine, saliva, cerebrospinal fluid, cervical secretions and bronchoalveolar lavage fluid. Most comparative clinical studies reveal increased chemokine levels in high-grade tumor proximal fluids rather than in low-grade tumors and benign conditions, indicating shorter survival periods. The data confirm peritumoral fluid chemokines as sensitive diagnostic and prognostic markers, as well as offer support for chemokines and their receptors as potential targets for antitumor therapy.

Targeting Cell Signalling Pathways: Novel Targets for Alzheimer’s Disease

Targeting Cell Signalling Pathways: Novel Targets for Alzheimer’s Disease

Microvesicle-mediated Wnt/β-Catenin Signaling Promotes Interspecies Mammary Stem/Progenitor Cell Growth

Signaling mechanisms that regulate mammary stem/progenitor cell (MaSC) self-renewal are essential for developmental changes that occur in the mammary gland during pregnancy, lactation, and involution. We observed that equine MaSCs (eMaSCs) maintain their growth potential in culture for an indefinite period, whereas canine MaSCs (cMaSCs) lose their growth potential in long term cultures. We then used this system to investigate the role of microvesicles (MVs) in promoting self-renewal properties. We found that Wnt3a and Wnt1 were expressed at higher levels in MVs isolated from eMaSCs compared with those from cMaSCs. Furthermore, eMaSC-MVs were able to induce Wnt/β-catenin signaling in different target cells, including cMaSCs. Interestingly, the induction of Wnt/β-catenin signaling in cMaSCs was prolonged when using eMaSC-MVs compared with recombinant Wnt proteins, indicating that MVs are not only important for transport of Wnt proteins, but they also enhance their signaling activity. Finally, we demonstrate that the eMaSC-MVs-mediated activation of the Wnt/β-catenin signaling pathway in cMaSCs significantly improves the ability of cMaSCs to grow as mammospheres and, importantly, that this effect is abolished when eMaSC-MVs are treated with Wnt ligand inhibitors. This suggests that this novel form of intercellular communication plays an important role in self-renewal.

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities.

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50–100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

Cannabinoid receptors in the kidney.

The endocannabinoid system modulates cell signaling targets that are essential for energy homeostasis. Endocannabinoids bind to G protein-coupled receptors in the central nervous system and periphery, including the kidney. Modulation of cannabinoid receptor 1 (CB1) and CB2 activity in the kidney in diabetes and obesity has been identified as potential therapeutic target to reduce albuminuria and renal fibrosis. This review will highlight the results of recent studies that have identified a role for CB1 and CB2 in normal and pathological renal conditions. CB1 and CB2 have been reported to play key roles in renal function and dysfunction. Recent studies have determined that antagonism of CB1 and agonism of CB2 in diabetic nephropathy and obesity associated kidney disease can reduce albuminuria, potentially by acting on both the glomeruli and tubules. Emerging studies have also identified a role for CB1 in renal diseases associated with fibrosis, with CB1 upregulated in multiple models of human nephropathies. Emerging studies using isolated cells, rodent models, and human studies have identified a critical role for the endocannabinoid system in renal function and disease. Thus, therapeutics that modulate the activity of CB1 and CB2 in renal disease could become clinically relevant.

Divergent effects of Porcupine and Wntless on WNT1 trafficking, secretion, and signaling

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmitoylation, trafficking, secretion, and signaling. We first investigated possible interactions between PORCN, WLS, and WNT1, by carrying out co-immunoprecipitation studies. These studies demonstrate the existence of a complex containing PORCN and WLS. They further show that PORCN and WLS compete for binding to WNT1. Then, we used gain-of-function studies to investigate the cooperation between PORCN and WLS as well as possible biochemical interactions between PORCN, WLS, and WNT1. Consistent with the proposed roles for PORCN and WLS, we show that overexpression of PORCN promotes palmitoylation of WNT1 while overexpression of WLS does not. Overexpression of PORCN enhances the ability of WLS to promote WNT1 trafficking to the cell surface as well as secretion, but decreases the ability of WLS to activate WNT1 signaling in target cell. These observations suggest that the levels of WNT1 on the cell surface and in the media are not the sole determinants of the activation of Wnt signaling in target cells.

Systems-wide Experimental and Modeling Analysis of Insulin Signaling through Forkhead Box Protein O1 (FOXO1) in Human Adipocytes, Normally and in Type 2 Diabetes

Insulin resistance is a major aspect of type 2 diabetes (T2D), which results from impaired insulin signaling in target cells. Signaling to regulate forkhead box protein O1 (FOXO1) may be the most important mechanism for insulin to control transcription. Despite this, little is known about how insulin regulates FOXO1 and how FOXO1 may contribute to insulin resistance in adipocytes, which are the most critical cell type in the development of insulin resistance. We report a detailed mechanistic analysis of insulin control of FOXO1 in human adipocytes obtained from non-diabetic subjects and from patients with T2D. We show that FOXO1 is mainly phosphorylated through mTORC2-mediated phosphorylation of protein kinase B at Ser(473) and that this mechanism is unperturbed in T2D. We also demonstrate a cross-talk from the MAPK branch of insulin signaling to stimulate phosphorylation of FOXO1. The cellular abundance and consequently activity of FOXO1 are halved in T2D. Interestingly, inhibition of mTORC1 with rapamycin reduces the abundance of FOXO1 to the levels in T2D. This suggests that the reduction of the concentration of FOXO1 is a consequence of attenuation of mTORC1, which defines much of the diabetic state in human adipocytes. We integrate insulin control of FOXO1 in a network-wide mathematical model of insulin signaling dynamics based on compatible data from human adipocytes. The diabetic state is network-wide explained by attenuation of an mTORC1-to-insulin receptor substrate-1 (IRS1) feedback and reduced abundances of insulin receptor, GLUT4, AS160, ribosomal protein S6, and FOXO1. The model demonstrates that attenuation of the mTORC1-to-IRS1 feedback is a major mechanism of insulin resistance in the diabetic state.

Topical drugs for the treatment of psoriasis

In recent years, great progress has been made in the pathogenesis of psoriasis, which has provided multiple new targets for the treatment of psoriasis, and ushered the treatment, especially topical treatment, of psoriasis to a new era. Some new targeted topical drugs with action mechanisms different from those of traditional drugs have been applied in clinical practice, which are mainly small-molecule inhibitors targeting cell signaling pathways or enzymes involved in the pathogenesis of psoriasis, such as the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, phosphodiesterase type 4 (PDE4), mitogen - activated/extracellular-signal - regulated kinase kinase (MEK), vascular endothelial growth factor receptor (VEGFR), tropomyosin receptor kinase A (TrKA), retinoic acid receptor-related orphan receptor γ (RORγ), and so on. At present, many phaseⅠ and Ⅱ clinical trials are conducted for these drugs. Some completed trials have shown that they have good effectiveness, safety and tolerance, providing more options for the topical treatment of psoriasis. Key words: Psoriasis; External application drugs; Small-molecule inhibitors; Signal transducing pathway; Enzyme

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts.

There is currently tremendous interest in developing anti-cancer therapeutics targeting cell signaling pathways important for both cancer cell metabolism and growth. Several epidemiological studies have shown that diabetic patients taking metformin have a decreased incidence of pancreatic cancer. This has prompted efforts to evaluate metformin, a drug with negligible toxicity, as a therapeutic modality in pancreatic cancer. Preclinical studies in cell line xenografts and one study in patient-derived xenograft (PDX) models were promising, while recently published clinical trials showed no benefit to adding metformin to combination therapy regimens for locally advanced and metastatic pancreatic cancer. PDX models in which patient tumors are directly engrafted into immunocompromised mice have been shown to be excellent preclinical models for biomarker discovery and therapeutic development. We evaluated the response of four PDX tumor lines to metformin treatment and found that all four of our PDX lines were resistant to metformin. We found that the mechanisms of resistance may occur through lack of sustained activation of adenosine monophosphate-activated protein kinase (AMPK) or downstream reactivation of the mammalian target of rapamycin (mTOR). Moreover, combined treatment with metformin and mTOR inhibitors failed to improve responses in cell lines, which further indicates that metformin alone or in combination with mTOR inhibitors will be ineffective in patients, and that resistance to metformin may occur through multiple pathways. Further studies are required to better understand these mechanisms of resistance and inform potential combination therapies with metformin and existing or novel therapeutics.

Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens.

Cancer stem cells in drug resistant lung cancer: Targeting cell surface markers and signaling pathways.

Lung cancer is the leading cause of cancer mortality worldwide. Despite advances in anti-cancer therapies such as chemotherapy, radiotherapy and targeted therapies, five-year survival rates remain poor (<15%). Inherent and acquired resistance has been identified as a key factor in reducing the efficacy of current cytotoxic therapies in the management of non-small cell lung cancer (NSCLC). There is growing evidence suggesting that cancer stem cells (CSCs) play a critical role in tumor progression, metastasis and drug resistance. Similar to normal tissue stem cells, CSCs exhibit significant phenotypic and functional heterogeneity. While CSCs have been reported in a wide spectrum of human tumors, the biology of CSCs in NSCLC remain elusive. Current anti-cancer therapies fail to eradicate CSC clones and instead, favor the expansion of the CSC pool and select for resistant CSC clones thereby resulting in treatment resistance and subsequent relapse in these patients. The identification of CSC-specific marker subsets and the targeted therapeutic destruction of CSCs remains a significant challenge. Strategies aimed at efficient targeting of CSCs are becoming increasingly important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. This review focuses on the current knowledge of cancer stem cell markers in treatment-resistant lung cancer cells and the signaling cascades activated by these cells to maintain their stem-like properties. Recent progress in CSC-targeted drug development and the current status of novel agents in clinical trials are also reviewed.

Bioinert Anodic Alumina Nanotubes for Targeting of Endoplasmic Reticulum Stress and Autophagic Signaling: A Combinatorial Nanotube-Based Drug Delivery System for Enhancing Cancer Therapy.

Although nanoparticle-based targeted delivery systems have gained promising achievements for cancer therapy, the development of sophisticated strategies with effective combinatorial therapies remains an enduring challenge. Herein, we report the fabrication of a novel nanomaterial, so-called anodic alumina nanotubes (AANTs) for proof-of-concept cancer therapy by targeting cell signaling networks. This strategy is to target autophagic and endoplasmic reticulum (ER) stress signaling by using thapsigargin (TG)-loaded AANTs cotreated with an autophagy inhibitor 3-methyladenine (3-MA). We first show that AANTs are nontoxic and can activate autophagy in different cell types including human fibroblast cells (HFF), human monocyte cells (THP-1), and human breast cancer cells (MDA-MB 231-TXSA). Treatment with 3-MA at a nontoxic dose reduced the level of autophagy induced by AANTs, and consequently sensitized breast cancer cells to AANTs-induced cellular stresses. To target autophagic and ER stress signaling networking, breast cancer cells were treated with 3-MA together with AANTs loaded with the prototype ER stress inducer TG. We demonstrated that 3-MA enhanced the cancer cell killing effect of AANTs loaded with TG. This effect was associated with enhanced ER stress signaling due to the combination effect of TG and 3-MA. These findings not only demonstrate the excellent biocompatibility of AANTs as novel biomaterials but also provide new opportunities for developing ER- and autophagy-targeted delivery systems for future clinical cancer therapy.

Competitive and compensatory effects of androgen signaling and glucocorticoid signaling

Androgens and glucocorticoids have competitive and compensatory effects in several physiological and pathophysiological processes. Although blood androgen levels affect blood glucocorticoid levels and vice versa, it does not fully explain the relationship between the effects of androgens and glucocorticoids. Androgens and glucocorticoids exert their functions through binding to androgen receptor (AR) and glucocorticoid receptor (GR), respectively. AR homodimer and GR homodimer bind to the androgen response element (ARE) and glucocorticoid response element (GRE), respectively, where they positively or negatively regulate transcription. AR/GR heterodimer can also form but whether it has a physiological role is unclear. Notably, some ARE/GRE sites are recognized by both AR and GR. This review focuses on the functional interventions between androgen signaling and glucocorticoid signaling in target cells that are involved in muscle atrophy, lipid metabolism in adipocytes and hepatocytes, and pancreatic β-cell death. Androgens and glucocorticoids exert opposite effects by differentially regulating key genes ( e.g. , insulin-like growth factor-1, atrogin-1, and thioredoxin-interacting protein) involved in these physiological processes. We also review functional compensation between these steroids in the development of castration-resistant prostate cancer in which glucocorticoids compensate for the castration-induced loss of AR function by activating key genes ( e.g. , serum/glucocorticoid-regulated kinase 1). The gene expressions regulated by androgens and glucocorticoids are regulated through at least three different mechanisms in target cells: (i) regulation of applicable ligand levels by modulation of steroid metabolite enzyme levels, (ii) regulation of each other's receptor levels, and (iii) competitive binding between AR and GR on ARE/GRE sites. Recent findings shed light on the complicated relationship between androgen signaling and glucocorticoid signaling in various cellular processes.

Platelets induce apoptosis via membrane-bound FasL

AbstractAfter tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

Pseudohypoparathyroidism type Ib in 2015

The term pseudohypoparathryoidism (PHP) refers to a group of rare genetic and epigenetic disorders characterized by resistance to the action of parathyroid hormone (PTH) that activates cAMP signaling in target cells. Together with pseudohypoparathyroidism, Albright hereditary osteodystrophy (AHO) and progressive osseous heteroplasia (POH) represent rare, related and deeply impairing disorders encompassing heterogeneous features, such as brachydactyly, ectopic ossifications, short stature, mental retardation and endocrine deficiencies due to resistance to the action of different hormones. The two main subtypes, PHP-Ia and PHP-Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster respectively, while mutations in the PRKAR1A and PDE4D genes (also involved in mediating cAMP signalling) have been demonstrated in patients with acrodysostosis, a disease of bone formation with characteristics similar to AHO. The molecular overlap among these disorders indicates the need for different classification models and seriously alters our understanding of the mechanisms through which GNAS defects, together with the new recently described defects involving other components of the cAMP signalling cascade, cause AHO-related disorders.

Unzipping androgen action through ZIP9: a novel membrane androgen receptor.

Nearly 5 decades ago, the existence of an androgen receptor (AR) molecule was originally identified by Shutsung Liao at the University of Chicago with concordant findings published by Nicholas Bruchovsky and Jean Wilson (1–3). These seminal studies used rat prostate tissue and specifically described a protein with high affinity for 5 -dihydrotestosterone (DHT) that accumulated in the nucleus upon binding, initiating RNA synthesis and explaining the mode of action for androgens within target cells.Subsequentstudiesbymultiple laboratoriesdetermined that AR regulates the transcription of androgen-responsive genes and plays a pivotal role in male reproductive tract development, sexually dimorphic brain activities, and multiple other target tissues. Importantly, AR has an essential role in prostate carcinogenesis and progression, with blockade of its activity used as a primary therapeutic target. Classical nuclear AR signaling (also known as genomic actions) in target cells involves the diffusion of testosterone through the cell membrane and into the cytoplasm, where it or its metabolite DHT binds to the AR, triggering conformational changes, dissociation from heat shock protein 90, dimerization, and translocation to the cell nucleus. There, the AR complex binds to DNA at androgen-response elements, recruits coregulators, and activates transcription of genes that are then translated into proteins. This classic or genomic mechanism of androgen action is relatively slow, requiring approximately 1–6 hours for alterations in gene transcriptional activity. It is noteworthy that as early as 1975, Liao also described rapid androgen actions occurring within 10 minutes of DHT administration that were independent of RNA synthesis, speculating that “androgens may affect known protein factors or others that are yet to be identified” (4). While research attention remain focused on nuclear steroid receptor signaling pathways, culminating in the cloning of steroid receptor genes in the 1980s, including that for AR (5, 6), evidence began to emerge in the 1990s that androgens are also capable of exerting rapid effects by interacting with AR in the cytoplasm or at the plasma membrane. Membrane-associated ARs were initially described in cell types such as T lymphocytes, monocytes, and osteoblasts to effect influx of calcium (7–9). Subsequent studies found that similar to membrane estrogen receptors, membrane ARs can also interactwithsignalingmolecules torapidlyactivatesignaling pathways, including the MAPK/ERK (10) and focal adhesion kinase 1 (FAK) /phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways (11, 12). ARs along the cell surface have also been shown more recently to regulate actin cytoskeletal reorganization and prostate-specific antigen secretion in prostate cancer cells (13). The genomic AR signaling pathway has long been the standard therapeutic target for prostate cancer, and initially blockade of nuclear AR activity is very effective in slowing prostate tumor growth and progression. However, tumors eventually become resistant to nuclear AR antagonists and resume growth and rapid metastatic progression. Nongenomic androgen signaling represents an alternative focus for the development of potential treatment strategies that could enhance tumor response to antiandrogen treatment, particularly in delaying the development of castrate-resistant prostate cancer (14). Progress on studies of nongenomic androgen signaling in prostate cancer cells has been hampered by the challenges associated with identification of novel membrane ARs in these

Extracellular Vesicles from Neural Stem Cells Transfer IFN-γ via Ifngr1 to Activate Stat1 Signaling in Target Cells

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

A novel method for simulating insulin mediated GLUT4 translocation.

Glucose transport in humans is a vital process which is tightly regulated by the endocrine system. Specifically, the insulin hormone triggers a cascade of intracellular signals in target cells mediating the uptake of glucose. Insulin signaling triggers cellular relocalization of the glucose transporter protein GLUT4 to the cell surface, which is primarily responsible for regulated glucose import. Pathology associated with the disruption of this pathway can lead to metabolic disorders, such as type II diabetes mellitus, characterized by the failure of cells to appropriately uptake glucose from the blood. We describe a novel simulation tool of the insulin intracellular response, incorporating the latest findings regarding As160 and GEF interactions. The simulation tool differs from previous computational approaches which employ algebraic or differential equations; instead, the tool incorporates statistical variations of kinetic constants and initial molecular concentrations which more accurately mimic the intracellular environment. Using this approach, we successfully recapitulate observed in vitro insulin responses, plus the effects of Wortmannin-like inhibition of the pathway. The developed tool provides insight into transient changes in molecule concentrations throughout the insulin signaling pathway, and may be employed to identify or evaluate potentially critical components of this pathway, including those associated with insulin resistance. In the future, this highly tractable platform may be useful for simulating other complex cell signaling pathways. Biotechnol. Bioeng. 2014;111: 2454-2465. © 2014 Wiley Periodicals, Inc.