Cell migration is negatively modulated by ABCA1

Temporal and spatial changes of membrane lipid distribution in the plasma membrane are thought to be important for various cellular functions. ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for the generation of high density lipoprotein. Recently, we reported that ABCA1 maintains an asymmetric distribution of cholesterol in the plasma membrane. Here we report that ABCA1 suppresses cell migration by modulating signal pathways. ABCA1 knockdown in mouse embryonic fibroblasts accelerated cell migration and increased activation of Rac1 and its localization to detergent-resistant membranes. Phosphorylation of MEK and ERK also increased. Inhibition of Rac1 or MEK-ERK signals suppressed cell migration in ABCA1 knockdown cells. Because our experimental conditions for cell migration did not contain cholesterol or lipid acceptors for ABCA1, cellular cholesterol content was not changed. These data suggest that ABCA1 modulates cell migration via Rac1 and MEK-ERK signaling by altering lipid distribution in the plasma membrane.     

Genes located in a chromosomal inversion are correlated with territorial song in white‐throated sparrows

The genome of the white‐throated sparrow (Zonotrichia albicollis) contains an inversion polymorphism on chromosome 2 that is linked to predictable variation in a suite of phenotypic traits including plumage color, aggression and parental behavior. Differences in gene expression between the two color morphs, which represent the two common inversion genotypes (ZAL2/ZAL2 and ZAL2/ZAL2^m), may therefore advance our understanding of the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified gene expression and terrirorial aggression, including song, in a population of free‐living white‐throated sparrows. We analyzed gene expression in two brain regions, the medial amygdala (MeA) and hypothalamus. Both regions are part of a ‘social behavior network’, which is rich in steroid hormone receptors and previously linked with territorial behavior. Using weighted gene co‐expression network analyses, we identified modules of genes that were correlated with both morph and singing behavior. The majority of these genes were located within the inversion, showing the profound effect of the inversion on the expression of genes captured by the rearrangement. These modules were enriched with genes related to retinoic acid signaling and basic cellular functioning. In the MeA, the most prominent pathways were those related to steroid hormone receptor activity. Within these pathways, the only gene encoding such a receptor was ESR1 (estrogen receptor 1), a gene previously shown to predict song rate in this species. The set of candidate genes we identified may mediate the effects of a chromosomal inversion on territorial behavior.     

A photoconversion model for full spectral programming and multiplexing of optogenetic systems

Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength‐ and intensity‐dependent photoconversion, signaling, and output gene expression for our two previously engineered light‐sensing Escherichia coli two‐component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open‐source “Light Plate Apparatus” device. In principle, the parameterized model should predict the gene expression response to any time‐varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross‐reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision‐making pathways integrate multiple input signals.     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Tumor‐propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24‐dependent and Yap/Taz‐dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

The P2Y6 receptor signals through Gαq/Ca2+/PKCα and Gα13/ROCK pathways to drive the formation of membrane protrusions and dictate cell migration

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y₆ receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y₆ during cell migration. Here, we demonstrated that P2Y₆ activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y₆ increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα_q/calcium/protein kinases C (PKC) and Gα₁₃/RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y₆ receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.     

Stimulation with Porphyromonas gingivalis enhances malignancy and initiates anoikis resistance in immortalized oral keratinocytes

The aim of this study was to get new insights into molecular processes involved in tumor propagation of immortalized oral keratinocytes induced by the keystone pathogen Porphyromonas gingivalis. Cell culture experiments with immortalized OKF6 cells were performed to analyze cellular effects caused by bacterial stimulation focusing on altered gene expression, signaling pathways, proliferation rate, cell viability, migration and invasion behavior, and on the development of antiapoptotic pathways. Gene and protein expression were analyzed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and protein arrays. Trypan blue staining was used to analyze proliferation and viability, transwell assays for cellular migration, Matrigel assays for invasion, and anoikis-assays for evaluating anoikis resistance. Stimulation of OKF6 cells with Porphyromonas gingivalis led to an alteration in the molecular repertoire of proteins which are involved in cell proliferation, epithelial–mesenchymal transition, stem cell formation, migration, invasion, and anoikis resistance. Higher proliferation rates were detected in conjunction with an activation of PI3K/Akt signaling and the mTOR-pathway. Additionally, inhibition of glycogen-synthase-kinase3-β led to stabilization of β-catenin and Snail, which resulted in a switch from predominant E-cadherin to N-cadherin expression and increased expression of the stem cell markers Oct3/4, Sox2, and Nanog. Enhanced biosynthesis and enzyme activity of matrix metalloproteinase-9 was accompanied by elevated invasion behavior. Finally, anoikis resistance was detected in stimulated keratinocytes by decreased apoptosis of nonadherent cells and elevated expression of epidermal growth factor receptor and c-Met. Hence, Porphyromonas gingivalis is able to induce a more aggressive tumor-like phenotype in immortalized oral keratinocytes, thus contributing to enhanced tumor features.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin‐releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin‐releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH‐R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real‐time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)‐C pathway, exposure of the cells to phorbol 12,13‐dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen‐activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH‐induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.     

Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data

Cellular behavior in response to stimulatory cues is governed by information encoded within a complex intracellular signaling network. An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function. We previously applied quantitative mass spectrometry methods to characterize the dynamics of tyrosine phosphorylation in human mammary epithelial cells with varying human epidermal growth factor receptor 2 (HER2) expression levels after treatment with epidermal growth factor (EGF) or heregulin (HRG). We sought to identify potential mechanisms by which changes in tyrosine phosphorylation govern changes in cell migration or proliferation, two behaviors that we measured in the same cell system. Here, we describe the use of a computational linear mapping technique, partial least squares regression (PLSR), to detail and characterize signaling mechanisms responsible for HER2-mediated effects on migration and proliferation. PLSR model analysis via principal component inner products identified phosphotyrosine signals most strongly associated with control of migration and proliferation, as HER2 expression or ligand treatment were individually varied. Inspection of these signals revealed both previously identified and novel pathways that correlate with cell behavior. Furthermore, we isolated elements of the signaling network that differentially give rise to migration and proliferation. Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model. A model based on these nine sites and trained solely on data from a low HER2-expressing cell line a priori predicted migration and proliferation in a HER2-overexpressing cell line. We identify the nine signals as a “network gauge,” meaning that when interrogated together and integrated according to the quantitative rules of the model, these signals capture information content in the network sufficiently to predict cell migration and proliferation under diverse ligand treatments and receptor expression levels. Examination of the network gauge in the context of previous literature indicates that endocytosis and activation of phosphoinositide 3-kinase (PI3K)-mediated pathways together represent particularly strong loci for the integration of the multiple pathways mediating HER2′s control of mammary epithelial cell proliferation and migration. Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.     

Synthetic conversion of a graded receptor signal into a tunable,reversible switch

The ability to engineer an all‐or‐none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network ‘switches’ have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n_H>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady‐state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal‐transduction pathways.